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Research ArticleArticle

Aspirin-Triggered 15-epi-Lipoxin A4 (ATL) Generation by Human Leukocytes and Murine Peritonitis Exudates: Development of a Specific 15-epi-LXA4 ELISA

Nan Chiang, Tomoko Takano, Clary B. Clish, Nicos A. Petasis, Hsin-Hsiung Tai and Charles N. Serhan
Journal of Pharmacology and Experimental Therapeutics November 1998, 287 (2) 779-790;

Abstract

Aspirin (ASA) triggers the formation of 15-epi-lipoxins (15-epi-LXs or ATL [ASA-triggered LX]), which are potent bioactive eicosanoids that may contribute to the therapeutic impact of ASA. To elucidate the role of these new compounds in vivo, it is essential to establish quick and sensitive detection methods. To this end, we prepared an enzyme-linked immunosorbent assay specific for 15-epi-LXA4that proved to be highly sensitive (IC50 ∼ 50 pg, minimum detection ∼ 3.5 pg) and stereoselective. The amounts of 15-epi-LXA4 generated by human neutrophils from peripheral blood of healthy volunteers using this enzyme-linked immunosorbent assay were in agreement with those values obtained by liquid chromatography. Formation of 15-epi-LXA4 was cell ratio-dependent during THP-1 (a monocytic leukemia cell line)-neutrophil interactions with ASA-treated cells, and 15-epi-LXA4 was not detected with either cell type alone. Generation of 15-epi-LXA4 was also examined in murine peritonitis with ASA administration. Exudates from ASA-treated mice showed increased production of 15-epi-LXA4 that was diminished by indomethacin, a blocker of ASA-dependent acetylation of prostaglandin G/H synthase. A cytochrome P450 inhibitor administered in the presence of ASA did not prevent 15-epi-LXA4 formation, which suggests that P450 does not significantly contribute to formation of 15-epi-LXA4 in this murine model. These results indicate that the new enzyme-linked immunosorbent assay is both sensitive and selective for 15-epi-LXA4 and that 15-epi-LXA4is produced by human leukocyte-leukocyte interactions. In addition, 15-epi-LXA4 is generated by inflammatory exudates when ASA is administered during murine peritonitis and when prostaglandin G/H synthase is upregulated and acetylated. This assay should provide rapid means to investigate 15-epi-LXA4 generation in both cellular and animal models.

Lipoxins belong to the eicosanoid family of bioactive lipid mediators that carry trihydroxytetraene structures as distinguishing features. They are generated in mammals predominantly by transcellular biosynthetic routes during cell-cell interactions, which is now recognized as an important means of both amplifying and generating new lipid-derived mediators (reviewed in Serhan, 1997). Two major transcellular routes of LX biosynthesis by LO interactions in human cell types are established. LXs generated by these two pathways carry their C-15 hydroxyl group mainly in the 15S-configuration, which is inserted by lipoxygenase-based mechanisms. LXs not only are formed in vitro in isolated cells but also are generated in humans and in experimental animals. Situations that lead to cell-cell adherence and inflammation in vivo can produce LXs within local microenvironments (reviewed in Serhan, 1997). At the nanomolar level, LXs display vasodilatory and immunoregulatory roles in in vitro and in vivo models. LXs inhibit, for example, both neutrophil and eosinophil chemotaxis (Lee et al., 1989;Soyombo et al., 1994). In coincubation systems, LXA4 inhibits PMN transmigration across both endothelial and epithelial cells (Colgan et al., 1993). Thus their immunoregulatory actions implicate them as endogenous “stop signals” acting on human leukocytes (Serhan, 1997).

Aspirin is the lead nonsteroidal anti-inflammatory drug (Weissmann, 1991). Besides its antithrombotic and anti-inflammatory action, low doses of aspirin have several newly recognized beneficial actions that include prevention of cardiovascular diseases (Savage et al., 1995) and decreasing incidence of lung, colon and breast cancer (Giovannucci et al., 1995; reviewed in Levy, 1997;Schreinemachers and Everson, 1994). They may also be relevant in treatment of human immunodeficiency virus (Macilwain, 1993). Although inhibition of PGHS could account for many of ASA’s pharmacological properties (Samuelsson, 1982), it is difficult to ascribe the newly recognized therapeutic effects of ASA solely to the inhibition of prostaglandin formation (Weissmann, 1991). Along these lines, a novel biosynthetic pathway was recently uncovered that is triggered by ASA treatment and costimulation of human leukocytes and either vascular endothelial cells or mucosal epithelial cells (Clària et al., 1996; Clària and Serhan, 1995). Briefly, ASA acetylation of PGHS-2 in endothelial or epithelial cells switches the enzyme’s catalytic activity from a prostaglandin synthase to anR-lipoxygenase also found with isolated enzymes (Lecomteet al., 1994; Mancini et al., 1994; Xiao et al., 1997). Hence, in either activated endothelial or epithelial cells, acetylated PGHS-2 generates 15R-HETE from endogenous stores of AA and leads to production of 15-epi-LXs viatranscellular biosynthesis in PMN (reviewed in Serhan, 1997). The 15-epi-LXs (ATL, aspirin-triggered LXs) have their C-15 hydroxyl group in the R configuration that is retained from the precursor 15R-HETE (Clàra et al., 1996; Clària and Serhan, 1995; Serhan, 1997).

The newly uncovered ATL have been examined in several experimental settings. It has already been established that 15-epi-LXA4is approximately twice as potent as native LXA4 in inhibiting neutrophil adhesion to endothelial cells (Clària and Serhan, 1995) and that 15-epi-LXB4 inhibits cell proliferation in vitro (Clària et al.,1996). Recently, we found that stable analogs of LXA4 and 15-epi-LXA4—specifically, analogs that resist rapid inactivation—applied topically to mouse ears dramatically inhibit leukocyte infiltration (Takano et al., 1997). These analogs also inhibit leukocyte rolling and adherence in vivo in the rat mesenteric microvasculature (Scalia et al., 1997). Given that ATL are generated via ASA-triggered transcellular biosynthesis and possess potent actions in events of interest in inflammation and proliferative disorders, they may contribute to some of the beneficial actions of ASA. Therefore, it is critical to establish cellular and animal models to test this hypothesis and develop methods with appropriate sensitivity and selectivity to elucidate biological settings involved in the generation and roles of ATL.

The antibody-based assays (radioimmunoassay or ELISA), which can detect picogram amounts of materials, have proved valuable tools in detecting eicosanoids (reviewed in Granström et al., 1987). Along these lines, we developed a LXA4 ELISA that detects LXA4 generated in vitro and in vivo (Levy et al., 1993). When the aspirin-triggered 15-epi-LX pathway was uncovered, we found that the LXA4 antiserum, which is selective when compared with other eicosanoids and LXA4 isomers (Levy et al.,1993), did not distinguish the chirality at carbon 15 between LXA4 and 15-epi-LXA4 (Clària and Serhan, 1995). Thus this LXA4 antiserum made possible the preliminary assessment of 15-epi-LXA4 in ASA-dependent fashion in rat kidney (Badr et al., 1996). Here, we report the development of a new 15-epi-LXA4-selective ELISA with high sensitivity and stereoselectivity that distinguishes 15-epi-LXA4 from native LXA4, and we demonstrate its utility for detecting 15-epi-LXA4generation by both cells and animal models.

Materials and Methods

Materials.

The following drugs and chemicals were kindly provided by or obtained from the sources indicated: THP-1 (human acute monocytic leukemia cell line) and Sf9 (Spodoptera frugiperda) cells (ATCC, Rockville, MD), cell culture media and reagents (Biowhittaker, Walkersville, MD), goat anti-rabbit IgG (Zymed, San Francisco, CA), 96-well polystyrene plates for ELISA (Costar, Cambridge, MA), anti-human PGHS-2 polyclonal antibody (Oxford Biomedical Research, Oxford, MI), extract-clean C18 cartridges (500 mg) (Alltech, Deerfield, IL), 3,3′,5,5′-tetramethyl benzidine (TMB), protein A beads, LPS (derived from Escherichia coli, serotype 055:B5), ionophore A23187, indomethacin, phenylmethyl sulfonyl fluoride (PMSF), TritonX-100, ethylenediaminetetraacetic acid (EDTA), leupeptin and aprotinin (Sigma Chemical Co., St. Louis, MO), AA (Cayman Chemical Co., Ann Arbor, MI), 15R-HETE, 15S-HETE, 5S-HETE, 12S-HETE, LXA4 and LXB4 (Cascade Biochem, Reading, Berkshire, England), 15-epi-LXA4-methyl ester, 15-(R/S)-methyl-LXA4 and 15-epi-LXB4-methyl ester prepared by total organic synthesis using described procedures (Nicolaou et al., 1991;Serhan et al., 1995), 17-ODYA (Biomol, Plymouth Meeting, PA), methyl formate (Eastman Kodak, Rochester, NY), aspirin (Spectrum Chemical, Cardena, CA), LXA4 ELISA kit (ELISA Technology Inc., Lexington, KY), and chemiluminescence substrate kits (Boehringer Mannheim, Indianapolis, IN). Recombinant baculovirus containing human PGHS-2 cDNA was a generous gift from Dr. Robert A. Copeland of DuPont Merck (Wilmington, DE).

Cell culture and isolation.

THP-1 cells were grown in RPMI supplemented with 10% fetal bovine serum and antibiotics in a 37°C incubator with 5% CO2. Human PMN were isolated from fresh peripheral blood of healthy donors who had denied taking aspirin or other medication for at least one week. PMNs were isolated by Ficoll-Hypaque gradient centrifugation and dextran sedimentation (Böyum, 1968) as described in Levy et al. (1993). Cells were suspended in PBS++ and contained 96 ± 3% PMN (n = 3), as enumerated by light microscopy.

Preparation of 15-epi-LXA4 antibody.

15-epi-LXA4 was coupled to KLH through the succinimide ester of 15-epi-LXA4 (Tai and Yuan, 1978) and used as the antigen to produce polyclonal antibody. 15-epi-LXA4 was also labeled with HRP through the succinimide ester of 15-epi-LXA4, which was then diluted (1:1000) and used to develop an ELISA for 15-epi-LXA4. Rabbits were immunized initially with 1 mg of 15-epi-LXA4-KLH conjugate through s.c. injections. They were then boosted twice monthly with 0.5 mg of the conjugate and bled via their ear veins one week after each boost. The blood was collected, and 1/10 volume of 3.8% sodium citrate was added to prevent clotting. It was then centrifuged at 2000 × g for 10 min. Clear supernatants were collected as crude sera and used to prepare the ELISA.

15-epi-LXA4 ELISA.

Polystyrene 96-well plates were precoated with about 1 μg of affinity-purified goat anti-rabbit IgG per well in 100 μl of coating buffer (0.1 M NaHCO3/Na2CO3, pH 9.6) for 2 hr at room temperature. Each plate was then blocked with 200 μl of 5% nonfat milk, 5% sucrose in EIA buffer (100 mM K2HPO4/KH2PO4, pH 7.4, containing 150 mM NaCl, 0.5% BSA and 0.025% proclin 300) for 1 hr at room temperature. After three washings with buffer consisting of 10 mM K2HPO4/KH2PO4, pH 7.4, containing 0.05% Tween 20 (i.e., wash buffer), the assay was initiated by adding diluted antisera (1:10,000 dilution, 50 μl), HRP-labeled 15-epi-LXA4 (100 μl) and serial dilutions of synthetic 15-epi-LXA4 or samples (50 μl) into each well. Each plate was gently shaken for 1 hr at room temperature. After three washings with wash buffer, the enzyme reaction was carried out by adding 200 μl of substrate (1.25 mM TMB and 6 mM H2O2 in 25 mM sodium citrate buffer, pH 3.5) for 30 min at room temperature. The absorbance changes at 650 nm were monitored using an ELISA Plate Reader (Bio-Tek Instruments, Inc., Winooski, VT). All samples were assayed in duplicate.

Immunoprecipitation of synthetic 15-epi-LXA4.

Immobilized 15-epi-LXA4 IgG was prepared by incubation of 0.5 ml of protein A beads with 15-epi-LXA4 antiserum in ELISA buffer (100 μl for 18 hr). The beads were washed and suspended in 0.5 ml of ELISA buffer. The synthetic 15-epi-LXA4 was then incubated with immobilized IgG for 1 hr at room temperature. The beads were rapidly pelleted, suspended in ELISA buffer (0.5 ml) and added to two volumes of cold methanol (1°C). The samples were then extracted using solid-phase C18 cartridges (Serhan, 1990) and taken to LC/MS/MS for further analysis.

LC/MS/MS of synthetic LXA4 and 15-epi-LXA4.

LC/MS/MS was performed on an LCQ (Finnigan MAT, San Jose, CA) quadrupole ion trap mass spectrometer system equipped with an electrospray atmospheric pressure ionization probe. Compounds were dissolved in methanol and injected into the HPLC component, which consisted of a SpectaSYSTEM P4000 quaternary gradient pump (Thermo Separation Products, San Jose, CA), a LUNA C18-2 (150 × 2 mm, 5 μm) column (Phenomenex, Torrance, CA), and a rapid spectra scanning SpectraSYSTEM UV2000 UV/VIS absorbance detector (Thermo Separation Products). This column was eluted isocratically with methanol/water/acetic acid (65:35:0.01, v/v/v) at 0.2 ml/min into the electrospray probe. The spray voltage was set to 5 kV and the heated capillary to 250°C. Over a 2-sec scan cycle, full-scan mass spectra (MS) were acquired by scanning between m/z 95-410 in the negative ion mode, followed by the acquisition of product ion mass spectra (MS/MS) of the most intense molecular anions (e.g.,[M-H] = m/z 351 for LXA4 and 15-epi-LXA4).

Cell Incubations.

For PMN incubations, freshly isolated PMN (20 × 106/ml) were incubated in Dulbecco’s phosphate-buffered saline (PBS++) with 5 μM of A23187 for 30 min at 37°C in the presence of 15R-HETE (10 μM) or vehicle (ethanol) alone (Clària and Serhan, 1995). All incubations were stopped at the indicated time intervals by addition of two volumes of cold methanol (1°C). For coincubation of THP-1 and PMN, THP-1 was incubated for 16 hr with LPS (1 μg/ml) at 37°C (Endo et al., 1996; Fu et al., 1990). The cells were then pelleted, suspended (10 × 106/ml) in PBS++ and then treated with ASA (300 μM) for 20 min at 37°C and coincubated with freshly isolated PMN at different cell ratios for 5 min at 37°C, followed by addition of 5 μM of A23187 and 20 μM of AA for 30 min at 37°C. These concentrations were selected on the basis of reported values. For example, ASA treatment switches human PGHS-2 oxygenase activity to anR-lipoxygenase activity that peaks at 20 min (Xiao et al., 1997). 15-epi-LXA4 generation in coincubation of cell types without P450 appears to be solely dependent on ASA acetylation of PGHS-2. This notion is further supported by the finding that statistically significant differences in 15-epi-LXA4levels were not observed with ASA concentrations from 100 to 500 μM (Clària and Serhan, 1995), which suggests that acetylation of PGHS-2 is the limiting event in this system and that acetylation of endothelial cell PGHS-2 in intact cells was saturated at these concentrations. Also, it was established that 15R-HETE production by acetylated PGHS-2 is both AA- and time-dependent. For example, 20 μM of AA gave maximum production of 15R-HETE at 3 min, which remained at equivalent levels for 5 to 30 min with recombinant PGHS-2 (Mancini et al., 1997). Thus these conditions [i.e., ASA (300 μM for 20 min) and AA (20 μM for 30 min)] were utilized for THP-1/PMN coincubation experiments to optimize 15-epi-LXA4 generation. Incubations were terminated and samples were then extracted with solid-phase C18 cartridges (Serhan, 1990). The methyl formate eluants from the solid-phase extraction were taken to dryness with a stream of N2, and the samples were next suspended in methanol/water (2:1; 100 μl) and taken for ELISA analysis. Serial dilutions of the samples were incubated with standard amounts of HRP-labeled 15-epi-LXA4 and 15-epi-LXA4 antiserum in 96-well plates precoated with goat anti-rabbit IgG.

Mouse peritoneal lavage.

Balb/c mice with an average weight of 20 g were individually injected within the peritoneum with LPS (1.25 mg/kg b.wt.). After 16 hr, the mice were treated with either 5 mg of indomethacin or vehicle (ethanol), again by i.p. injection, for 30 min before treatment with ASA (125 mg/kg b.wt.) and 0.7 mg of 17-ODYA or ASA alone via i.p. injection for 30 min. In vivo ASA treatment at a dose of 150 mg/kg s.c. or 30 to 300 mg/kg p.o. showed almost complete inhibition of TXB2 production in mouse (Anton et al., 1990; Molinari et al.,1987). At an i.p. dose of 25 to 100 mg/kg, ASA was also shown to inhibit photochemically induced platelet aggregation in pial microvessels of mouse in vivo (el-Sabban and Radwan, 1997). 17-ODYA is a potent inhibitor of P450 eicosanoid metabolism that does not inhibit either cyclooxygenase or LO activity (Muerhoff et al., 1989). Addition of 17-ODYA (5 μM, 20 min) to A549 cells resulted in about 50% reduction in 15-HETE generation in vitro (Clària et al., 1996). In addition, 17-ODYA was shown to inhibit the metabolism of AA by cytochrome P450 in renal cortical microsomes of rats in a concentration-dependent fashion with IC50 < 100 nM. Even at very high concentrations (5 and 25 μM), 17-ODYA did not inhibit the production of either PGE2 or PGF2α (Zou et al., 1994). The results of these experiments guided the amounts of inhibitors and parameters selected for the in vivo experiments. To this end, peritonitis was induced by i.p. injection of 2 ml of 2% casein, which was prepared as described in Yamaki and Oh-ishi (1990). The mice were sacrificed 4 hr later, and peritoneal lavages were collected. For some experiments, the lavage was added directly to two volumes of cold methanol (1°C). In other experiments, the lavages were treated further with indomethacin (300 μM) or vehicle (DMSO) for 5 min and with ASA (300 μM) and 17-ODYA (5 μM) or ASA alone for 5 min before the addition of ionophore A23187 (5 μM) for 30 min at 37°C. The incubations were individually added to two volumes of cold methanol (1°C) and allowed to precipitate at 4°C for 30 to 60 min. The samples were then extracted with solid-phase C18 cartridges, vide supra.

Immunoblots for PGHS-2.

THP-1 cells were exposed to LPS (1 μg/ml) for 16 hr at 37°C. Balb/c mice with an average weight of about 20 g were exposed to LPS (1.25 mg/kg b.wt.) for 16 hr by i.p. injection. Cell pellets from THP-1 incubations as well as mouse peritoneal lavage samples were obtained after centrifugation at 1000 rpm for 10 min at room temperature. Individual cell pellets were washed with PBS++, freeze-thawed three times in liquid nitrogen and suspended in lysis buffer consisting of 50 mM Tris/HCl, pH 7.5, containing 10 mM EDTA, 1% Triton X-100, 1 mM PMSF, 20 μM leupeptin and 10 μg/ml aprotinin. Equal amounts of protein were subjected to SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) microporous membrane by electroblotting. Membranes were blocked in 5% nonfat milk in TBST (0.9% NaCl and 0.05% Tween-20 in 20 mM Tris/HCl, pH 7.4) and probed with an anti-human PGHS-2 polyclonal antibody (1:100 dilution) for 1 hr. After washing three times with TBST, membranes were incubated with HRP-linked goat anti-rabbit IgG (1:10,000 dilution) for 1 hr, and the immunoreactive bands were developed by incubating with chemiluminescence substrates and visualized by exposure to an X-ray film.

Statistical analysis.

Results were expressed as the mean ± S.E.M. from n = 3. Statistical evaluation of the results was carried out using Student’s t test, and P values < .05 were considered statistically significant.

Results

ELISA sensitivity and selectivity.

To develop a sensitive and selective ELISA for 15-epi-LXA4, rabbits (denoted numbers 5347 and 5348) were immunized with KLH-linked 15-epi-LXA4, and antisera were collected and tested for their ability to recognize 15-epi-LXA4, which was prepared by total organic synthesis. After conditions for ELISA were optimized (see “Materials and Methods”), concentration dependence was evaluated using serial dilutions of unlabeled 15-epi-LXA4 as a competitor as in figure 1. Fifty percent inhibition of maximum binding with HRP-linked 15-epi-LXA4(IC50) was ∼0.14 pmol (50 pg) and ∼0.21 pmol (75 pg) of 15-epi-LXA4 for antisera 5347 and 5348, respectively. The minimum amounts detectable (∼80% of maximum binding) were ∼10 fmol (3.5 pg) and ∼21 fmol (7.5 pg) of 15-epi-LXA4 for 5347 and 5348, respectively (not shown). Because antiserum 5347 displayed higher affinity toward 15-epi-LXA4, it was selected for further experiments.

Figure 1
Figure 1

Specificity of 15-epi-LXA4 antiserum and direct comparison with LXA4 antiserum. 15-epi-LXA4 (1–1,000 pg), several lipoxin analogs (panel A) or individually related HETEs (panel B) were incubated with standard amounts of HRP-labeled 15-epi-LXA4 and 15-epi-LXA4 antiserum in 96-well plates precoated with goat anti-rabbit IgG as described in “Materials and Methods.” On the vertical axis, % B/B0 represents the bound enzyme activity in the presence (B) of unlabeled compound relative to its absence (B0). All data are expressed as the mean ± S.E.M. from n = 3.

To determine the antibody’s selectivity, we examined, by ELISA, 1 pg to 1 ng of several lipoxin-related structures (fig. 1A) as well as individually related HETEs (fig. 1B) for their ability to interact with the antibody. ELISA was also performed in the presence of high concentrations of each compound (up to 100 ng) to determine IC50 values and cross-reactivity (table1). The methyl ester of 15-epi-LXA4 was almost as potent as 15-epi-LXA4in interacting with the antibody (∼83% of cross-reactivity). Among the other closely related eicosanoids, cross-reactivity was found to be 1.25% for the biosynthetic precursor 15R-HETE, followed by 0.63% for native LXA4 (which differed only in chirality at carbon 15 alcohol in the S configuration), 0.25% for 5S-HETE and 15-R/S-methyl-LXA4 (in which a methyl group was introduced at carbon 15), 0.17% for 15-epi-LXB4 methyl ester, and 0.1% for LXB4and 15S-HETE (table 1). Substantial cross-reactivity was not found for 12S-HETE. Thus these findings indicate that the new anti-15-epi-LXA4 antisera were highly stereoselective toward 15-epi-LXA4 in recognizing the C-15 hydroxyl group at an R configuration.

View this table:
Table 1

Cross-reactivity of 15-epi-LXA4 antiserum to lipoxins and related eicosanoids

LC/MS/MS of synthetic and immunoprecipitated 15-epi-LXA4.

Because the anti-LXA4antisera recognized both 15-epi-LXA4 and LXA4, with a preference for 15-epi-LXA4, it was essential also to establish and confirm the physical properties of the synthetic materials used to generate the antisera. Liquid chromatography/mass spectrometry analysis gave UV chromatograms when monitored at 300 nm with single major peaks with retention times of 14.7 and 15.7 min for LXA4 and 15-epi-LXA4, respectively, using a LUNA C18-2 column (see “Materials and Methods”). The two epimers do not resolve as free acids with some HPLC columns. The present column and mobile phase made possible about 1 min of separation. The major anion in the MS spectra for these retention times was m/z351 (fig. 2A and C, cleavage sitea), which represented [M-H]− for both LXA4 and 15-epi-LXA4. Each MS/MS spectrum of LXA4 and 15-epi-LXA4 revealed essentially the same fragmentation patterns (fig. 2, B and D). Prominent daughter ions were observed for both at m/z 333 [351-H2O]; 315 [351-H2O, -H2O]; 307 [351-CO2] (see fig. 2, A and C, cleavage siteb); 289 [351-H2O, -CO2]; 271 [351-H2O, -H2O, -CO2]; 251 [351-CHO(CH2)4CH3] (see fig. 2, A and C, cleavage site d); 235 [351-CHO(CH2)3COOH] (see fig. 2, A and C, cleavage site c′); 233 [d-H2O]; 215 [d-H2O, -H2O]; 207 [d-CO2]; 189 [d-H2O, -CO2]; 135 [d-CHO(CH2)3COOH] and 115 [CHO(CH2)3COO−] (see fig. 2, A and C, cleavage site c). These ions are consistent with those recently reported for LXA4 analyzed by electrospray/collision-induced dissociation mass spectrometry (Griffiths et al., 1996). Successive MS/MS analysis revealed statistically significant differences in the intensities of certain daughter ions in these two epimers, particularly for m/z333, 251, 215 and 115 (table 2). Conformational differences between the molecules, arising from opposite stereochemistries at carbon 15, are likely to influence the relative probabilities for each fragmentation, and these properties may therefore serve as an additional means for identification of these compounds (determination of UV absorbance, retention times and intensities of selective MS/MS fragments; see table 2).

Figure 2
Figure 2

LC/MS/MS of synthetic LXA4 and 15-epi-LXA4. Mass spectra of synthetic LXA4(panel A) and 15-epi-LXA4 (panel C) and MS/MS analysis of LXA4 (panel B) and 15-epi-LXA4 (panel D) were performed with a Finnigan LCQ LC/MS/MS as described in “Materials and Methods.”

View this table:
Table 2

LXA4 and 15-epi-LXA4 MS/MS daughter ion relative intensities

After the fragmentation was evaluated for 15-epi-LXA4, LC/MS/MS analysis was used next to determine whether 15-epi-LXA4 antiserum interacts with 15-epi-LXA4 in the conditions used for ELISA assay development. To this end, synthetic 15-epi-LXA4 (100 ng) was incubated with anti-15-epi-LXA4 antiserum-bound protein A beads in ELISA buffer 0.5 ml for 1 hr at room temperature. The protein A beads with the immunoprecipitated materials were centrifuged (14,000 rpm, 2 min), extracted (as described in “Materials and Methods”) and taken for analysis by LC/MS/MS as above. The major signature ions for 15-epi-LXA4 were observed (data not shown), which indicates that the anti-15-epi-LXA4 antiserum did indeed recognize and interact with 15-epi-LXA4 within the conditions of the assay.

Generation of 15-epi-LXA4 by activated human PMN.

To examine the ability of this ELISA to quantitate 15-epi-LXA4 generated by cellular sources and to qualify its potential uses, we stimulated isolated suspensions of human peripheral blood PMN (107 cells) in the presence or absence of 15R-HETE. Upon activation, approximately 40 ng of 15epi-LXA4 was generated by 107 PMN exposed to 15R-HETE (fig. 3), whereas only less than ∼1 ng of 15-epi-LXA4 was found in the absence of the exogenous precursor 15R-HETE with PMN from peripheral blood of healthy individuals. These values (i.e., ∼40 ng/107 PMN) were in agreement with recently reported values (Clària et al., 1996;Clària and Serhan, 1995) obtained with isolated peripheral blood PMN from healthy volunteers, where 15-epi-LXA4 production was quantitated by combined RP-HPLC and ELISA analyses. Given the sensitivity of 15-epi-LXA4 ELISA, we could detect 15-epi-LXA4 from less than 107 PMN, and it proved to be linear in this range (i.e., dependent on cell number). This cell incubation number, 107 PMN, was selected for these experiments to ensure statistically significant values that facilitated quantitation and established the time course of 15-epi-LXA4 generation, which minimized the impact of individual donor variations with lower cell numbers. Thus the present ELISA proved useful in detecting 15-epi-LXA4 generated by cellular sources.

Figure 3
Figure 3

Generation of 15-epi-LXA4 by activated PMN. Human PMN (10 × 106) were incubated with 5 μM of A23187 at 37°C for 30 min in the presence of 15R-HETE (10 μM) or vehicle alone. For the kinetics of 15-epi-LXA4production (inset), incubations were collected at indicated time-points, and cells were separated from supernatant by rapid microcentrifugation. Incubations were terminated by adding two volumes of ice-cold methanol, and the samples were purified by extraction with solid-phase C18 cartridges. Serial dilutions of the samples were incubated with standard amounts of HRP-labeled 15-epi-LXA4and 15-epi-LXA4 antiserum in 96-well plates for ELISA analysis as described in “Materials and Methods.” The values are expressed as the mean ± S.E.M. from n = 3. Statistical difference was obtained (*P < .01) by comparing values from incubations with stimulated PMN alone and incubations with stimulated PMN plus 15R-HETE.

To determine the time course of 15-epi-LXA4 generation by PMN, cell pellets were collected and separated from supernatants by rapid centrifugation and assessed for amount of 15-epi-LXA4. Statistically significant values for 15-epi-LXA4 were obtained within 2.5 min (P = .04) after addition of stimulus to PMN in suspension and remained elevated from 2.5 to 20 min. The amount of 15-epi-LXA4 observed at 2.5 min did not prove to be statistically different from that obtained at 20 min (P = .16). Also, 15-epi-LXA4 remained in the supernatant throughout the time course (fig. 3, inset). The lack of statistical significance between these points probably reflects individual donor variability for each time course. Together, these findings indicate that 15-epi-LXA4 was rapidly generated and released from the cells. Moreover, 15-epi-LXA4 was neither lost from the supernatant nor associated with cell pellets during these incubations.

Generation of 15-epi-LXA4 by coincubation of PMN and THP-1 cells.

Both LX and ATL are predominantly generatedvia transcellular biosynthesis during cell-cell interaction in mammalian tissues (reviewed in Serhan, 1997). Only a few individual cell types are known to generate lipoxins as a product of a single cell origin, such as activated macrophages isolated from rainbow trout (Pettitt et al., 1991) or endogenously primed PMN isolated from asthmatic patients (Chavis et al., 1996). It was therefore of interest to determine whether single cell types, which possess both PGHS-2 and 5-LO, have the ability to generate 15-epi-LXA4. Along these lines, the production of 15-epi-LXA4 by THP-1 cells was evaluated using this new ELISA (fig. 4). PGHS-2 was up-regulated in the THP-1 cells by exposure to LPS (1 μg/ml) for 16 hr (figs. 4 and 5). LPS elicits a marked increase in PGHS-2 mRNA as well as protein levels in macrophages (Morham et al., 1995). Of interest, significant amounts of 15-epi-LXA4 were not detected in LPS-treated THP-1 cells incubated in the presence of ASA and exogenous AA when compared with values obtained with reagents alone (ASA, AA and A23187). It was recently shown that expression of 5-LO can be up-regulated by GM-CSF in human monocytes (Ring et al., 1996). However, significant increases in 15-epi-LXA4 production were not observed in THP-1 cells treated with GM-CSF (1 ng/ml) for 48 hr and LPS (1 μg/ml) for 16 hr, although 5-LO transcript levels were increased after this treatment (data not shown). Thus the presence of both 5-LO and PGHS-2 within a single cell type was not sufficient to generate 15-epi-LXA4.

Figure 4
Figure 4

15-Epi-LXA4 generation by costimulation of human PMN and THP-1 cells. THP-1 cells (10 × 106) were incubated with LPS (1 μg/ml) for 16 hr and treated with ASA (300 μM) for 20 min before coincubation with or without PMN at a cell ratio of 1:6 (THP-1/PMN) for 30 min at 37°C in the presence of A23187 (5 μM) and AA (20 μM). The coincubations were performed at different cell ratios in the presence or absence of AA (20 μM) or A23187 (5 μM) (inset). The incubations were terminated, and the samples were extracted for ELISA analysis as in figure 1. The values are expressed as the mean ± S.E.M. from n = 3. Statistical differences were obtained when compared to values from incubations at a cell ratio of 1:0 (*P < .01, **P = .01) and compared with values from incubations at a cell ratio of 1:6 with vehicle alone (+P < .01, ++P = .04).

Figure 5
Figure 5

PGHS-2 is expressed by THP-1 cells and mouse peritoneal lavage cells: Western blot analysis. Mice were injected with LPS (1.25 mg/kg b.wt.). THP-1 cells were treated with LPS (1 μg/ml) at 37°C. Sixteen hours later, the THP-1 cells and cells from peritoneal lavage were collected and suspended in lysis buffer. The whole-cell lysates (20 μg/lane) were subjected to SDS-PAGE and transferred to PVDF membrane by electroblotting. The blot was probed using an anti-human PGHS-2 polyclonal antibody (1100 dilution) and developed as described in “Materials and Methods.” Lane 1 contains cell lysates from untreated peritoneal lavage, Lane 2 the cell lysates from LPS-treated peritoneal lavage. Lane 3 contains the untreated THP-1 cells, Lane 4 the LPS-treated THP-1 cells. Molecular weight markers are indicated by arrows.

In sharp contrast, 15-epi-LXA4 was generated during coincubation of human leukocytes in the presence of stimulated PMN at a cell ratio of 1:6 (THP-1/PMN). Here, generation of 15-epi-LXA4 was increased to ∼5 ng/107 THP-1 (P = .01 was obtained for a cell ratio of 1:6 vs. 1:0 with A23187 alone and P = .04 compared with a cell ratio of 1:6 with vehicle only) (fig. 4 inset), and it proved to be generated from endogenous sources of substrate. By direct comparison, in the presence of exogenous AA, 15-epi-LXA4 generation was ∼19 ng/107 THP-1 (∼4-fold increase), a finding that supports AA as a precursor for 15-epi-LXA4 biosynthesis. In addition, generation of 15-epi-LXA4 was observed to be cell ratio-dependent in both the presence and the absence of exogenous AA, and it required costimulation (fig. 4 inset). It was previously shown that 15-epi-LXA4 generation in HUVEC/PMN and A549/PMN coincubations reached maximum levels at a cell ratio between 1:5 and 1:10. In the present experiments, a THP-1/PMN cell ratio of 1:6 was chosen because it approximated the monocyte/neutrophil ratio in human peripheral blood of healthy individuals. At this cell ratio, statistically significant values for 15-epi-LXA4 were obtained in both the presence and the absence of exogenous AA compared with a cell ratio of 1:0 (P < .01 and P = .01, respectively) and compared with a cell ratio of 1:6 with vehicle only (P < .01 and P = .04, respectively). Thus 15-epi-LXA4 was generated during THP-1 and PMN stimulation mainly viatranscellular biosynthesis during heterotypic peripheral blood cell-cell interactions.

Generation of 15-epi-LXA4 by mouse peritoneal exudates.

To determine whether 15-epi-LXA4 can be detected with animal models, experiments were carried out using a mouse peritonitis model. In this model, PGHS-2 protein levels were shown to be up-regulated by i.p. injection of LPS. Peritonitis was induced by i.p. injection of casein (as in Yamaki and Oh-ishi, 1990). In these experiments, up-regulation of PGHS-2 was also demonstrated by Western blot analysis, and the immunoreactive bands were observed at ∼70 kDa in peritoneal lavage samples from LPS-treated mice (fig. 5, lanes 1 and 2). The molecular sizes of the PGHS-2 proteins were found to be slightly different between mouse peritoneal cell lysates and human THP-1 cells (fig. 5, lane 4). Because the size of PGHS-2 protein in THP-1 cells was the same as that in Sf9 cells overexpressing human PGHS-2 (data not shown), it is possible that PGHS-2 from human and mouse were glycosylated to a different extent. Along these lines, it has been demonstrated that mouse PGHS-2 has four N-glycosylation sites and that their molecular sizes varies from 65 to 74 kDa as a result of N-glycosylation heterogeneity (Otto et al., 1993).

Four hours after leukocyte infiltration was initiated, approximately 25 × 106 cells were obtained from peritoneal lavage of each mouse. The leukocyte populations represented ∼73% PMN and 10% monocytes (and/or macrophages), respectively, as determined by H/E staining and enumeration by light microscopy. To test whether ASA treatment of the mice results in the generation of 15-epi-LXA4 during an inflammatory event, ASA was administered by i.p. injection (see experimental timeline, upper panel of fig. 6). The collected peritoneal exudates from each mouse were incubated in the presence or absence of ionophore A23187 without addition of exogenous substrates, and samples from individual mice were analyzed separately. Without administration of ASA, approximately 0.5 ng of 15-epi-LXA4 per 5-ml lavage was associated with peritoneal exudates from each mouse. Given one or two doses of ASA (see experimental timeline of fig. 6), the mean values for 15-epi-LXA4 production were about 1.5 and 1.8 ng, respectively, per 5-ml peritoneal lavage per mouse (fig. 6). Naive animals (without any treatment) gave very low levels (<0.2 ng/5-ml peritoneal lavage per mouse) of 15-epi-LXA4 (data not shown). The physiological relevance of this value obtained in the absence of experimental challenge is currently not known.

Figure 6
Figure 6

Peritoneal inflammatory exudates from ASA-treated mice generate 15-epi-LXA4. Mice were injected with LPS (1.25 mg/kg b.wt.) to induce PGHS-2, and 16 hr later they were treated with vehicle or (panel a) one bolus dose of ASA (0.125 g/kg b.wt.) by i.p. injection for 30 min before casein-initiated induction of neutrophil infiltration for 4 hr. b) Consecutive doses of ASA were given in a separate group of animals that received ASA (0.125 g/kg b.wt.) by i.p. injection 30 min before casein injection and before sacrificing the mice. The lavage exudates were collected and stimulated with 5 μM A23187 (30 min, 37°C). The values are expressed as the mean ± S.E.M. from three independent experiments. The values from both treatment groups were statistically different (*P = .05 for group a, **P = .03 for group b) from those obtained with animals that did not receive ASA (vehicle). The samples were extracted as described in “Materials and Methods.” b.wt., body weight; IP, i.p. injection.

Because LPS up-regulates PGHS-2 (Herschman, 1996) and was shown to induce neutrophil recruitment into mouse bronchoalveolar lavagein vivo (Gonçalves de Moraes et al., 1996), we tested the possibility that animals treated with LPS could give rise to 15-epi-LXA4 in the absence of casein. Our results showed a low level of 15-epi-LXA4 generation in these LPS-treated animals in the presence or absence of ASA (0.25 and 0.3 ng, respectively, per 5-ml peritoneal lavage per mouse), which suggests that LPS alone was not sufficient to elicit neutrophil infiltration into peritoneal lavage (data not shown). Casein-induced neutrophil infiltration and ASA are required in this scenario to generate statistically significant levels of 15-epi-LXA4. In addition, because casein alone does not induce PGHS-2 (Kuwamotoet al., 1997), it is not likely to trigger 15-epi-LXA4 generation (in the absence of LPS or other PGHS-2 inducers). Thus these results demonstrate that ASA administration in a model of peritonitis gives inflammatory exudates that, when stimulated, generate 15-epi-LXA4 in appreciable levels from endogenous sources of mouse inflammatory cells.

To address the potential routes involved in 15-epi-LXA4production by inflammatory peritonitis exudates, we carried out additional treatments with the exudates. It was of particular interest to find that further exposure of peritoneal lavages ex vivoto ASA (300 μM) resulted in a 5- to 10-fold increase in the amounts of 15-epi-LXA4 generated (e.g., 10 ng/5 ml or ∼4 ng/107 lavage cells) by the peritoneal lavage exudate cells taken from each mouse. Indomethacin, which blocks PGHS-2 acetylation (Mancini et al., 1997), was examined for its effect on 15-epi-LXA4 generation. The results shown in fig.7 indicate that the formation of 15-epi-LXA4 was significantly decreased (P < .01 compared with ASA treatment alone; also P = .02 compared with ASA plus ODYA treatment) after i.p. injection as well as treatment of the exudates with indomethacin (5 mg and 300 μM, respectively). Because the generation of 15-epi-LXA4 during epithelial or adenocarcinoma cell (A549) and PMN interactions involves two separate pathways to produce 15R-HETE, namely ASA-acetylated PGHS-2 and cytochrome P450 (Clària et al., 1996), we assessed the potential contribution of P450 to 15-epi-LXA4production by the mouse exudates. To this end, individual mice were injected i.p. with 17-ODYA, and collected exudates were treatedex vivo with 17-ODYA, a specific cytochrome P450 inhibitor (Muerhoff et al., 1989), together with ASA (see experimental timeline in fig. 7). The results showed that 17-ODYA did not significantly reduce 15-epi-LXA4 generation in this tissue.

Figure 7
Figure 7

Indomethacin but not 17-ODYA inhibits 15-epi-LXA4 generation by mouse peritoneal exudates. Mice were injected with LPS (1.25 mg/kg b.wt.). Sixteen hours later, they were treated with ASA (0.125 g/kg b.wt.) and with 5 mg indomethacin, ASA (0.125 g/kg b.wt.) and 0.7 mg 17-ODYA or with ASA alone before induction of PMN infiltration by i.p. injection of casein. The peritoneal lavage samples (18 × 106 PMN/mouse) were incubated further with 300 μM ASA and indomethacin, 300 μM ASA and 5 μM 17-ODYA or ASA alone and stimulated with A23187 (5 μM) as described in “Materials and Methods.” The reactions were stopped and the samples extracted. The values are expressed as the mean ± S.E.M. from three independent experiments. Values for indomethacin plus ASA treatment were statistically different from ASA treatment alone (*P < .01) and ASA plus ODYA (+P = .02). b.wt., body weight; IP, i.p. injection.

Discussion

Earlier, we developed a LXA4 ELISA that was both sensitive and selective for LXA4 when directly compared with other eicosanoids including cross-reactivity with LXB4(Levy et al., 1993). Of particular interest, this antibody was later found to recognize both the recently identified 15-epi-LXA4 and native LXA4 (data not shown), which have been found to be generated by different biosynthetic routes, 15-epi-LXA4 giving greater biopotencies in several systems. The physical properties of these biologically derived epimers are very similar. Indeed, MS/MS analysis of LXA4 and 15-epi-LXA4 indicated that both give essentially identical ions upon fragmentation (fig. 2B and D), differing only in the relative intensities of certain daughter ions (table 2). Therefore, we developed a new ELISA using a 15-epi-LXA4 antibody that recognizes the newly identified 15-epi-LXA4 (IC50 ≈ 50 pg, minimum detection = 3.5 pg) in the picogram range (see fig.1). This antibody also proved to be stereoselective toward 15-epi-LXA4 in that it recognizes the carbon 15 alcohol in the R configuration and distinguishes between LXA4 and 15-epi-LXA4.

Antisera used in the present experiments were raised with synthetic compounds of absolute stereochemistry. The specificity of anti-15-epi-LXA4 antiserum was clearly demonstrated with eicosanoids that lacked either a C-15 alcohol group (i.e.,5S-HETE and 12S-HETE) or the tetraene structure (i.e., 15R-HETE and 15S-HETE). Also, antibody recognition was reduced when a methyl group was introduced at C-15 to replace hydrogen (i.e.,15-R/S-methyl-LXA4) at carbon 15. Eicosanoids carrying a 15S-alcohol group (i.e.,LXA4, LXB4 and 15S-HETE) were not recognized by this antibody (fig. 1, A and B). In earlier reports, 15-epi-LXA4 was identified by RP-HPLC combined with gas chromatography analyses of derivatized materials, which distinguish 15-epi-LXA4 from native LXA4 by their different retention times in each chromatography system (Clària et al., 1996; Clària and Serhan, 1995). In order to achieve base-line separation, methyl esters of both LXA4 and 15-epi-LXA4 and derivatization were required. Derivatization could lead to quantity losses of the compounds of interest during sample work-up. The present results indicated that this ELISA is at least 200 times more sensitive than UV-monitored RP-HPLC (minimum UV detection ∼ 1 ng). Thus the present ELISA proved to be highly sensitive and stereoselective and is a rapid and relatively inexpensive procedure for monitoring 15-epi-LXA4.

Results from kinetic experiments performed to evaluate 15-epi-LXA4 production by PMN suggest that the activation of 5-LO in PMN displays a similar time course in converting either AA (Krump and Borgeat, 1994; Pouliot et al., 1994) or 15R-HETE (fig. 3). PMN stimulated with the divalent cation ionophore A23187 gives a sustained release of both arachidonate and leukotriene B4 formation evident within 1 min that plateaus by 3 min with washed PMN in suspension (Krump and Borgeat, 1994). This time course is similar in the case of 15-epi-LXA4generation by PMN (fig. 3 inset).

Generation of 15-epi-LXA4 by THP-1 cells and leukocyte-leukocyte interactions was also investigated. Because THP-1 cells possess both PGHS-2 and 5-LO, it was possible in theory (see fig.8) that this cell type could generate 15-epi-LXA4 without requiring cell-cell interactions and transcellular biosynthesis. THP-1 cells incubated alone did not, however, produce detectable amounts of 15-epi-LXA4 (fig.4). In this particular cell line, it is possible that functional 5-LO is not located at appropriate intracellular sites within the THP-1 cells to interact with PGHS-2-derived endogenous 15R-HETE. In sharp contrast, in the presence of human PMN that possess functional 5-LO, 15-epi-LXA4 generation was increased by more than 10-fold after ASA treatment (fig. 4). This value for 15-epi-lipoxin generation is in the range of, and comparable to, those generated during either PMN-vascular endothelial or PMN-airway epithelial cell interactions (Clària et al., 1996; Clària and Serhan, 1995).

Figure 8
Figure 8

Proposed scheme for generating 15-epi-LXA4 by murine inflammatory exudates. Arachidonic acid is converted to 15R-HETE predominantly by ASA-acetylated PGHS-2 in macrophage and epithelial cells. Cytochrome P450 might make only a minor contribution to 15R-HETE generation in the mouse. 15R-HETE then undergoes transcellular conversion by 5-LO in PMN to 15-epi-LXA4via a 15-epi-5(6)-epoxytetraene intermediate.

15-epi-LXA4 was also generated from endogenous sources of AA (11–14 nM) within levels required to evoke bioactivity (Clària et al., 1996; Clària and Serhan, 1995;Serhan et al., 1995), because 15-epi-LXA4inhibits PMN adhesion to vascular endothelial cells at levels as low as 0.1 nM in vivo (Clària and Serhan, 1995; also seeScalia et al., 1997). Together, these are the first results demonstrating that 15-epi-LXA4 not only is generated during endothelial or epithelial cell-leukocyte (neutrophil) interactions, but also is generated during leukocyte-leukocyte interactions, as exemplified here by results obtained with THP-1 cells (a monocytic leukemia cell line) activated with human PMN.

It is of interest that 15-epi-LXA4 was generated by exudate inflammatory cells taken from a casein-induced murine peritonitis model (fig. 6). In this complex inflammatory milieu, 15-epi-LXA4was formed and was probably the product of PMN carrying 5-LO activity (PMN represent 73% of total lavage cells) interacting with acetylated PGHS-2 of macrophages. LPS induces PGHS-2 activity in mouse peritoneal macrophages (Reddy and Herschman, 1994). It also induces TNF-α gene expression (Goldfeld et al., 1990), which could have contributed to the up-regulation of PGHS-2. The macrophages represented ∼10% of the total cells within the inflammatory exudates. In theory, it is also possible that epithelial or other cell types of the peritoneal cavity could have contributed 15R-HETE to produce 15-epi-LXA4. Upon stimulation of exudate cells from ASA-treated mice, 15-epi-LXA4 was clearly generated in an ASA-dependent fashion (fig. 6). Here, small amounts of 15-epi-LXA4 were detected without ASA treatment of the mice; they could have been the product of PGHS-2 acetylated by potential endogenous acetylating agents (see fig. 8). It is well known that acetylation of histone, as a part of post-translational modification, is important for transcription, assembly of chromatin and maintenance of cell differentiation (Wade et al., 1997). Thus other PGHS-2 endogenous acetylating routes may exist within the mouse peritoneal cavity, or, of equal likelihood, small amounts of 15R-HETE could be produced by origins other than PGHS-2 or P450 that remain to be identified (vide infra).

LPS was used in THP-1/PMN coincubations and in a mouse peritonitis model to induce PGHS-2 (fig. 5, lanes 2 and 4). LPS was shown to induce PGHS-2 activity in human monocytes, reaching maximum values within 12 to 24 hr after treatment (Endo et al., 1996; Fu et al., 1990), and a similar induction time course was observed in mouse peritoneal macrophages (Reddy and Herschman, 1994). PGHS-2 can also be up-regulated by other stimuli, such as growth hormones, cytokines, serum and phorbol esters (Herschman, 1996), in a wide range of cell types. It was shown that, in HUVEC/PMN and A549/PMN coincubations, formation of 15-epi-LXA4 was enhanced by IL-1β, which induced PGHS-2 in HUVEC and A549 cells (Clàriaet al., 1996; Clària and Serhan, 1995). In addition, IL-1β and TNF-α were shown to induce PGHS-2 in HT-29 C1.19A cells (human enterocytes), which, in the presence of ASA, generated 15R-HETE (a precursor of 15-epi-LXA4biosynthesis) (Gronert et al., 1998). Therefore, it appears that other specific cytokines that can regulate PGHS-2 appearancein vivo (i.e., TNF-α and IL-1β) should also trigger 15-epi-LXA4 generation in THP-1/PMN coincubations as well as in murine peritonitis induced by cytokines when ASA is administered, because acetylation of PGHS-2 is a required step in this pathway for 15R-HETE formation.

In the mouse, several other origins of substrate may contribute to 15-epi-LXA4. For example, it is also possible that racemic 15-HETE was generated either by epithelial cytochrome P450 (Capdevilaet al., 1986) or via nonenzymatic pathways involving reactive oxygen species released by activated PMN (Fridovich and Porter, 1981). Without stimulation of the inflammatory exudates, the amounts of 15-epi-LXA4 generated by ASA-treated mice were in a relatively low range (0.3–0.6 ng, 0.17–0.34 nM in each individual mouse) (data not shown). These values could reflect the long exposure of the mouse peritoneum to high levels of LPS in vivo that could induce the production of proinflammatory cytokines or other factors that might uncouple and/or metabolically inactivate 15-epi-LXA4 transcellular biosynthesis. Eicosanoids are local mediators and are well known to be rapidly generated and degraded by further transformation. Nevertheless, 15-epi-LXA4 was detected within peritonitis samples, yet with additional ex vivo treatment of exudates with ASA, the amounts of 15-epi-LXA4 were increased to ∼10 ng per 5 ml of the peritoneal lavage from each mouse (fig. 7). Because further ASA treatment enhanced the amounts of 15-epi-LXA4 generated, the levels found in vivo may reflect incomplete PGHS-2 acetylation by ASA and/or degradation of 15-epi-LXA4 by further conversion and metabolism or, which is more likely, absorption of the compound by the surrounding peritoneal tissues during the time course of the experimental peritonitis. It should also be noted that ASA undergoes rapid hydrolysis and deacetylation in aqueous medium and may have done so, to some extent, in the present experiment when ASA was injected into the mouse peritoneal cavity. Given these considerations, 15-epi-LXA4 was produced and generated in an ASA-dependent fashion from endogenous sources within a range commensurate with its currently known bioactions.

We also tested the relative contributions of ASA-acetylated PGHS-2 and/or P450 to the production of 15-epi-LXA4 in the mouse inflammatory exudates (fig. 7) for comparison with the pathways observed with human tissues (Clària et al., 1996;Clària and Serhan, 1995). Oxygenation of AA by cytochrome P450 results in ∼40% R and 60% S configuration of 15-HETE (Capdevila et al., 1986). In addition, cytochrome P450 contributes to 15-HETE generation by human adenocarcinoma-derived A549 cells (Clària et al., 1996) and results in 15-epi-LXA4 generation during A549-PMN costimulation. In contrast, in this mouse model, 15-epi-LXA4 formation was not inhibited by the P450 inhibitor (17-ODYA). The present results also show that indomethacin, which specifically blocks PGHS-2 acetylationin vitro (Mancini et al., 1997), inhibited 15-epi-LXA4 production by mouse tissues, which suggests that 15-epi-LXA4 formation in this scenario occurs mainlyvia cyclooxygenase-dependent mechanisms. Taken together, our results suggest that 15-epi-LXA4 formation by mouse peritoneal inflammatory exudates occurs predominantly via an ASA- and PGHS-2-dependent route (figs. 7 and 8). This is the first demonstration of 15-epi-LXA4 generation in a model of murine peritonitis.

It was previously shown that lipoxins are generated in several species of experimental animals. For example, LXA4 is generated in mouse kidney with glomerulonephritis in a P selectin-dependent fashion (Mayadas et al., 1996). Also, LXA4 and LXB4 are both formed in ischemic rat brain (Kim and Tominaga, 1989). LXA4 was recently found to regulate LTB4-mediated delayed hypersensitive reactions in guinea pig (Feng et al., 1996) and to inhibit infiltration of neutrophils to glomerulonephritic kidney in rats (Papayianni et al., 1995). For the newly identified 15-epi-LXA4, we have reported in preliminary experiments that 15-epi-LXA4was generated in rat kidney in ASA-dependent fashion (Badr et al., 1996). Together with the findings of the present studies, namely that 15-epi-LXA4 was generated in a model of murine peritonitis, it is likely that 15-epi-LXA4 can also be generated upon ASA treatment in other experimental animals in vivo where cell-cell interactions are accelerated and PGHS-2 is up-regulated (e.g., during the inflammation process). These results also raise the possibility that other PGHS-2 endogenous acetylating routes may exist in the mouse peritoneal cavity that give rise to 15-epi-LXA4 (see fig. 8).

In summary, we developed a new ELISA for 15-epi-LXA4 that proved highly sensitive as well as stereoselective, compared with its epimer LXA4 at the level required to interact selectively with 15-epi-LXA4. Utilizing this ELISA, we established that 15-epi-LXA4 generation proceeds viatranscellular biosynthesis during heterotypic leukocyte-leukocyte interactions. Furthermore, this is the first evidence for 15-epi-LXA4 generation by inflammatory exudate cells from a murine model with LPS and casein-induced peritonitis. Together, these findings document useful means to investigate 15-epi-LXA4generation further.

Acknowledgments

We thank Mary Halm Small for skillful assistance in preparation of this manuscript and Dr. Joan Clària for carrying out the LXA4 ELISA.

Footnotes

  • Send reprint requests to: Charles N. Serhan, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115.

  • ↵1 This work was supported in part by grants no. GM38765 and DK50305 (C.N.S.) from the National Institutes of Health.

  • ↵2 Present address: Nephrology Division, Royal Victoria Hospital, 3775 University Street, Montreal, Quebec, Canada H3A 2B4.

  • Abbreviations:
    AA
    arachidonic acid
    ASA
    aspirin (acetylsalicylic acid)
    ATL
    aspirin-triggered lipoxins
    ELISA
    enzyme-linked immunosorbent assay
    GM-CSF
    granulocyte monocyte colony-stimulating factor
    HETE
    hydroxy eicosatetraenoic acid
    5S-HETE
    (5S)-5-hydroxy-8,11,14-cis-6-trans-eicosatetraenoic acid
    12S-HETE
    (12S)-12-hydroxy-5,8,14-cis-10-trans-eicosatetraenoic acid
    15S-HETE
    (15S)-15-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid
    15R-HETE
    (15R)-15-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid
    HRP
    horseradish peroxidase
    LC/MS/MS
    liquid chromatography tandem mass spectrometry-mass spectrometry
    LO
    lipoxygenase
    LPS
    lipopolysaccharide
    LTB4 (leukotriene B4)
    5S,12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid
    LX
    lipoxin
    15-epi-LXA4
    5S,6R,15R-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid
    15-epi-LXB4
    5S, 14R,15R-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid
    15-R/S-methyl-LXA4
    5S,6R,15(R/S)-trihydroxy-methyl-7,9,13-trans-11-cis-eicosatetraenoic acid
    LXA4
    5S, 6R,15S-trihydroxyl-7,9,13-trans-11-cis-eicosatetraenoic acid
    LXB4
    5S, 14R,15S-trihydroxyl-6,10,12-trans-8-cis-eicosatetraenoic acid
    ODYA
    17-octadecynoic acid
    PGHS
    prostaglandin G/H synthase
    PMN
    neutrophil(s)
    RP-HPLC
    reverse-phase high-pressure liquid chromatography
    KLH
    keyhole limpets hemocyanin
    THP-1
    human acute monocytic leukemia cell line. PVDF, polyvinylidine difluoride
    SDS-Page
    sodium dodecyl sulfate-polyacrylamide gel electrophoresis
    • Received January 8, 1998.
    • Accepted June 1, 1998.

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Aspirin-Triggered 15-epi-Lipoxin A4 (ATL) Generation by Human Leukocytes and Murine Peritonitis Exudates: Development of a Specific 15-epi-LXA4 ELISA

Nan Chiang, Tomoko Takano, Clary B. Clish, Nicos A. Petasis, Hsin-Hsiung Tai and Charles N. Serhan
Journal of Pharmacology and Experimental Therapeutics November 1, 1998, 287 (2) 779-790;
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