Lipoxin B₄ regulates human monocyte/neutrophil adherence and motility: design of stable lipoxin B₄ analogs with increased biologic activity

^a Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesia, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA
^b Department of Chemistry, Loker Hydrocarbon Institute, University of Southern California, Los Angeles, California 90089-1661, USA

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Lipoxins are biologically active products of arachidonic acid that are formed via cell–cell interactions, particularly those involving leukocytes. Lipoxin A₄ and lipoxin B₄ (LXB₄), within similar concentration ranges, each inhibit human neutrophil, activate monocyte adherence and motility, and are rapidly converted by initial dehydrogenation to other inactive metabolites by human monocytes. Here, we exposed LXB₄ to isolated recombinant 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and found that it was a good substrate for the enzyme (K_m=6.9 µM); we identified the major product as 5-oxo-LXB₄ via physical methods including liquid chromatography/tandem mass spectrometry. This is the first evidence of 15-PGDH converting a substrate hydroxyl group at a position other than the -6 carbon. Based on these observations, several LXB₄ analogs were designed and prepared by total organic synthesis to test as stable mimetics: 5(S)-methyl-LXB₄-me, 5(R)-methyl-LXB₄-me, and 15-epi-LXB₄-me (the aspirin-triggered form of LXB₄). Both 5(S)-methyl-LXB₄-me and 5(R)-methyl-LXB₄-me were resistant to rapid conversion. In addition, actions of the stable analogs were evaluated separately with human mono-cytic cells and neutrophils, and 5(S)-methyl-LXB₄-me was more potent (nM range) than LXB₄ for both cell types. In contrast, 5(R)-methyl-LXB₄-me was potent in inhibiting neutrophil transmigration across endothelial monolayers, but did not stimulate monocyte adherence. These results indicate that LXB₄ analogs can be designed to resist rapid transformation and retain bioactivity with both monocytes and neutrophils. Moreover, they suggest that LXB₄ stable analogs are useful tools to selectively evaluate the modes of actions of LXB₄ with different tissues.—Maddox, J. F., Colgan, S. P., Clish, C. B., Petasis, N. A., Fokin, V. V. Serhan, C. N., Lipoxin B₄ regulates human monocyte/neutrophil adherence and motility: design of stable lipoxin B₄ analogs with increased biologic activity. FASEB J. 12, 487–494 (1998)

Key Words: eicosanoids • leukocytes • inflammation • lipid mediators

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
EICOSANOIDS ARE lipid mediators that have a multifaceted effect on immune system functions, including up- and down-regulation of leukocyte activity. The lipoxygenases involved in eicosanoid production, including 5-, 12-, and 15-lipoxygenase, are known to exist in both plant and animal tissues, but only in the human phagocytic system are their functions well defined, producing the proinflammatory leukotrienes and the antiinflammatory lipoxins (LX)² (1). Lipoxin A₄ (LXA₄), which carries hydroxyl groups at carbons 5S, 6R, and 15S; and lipoxin B₄ (LXB₄), which carries hydroxyl groups at carbons 5S, 14R, and 15S, are bioactive eicosanoids that possess a tetraene structure and are formed from dual lipoxygenation of arachidonic acid (2). In addition, as recently reported by this laboratory, aspirin-triggered LX are produced from the sequential actions of aspirin-treated cyclooxygenase 2, forming 15(R)-hydroxyeicosatetraenoic acid, and 5-lipoxygenase, which results in LX carrying the hydroxyl group at carbon 15 in the R-position instead of the native 15(S) form of the molecules (3). LX are generated during cell–cell interactions in vitro (reviewed in ref 4) and have been documented in vivo from humans with asthma (5) and rheumatoid arthritis (6). LXB₄ has not been studied as extensively as LXA₄; it is chemically and biologically less stable and isomerizes rapidly, and therefore is more difficult to handle. There are, nevertheless, specific and potent subnanomolar–nanomolar range actions attributed to LXB₄, including stimulating proliferation and differentiation of granulocyte-monocyte colonies from human mononuclear cells (7), increasing the length of the S phase in the cell cycle and enhancing nuclear protein kinase C activity of Friend erythroleukemia cells (8), and sleep induction (9).

In addition to its specific actions, LXB₄ also shares actions with LXA₄; for example, LXA₄ and LXB₄ selectively stimulate human peripheral blood monocytes (10), enhance growth of myeloid progenitor cells (11), and inhibit human neutrophil (PMN) transmigration and adherence (3, 12) (recently reviewed in ref 13). Human monocytic cells rapidly convert LXA₄ and LXB₄ to inactive oxo- and dihydro-products whereas PMNs, within physiologic concentrations, do not significantly further metabolize LX. The initial dehydrogenation of LX to form oxo-LX is likely via the action of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which is present in human monocytes. This enzyme generates 15-oxo-LXA₄, but the enzyme that converts LXB₄ to its biologically inactive product, 5-oxo-LXB₄, has not been specifically identified (10). The catalytic activity with LXA₄ was confirmed with recombinant enzyme; LXA₄ analogs that resist this rapid conversion and inactivation were synthesized (14). These LXA₄ stable analogs were subsequently shown to be more potent than native LXA₄ in stimulating human monocytes in vitro, in the concentration range from 0.1 to 100 nM (15), and also displayed very potent in vivo anti-inflammatory activity in a mouse ear inflammation model (16). Successful design and synthesis of LXA₄ analogs that are more resistant to inactivation and more potent than native LXA₄ led to the consideration of criteria essential to the design of synthetic LXB₄ stable analogs. Here, we report the first characterization of LXB₄-derived products from recombinant dehydrogenase by liquid chromatography/tandem mass spectrometry (LC/MS-MS) as well as the rational design of novel bioactive LXB₄ synthetic analogs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
LXB₄ and analog analysis
LXB₄ was purchased from Cascade Biochem Ltd. (Berkshire, U.K.) and synthetic analogs were prepared and characterized, including NMR spectroscopy, by using methods similar to those in ref 14). The physical values for these compounds were as follows: 15-epi-LXB₄-me: ¹H-NMR (360 MHz, C₆D₆), 6.65 (m, 2H), 6.27 (dd, J=15.3 and 10.6 Hz, 1H), 6.13 (dd, J=14.7 and 10.5 Hz, 1H), 5.93 (m, 2H), 5.56 (dd, J=14.6 and 6.4 Hz, 2H), 3.96 (m, 1H), 3.76 (t, J=7.1 Hz, 1H), 3.42 (s, 3H), 3.27 (m, 1H), 2.14 (t, J=7.3 Hz, 2H), 1.63 - 1.24 (m, 12H), 0.85 (t, J=7.5 Hz, 3H). ¹³C-NMR (125 MHz, C₆D₆), 173.29, 137.99, 133.88, 133.22, 132.63, 129.66, 129.59, 128.75, 125.47, 76.04, 74.83, 72.01, 51.28, 36.80, 33.83, 32.17, 25.66, 22.96, 21.00, 14.17. UV: _max = 288, 301, and 315 nm. 5(R/S)-methyl-LXB₄-me: ¹H-NMR (500 MHz, C₆D₆), 6.78-6.68 (m, 1H), 6.22 (dd, J=14.9 and 10.9 Hz, 1H), 6.12 (dd, J=14.9 and 10.9 Hz, 1H), 5.99 (d, J=10.1 Hz, 2H), 5.62 (dd, J=14.9 and 6.4 Hz, 2H), 3.97 (dd, J=8.8 and 5.9 Hz, 1H), 3.85 (dd, J=10.5 and 4.3 Hz, 1H), 3.45-3.41 (m, 1H), 3.31 (s, 3H), 2.09 (t, 2H), 1.81 - 1.38 (br m, 12H), 1.30 (s, 3H), 0.87 (t, 3H), 0.45 (br s, 3H). ¹³C-NMR (125 MHz, C₆D₆), 174.30, 138.75, 133.37, 132.78, 132.71, 129.87, 129.50, 128.72, 125.20, 75.67, 74.10, 72.36, 52.50, 41.52, 41.52, 34.25, 32.14, 31.62, 28.43, 25.44, 22.63, 19.31, 14.09. UV: _max = 288, 301, and 314 nm. Concentrations of LXB₄ analogs were determined using an extinction coefficient of 50,000.

Conversion by 15-PGDH
Purified recombinant 15-PGDH was a generous gift from Dr. H. H. Tai (University of Kentucky, Lexington). Enzyme activity was determined by measuring the formation of NADH from NAD⁺ spectrophotometrically at 340 nm as in ref 17. Briefly, 0.5 µg of 15-PGDH and potential substrates (2–30 µM) were added to buffer containing Tris-HCl (0.1 M, pH 9.0) and NAD⁺ (0.5 mM) in a total volume of 0.75 ml; increments in absorption at 340 nm were recorded every 15 s for 3 min. Reaction rates were calculated via zero order regression curves computer-fitted to the change in absorption as a function of time for the first 60 s.

Liquid chromatography/tandem mass spectrometry
LC/MS-MS was performed on an LCQ quadrupole ion trap mass spectrometer (Finnigan MAT, San Jose, Calif.) equipped with an electrospray atmospheric pressure ionization probe. The high-performance liquid chromatography (HPLC) component consisted of a SpectraSYSTEM P4000 quaternary gradient pump (Thermo Separation Products, San Jose, Calif.), a Prodigy ODS-3 (250x2 mm, 5 µm) column (Phenomenex, Torrance, Calif.), and a rapid spectra scanning SpectraSYSTEM UV2000 UV/VIS absorbance detector (Thermo Separation Products). The column was eluted isocratically with methanol/water/acetic acid (65:35:0.01) at 0.2 ml min^-1 into the electrospray probe. The spray voltage was set to 8 kV and the heated capillary to 250°C. Over a 2.4 s scan cycle, full-scan mass spectra (MS) were acquired by scanning between m/z 95 and 450 in the negative ion mode, followed by the acquisition of product ion mass spectra (MS-MS) of the most intense molecular anion (e.g., [M-H]^- m/z 351 for LXB₄).

Laminin adherence
These assays were performed with the human acute monocytic leukemia cell line, THP-1 (American Type Culture Collection, Rockville, Md.), as described in ref 10. Adherence of THP-1 cells with exposure to vehicle alone was 6.3–8.3% of total cells added.

PMN transmigration
Human PMN were isolated from healthy volunteers by standard procedures (18). The isolated cells contained 96 ± 3% PMN that were >97% viable as determined by trypan blue exclusion. Human umbilical vein endothelial cells (HUVEC) were cultured and a PMN transendothelial migration assay was performed in essentially the same manner as the PMN trans-epithelial migration in ref 14. Transmigration was quantitated by assaying for the PMN azurophilic granule marker enzyme myeloperoxidase (MPO) (19). After each assay, nonadherent PMNs were washed from the surface of the monolayer, and the number of PMNs that completely transversed the monolayer was determined from a standard curve of MPO activity.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Conversion of LXB₄ by dehydrogenation. LXB₄ is rapidly converted by human monocytic cell types via initial dehydrogenation at carbon 5 to oxo-and dihydro-products, which are biologically inactive with monocytes (10). Similar dehydrogenation of LXA₄ at carbon 15 is catalyzed by 15-PGDH (14). Therefore, LXB₄ was tested as a substrate with purified recombinant enzyme and proved to be converted by 15-PGDH, as determined by NADH formation ( Fig. 1A). The methyl ester of LXB₄ was a better substrate than LXB₄ free acid, as was found with LXA₄ (14); although the initial reaction rate of substrate conversion was slower than with prostaglandin E₂ (PGE₂), the K_m was similar to the reported value (20) (3.9 µM for PGE₂ vs. 6.9 µM for LXB₄; Fig. 1B). The formation of NADH indicated that LXB₄ was converted, but which of the three alcohol groups (at carbons 5, 14, and 15) was susceptible could not be determined by this level of analysis; therefore, LXB₄ incubations with 15-PGDH were directly analyzed via LC/MS-MS to specifically identify LXB₄-derived products.

View larger version (16K): [in this window] [in a new window]   Figure 1. Conversion of LXB4 by recombinant 15-PGDH. A) Time course of NADH formation was monitored at A340 with 4 µM PGE2, LXA4-methyl ester, LXB4-methyl ester, or vehicle (0.5% EtOH) and 0.5 µg 15-PGDH, as described in Experimental Procedures. B) Lineweaver-Burk plot of LXB4-me with 15-PGDH. Values for reaction velocity were determined from the rate of NADH formation at 0–60 s after substrate addition.

The UV chromatograms recorded at 300 and 335 nm showed evidence of two products from LXB₄, with retention times of approximately 20.6 and 21.2 min (data not shown). The 3-dimensional MS plot showed a peak at 14.8 min with an m/z value of 351, and another at 21.4 min with an m/z value of 349, corresponding to the [M-H]^- anions of LXB₄ and oxo-LXB₄ products, respectively ( Fig. 2A). The MS of the material eluted at 14.8 min gave the m/z 351 [M-H]^- base peak of LXB₄ as the predominant ion ( Fig. 2B). The MS-MS analysis of LXB₄ ( Fig. 2C) revealed major ions at m/z 333 [351-H₂O], 315 [351-2xH₂O], 289 [351-H₂O, -CO₂], 261 [351- H₂O, -CH₃(CH₂)₃CH₃], 251 [351-CHO(CH₂)₄CH₃], 233 [351-H₂O, -CHO(CH₂)₄CH₃], 221 [351- CHOCHOH(CH₂)₄CH₃], 207 [351-CO₂, -CHO-(CH₂)₄CH₃], 163 [351-CO₂, -CH₂COHCHOH-(CH₂)₄CH₃], and 129 [CH₃CO(CH₂)₃COO^-]. Native LXB₄ was characterized recently via electrospray/collision-induced dissociation (CID) MS-MS (21), but has not been evaluated by the ion trap tandem mass spectrometry technology used in the present experiments. The observed ions are largely consistent with the fragmentation pattern and ion chemistry reported with CID MS-MS (cf. ref 21); however, the m/z 163 daughter ion observed with the ion trap method was not a major product by CID MS-MS. This ion most likely resulted from both the loss of CO₂ from [M-H]^- and fragmentation at the bond between carbons 12 and 13 for a loss of CH₂COHCHOH-(CH₂)₄CH₃.

View larger version (37K): [in this window] [in a new window]   Figure 2. LC/MS-MS analysis of LXB4 and the major LXB4 metabolite. Recombinant 15-PGDH (1.0 µg) was incubated with LXB4 (100 ng) for 20 min at 37°C and aliquots were directly injected onto LCQ as described in Experimental Procedures. A) Three-dimensional MS chromatogram of LXB4 and products from RP-HPLC monitored between m/z 345 and 356. B) Mass spectrum of LXB4 with inset of LXB4 structure. C) MS-MS spectrum of LXB4. D) Mass spectrum of major LXB4 metabolite, 5-oxo-LXB4, with inset of structure. E) MS-MS spectrum of 5-oxo-LXB4.

The MS for the major product of LXB₄ at a retention time of 21.4 min displayed a base peak at m/z 349 ( Fig. 2D), which was consistent with the [M-H]^- anion of oxo-LXB₄. The MS-MS analysis of this ion revealed major ions at m/z 331 [349-H₂O], 313 [349-2xH₂O], 287 [349-H₂O, -CO₂], 269 [349- 2xH₂O, -CO₂], 259 [349-H₂O, -CH₃(CH₂)₃-CH₃], 233 [251-H₂O], 205 [349-CO₂, -CHO(CH₂)₄-CH₃], 161 [349-CO₂, -CH₂COHCHOH(CH₂)₄CH₃], and 129 [CH₃CO(CH₂)₃COO^-] ( Fig. 2E). As observed for the m/z 163 daughter ion from LXB₄, the ion at m/z 161 may have resulted from a loss of CO₂ and CH₂COHCHOH(CH₂)₄CH₃ from [M-H]^-. The reduction in mass of the remaining fragment by two units supports the proposed oxidation of the hydroxyl group at the 5-position. This fragment and the others noted above are consistent with the identification of this compound as 5-oxo-LXB₄. In addition, a second minor LXB₄ metabolite that eluted with 5-oxo-LXB₄, giving a doublet peak in the UV-monitored chromato-gram, was formed in approximately one-fourth of the quantity of 5-oxo-LXB₄. The MS and major ions in the MS-MS analyses were consistent with identification of this minor product as 15-oxo-LXB₄ (data not shown). The UV spectra of the major and minor metabolites were different in that the 5-oxo-LXB₄ gave a UV ^MeOH_max at ~335 nm and the 15-oxo-LXB₄ displayed a ^MeOH_max at 300 nm. There was no evidence from either the UV or MS chromatograms or from MS-MS analyses that 14-oxo-LXB₄, dioxo-LXB₄ (e.g., 5,15 dioxo-LXB₄), or double bond reduction products were formed. Thus, 15-PGDH appears to attack only the primary trihydroxytetraene structure at the carbon 5 or carbon 15 position, with an approximately 4 to 1 preference for the carbon 5 alcohol. Knowledge of the structural features and forms of these oxo-LXB₄ metabolites will support development of appropriate methods for their analysis in vivo.

Design of LXB₄ analogs
Identification of the major product of LXB₄ as 5-oxo-LXB₄, as well as previous evidence of rapid inactivation of LXB₄ by monocytic cells (cf. ref 10), led to the design and total organic synthesis of analogs with bulky substitutions placed at the carbon 5 position to protect and/or prevent catalysis at this alcohol group ( Fig. 3). Because aspirin-triggered LXB₄ (15-epi-LXB₄) (22) had not yet been examined with human monocytes for either biologic activity or further metabolism, the methyl ester was also prepared for purposes of direct comparison. These analogs were first evaluated as substrates for recombinant 15-PGDH and were compared directly with enzymatic conversion of LXB₄-me. In parallel incubations, analogs with a methyl group at the carbon 5 position—5(S)-methyl-LXB₄-me and 5(R)-methyl-LXB₄-me—showed very little susceptibility to 15-PGDH ( Fig. 4). Protection was also afforded to LXA₄ when a methyl group was placed at the carbon 15 position (14). In contrast, 15-epi-LXB₄-me was a slightly better substrate for the enzyme than LXB₄-me ( Fig. 4), and was converted exclusively at the carbon 5 alcohol to 5-oxo-15-epi-LXB₄, as determined via LC/MS-MS (data not shown). This increased rate of conversion of 15-epi-LXB₄-me as compared with LXB₄-me may reflect a preference by the enzyme for the 14R, 15R diol configuration. This is in sharp contrast to LXA₄ vs. 15-epi-LXA₄, where 15-epi-LXA₄ was less susceptible to conversion than the 15S-containing, or native, LXA₄ (14). Together, these results indicate that the actions of the dehydrogenase are stereospecific with LXB₄ and that the 5-methyl-containing analogs were protected from this dehydrogenation.

View larger version (25K): [in this window] [in a new window]   Figure 3. Structures of lipoxin B4 and synthetic analogs designed for these experiments.

View larger version (22K): [in this window] [in a new window]   Figure 4. Conversion of LXB4 synthetic analogs by recombinant 15-PGDH. Time course of NADH formation was monitored at A340 using 4 µM of each analog as described in Experimental Procedures.

Because 5(S)-and 5(R)-methyl-LXB₄-me resisted rapid conversion, their potential as inhibitors of enzyme activity with other substrates was examined ( Table 1). Indeed, these analogs did inhibit the enzymatic activity for LXB₄-me by 30% when tested at equimolar concentrations. In contrast, they did not inhibit PGE₂ conversion even when present at a fivefold greater concentration than PGE₂. These findings suggest that PGE₂ is a preferred substrate for this 15-PGDH and that LXB₄ will be converted only when PGE₂ is absent from the local milieu.

The results presented here are the first documentation of a 15-PGDH substrate that is metabolized at a hydroxyl group at a site other than the -6 carbon. 15-PGDH can attack hydroxyl groups of eicosanoids other than carbon 15—specifically, the 12 hydroxyl of 12-hydroxy-5,8,10-heptadecatrienoic acid (23), which is also in the -6 position. LXA₄ is converted by 15-PGDH; even though LXA₄, in addition to a carbon 15 hydroxyl, also carries an alcohol group at carbon 5, this position was not subject to attack (14). Thus, LXB₄ and LXA₄ are both substrates for 15-PGDH, and this recombinant enzyme provides a useful model dehydrogenase for conversion of LX and physical evaluation of LX products. The dehydrogenase is present in human monocytes (10) and is likely to be the primary enzyme involved in further metabolism of LX by these cells; however, this does not exclude the possibility of additional, as yet unidentified, dehydrogenases related to this enzyme in other cells and/or tissues to handle LX inactivation exclusively.

LXB₄ analogs stimulate monocytic cell adherence
LXB₄ stimulates adherence of both monocytes and THP-1 cells to laminin and is rapidly inactivated via dehydrogenation and reduction in these cells (10). To determine whether the analogs decrease, retain, or increase bioactions, we evaluated each analog in comparison to LXB₄. As shown in Fig. 5A, 5(S)-methyl-LXB₄-me was the most effective analog in stimulating THP-1 cell adherence to laminin and was more potent than native LXB₄ (which showed no statistically significant difference from carboxy methyl ester of LXB₄ in this bioassay; data not shown). 5(R)-methyl-LXB₄-me and 5(R/S)-methyl-8,9-acetylenic-LXB₄-me were much less potent than LXB₄ in activating cell adherence ( Fig. 5A). 15-epi-LXB₄-me (aspirin-triggered LXB₄)-induced adherence to laminin was essentially equivalent to LXB₄ ( Fig. 5B). Recently this laboratory found that biologically derived 15-epi-LXB₄ was more potent than LXB₄ in inhibiting adenocarcinoma cell growth (22). The rank order and potency of 15-epi-LXB₄ and LXB₄ in THP-1 cells vs. the adenocarcinoma line may reflect different sites of action and/or different metabolism profiles between the two cell types. Nevertheless, when taken together these results suggest that in addition to rapid inactivation of LXB₄ by 5-oxo formation with monocytic cells, there is a stereospecific preference for the 5(S) hydroxyl in stimulating this cell type and a need to maintain the tetraene structure for bioactivity.

View larger version (20K): [in this window] [in a new window]   Figure 5. LXB4 synthetic analogs increase THP-1 cell adherence to laminin. THP-1 cells were incubated (3x105 cells in 100 µl) with each compound for 40 min at 37°C. Values represent means ± SE for three to five separate experiments performed in quadruplicate and are expressed as the percent adherence above that with cells exposed to vehicle alone. A) LXB4 and stable analogs of LXB4. *P < 0.05 vs. LXB4, #P < 0.05 vs. 5(S)methyl-LXB4me. B) LXB4 and aspirin-triggered LXB4 analog (15-epi-LXB4).

LXB₄ analogs inhibit PMN migration
LXB₄ inhibits PMN transmigration across endothelial cells (12), and so we also determined the activity of these LXB₄ analogs in this coincubation system. LXB₄ is not converted to additional metabolites by either PMN or PMN–endothelial cell coincubations (13); accordingly, the rank order of activity of the stable analogs and native LXB₄ with these cell types might have proved different from monocytic cells. Thus, the 5(S)- and 5(R)-methyl-LXB₄-me analogs were evaluated as inhibitors of LTB₄-driven PMN transmigration across HUVEC monolayers, and both induced a similar concentration-dependent inhibition of migration (10^-9–10^-6 M) that was significantly greater than LXB₄ (P<0.05; Fig. 6). This demonstrates a difference in the structure–activity relationship of the analogs between THP-1 cell adherence and PMN inhibition of transmigration, specifically regarding the actions of 5(R)-methyl-LXB₄-me.

View larger version (19K): [in this window] [in a new window]   Figure 6. Effect of LXB4 synthetic analogs on human PMN transmigration across HUVEC monolayers. PMNs (2x106/monolayer) were exposed to vehicle (0.3% EtOH in HBSS), LXB4, 5(S)methyl-LXB4, or 5(R)methyl-LXB4 at the concentrations indicated for 15 min at 37°C. PMN were layered on HUVEC monolayers and driven to transmigrate by a 10 nM gradient of LTB4 for 90 min at 37°C. Values represent means ± SE for three separate experiments performed in triplicate, and are expressed as the percent inhibition of migration compared to PMN exposed to vehicle alone. *P < 0.05 vs. LXB4.

Precedence for LX receptors was established with the identification and cloning of a seven-transmembrane receptor for LXA₄ from PMN and monocytes (15, 24). The site (or sites) of action of LXB₄ have yet to be elucidated, in part because synthesis of a suitable LXB₄ radiolabel with high specific activity proved difficult (C. N. Serhan, personal communi~cation). New approaches to radiolabeling LXB₄ through the use of stable analogs documented here could aid in the identification of LXB₄ receptors on PMNs, monocytes, and the airway epithelial adenocarcinoma cell line. LXB₄ activity is sensitive to pertussis toxin in both PMN (13) and THP-1 cells (data not shown), implicating a G-protein-linked cell surface receptor. If LXB₄ acts via surface receptors, multiple forms of the receptor may exist in different cell types and/or tissues, as with prostaglandin E receptors (25).

Recent evidence demonstrated anti-inflammatory actions for stable synthetic analogs of LXA₄ that were more potent than dexamethasone in mouse ear inflammation models (16). These in vivo findings correlated with results from cellular-based bioassays with PMN and monocytes (14, 15). The LXB₄ stable analogs, reported here for the first time, were also tested by using a mouse ear inflammation model of vascular permeability; the results showed that 5(S)-methyl-LXB₄-me is a potent anti-inflammatory agent when applied topically (26). Thus, the ability of LX to inhibit PMN infiltration and selectively stimulate recruitment of monocytes at sites of tissue injury may contribute to orchestrating wound healing, clearly a unique action of this class of compounds in leukocyte function (reviewed in refs 13, 27).

In summary, LXB₄ metabolism by 15-PGDH was characterized and the major products were identified via LC/MS-MS, which permitted the design and synthesis of stable analogs of LXB₄. These novel LXB₄ analogs resisted this transformation and selectively maintained bioactivity observed for LXB₄ with both PMN and monocytic cells. These results establish LXB₄ analogs as potent modulators of leukocyte trafficking and enable further evaluation of their actions. Moreover, these results can aid not only in identifying the site (or sites) of action of LXB₄, but can also be applied to potential in vivo therapeutic applications of this class of LX stable analogs.

	ACKNOWLEDGMENTS

 
This work was supported in part by National Institutes of Health Grants R01-GM38765 and P01-DK50305 (to C.N.S.) and a research grant from Schering Berlex AG (to C.N.S. and N.A.P.). J.F.M. was a Postdoctoral Fellow of the National Arthritis Foundation. We also thank Mary Small for expert assistance in the preparation of the manuscript.

	FOOTNOTES

 
¹ Correspondence: Center for Experimental Therapeutics, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA. E-mail: cnserhan{at}zeus.bwh.harvard.edu

² Abbreviations: HUVEC, human umbilical vein endothelial cells; LC, liquid chromatography; leukotriene B₄ (LTB₄), (5S,12R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid; lipoxin A₄ (LXA₄), (5S,6R,15S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid; lipoxin B₄ (LXB₄), (5S,14R,15S)-trihydroxy-6,8,12-trans-10-cis-eicosatetraenoic acid; 15-epi-lipoxin B₄-methyl ester (15-epi-LXB₄-me), (5S,14R,15R)-trihydroxy-6,8,12-trans-10-cis-eicosatetraenoic acid, methyl ester; 5(R)-methyl-LXB₄-me, (5R,14R,15S)-trihydroxy-5(S)-methyl-6,8,12-trans-10-cis-eicosatetraenoic acid, methyl ester; 5(S)-methyl-LXB₄-me,(5S,14R,15S)-trihydroxy-5(R)-methyl-6,8,12-trans-10-cis-eicosatetraenoic acid, methyl ester; 5(R/S)-methyl-8,9-acetylenic-LXB₄-me, (5R/S,14R,15S)-trihydroxy-5-methyl-8,9-acetylenic-6,8,12-trans-10- cis-eicosatetraenoic acid, methyl ester; MPO, myeloperoxidase; MS-MS, tandem mass spectrometry; 15-PGDH, 15-hydroxyprostaglandin dehydrogenase; PGE₂, prostaglandin E₂; PMN, polymorphonuclear leukocyte; SIM, selected ion monitoring; LC/MS-MS, liquid chromatography/tandem mass spectrometry; HPLC, high-performance liquid chromatography; CID, collision-induced dissociation.

Received for publication September 26, 1997. Accepted for publication December 11, 1997.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

1. Yamamoto, S., Suzuki, H., and Ueda, N. (1997) Arachidonate 12-lipoxygenases. Prog. Lipid Res. 36, 23–41[Medline]
2. Samuelsson, B., Dahlén, S. E., Lindgren, J. Å., Rouzer, C. A., and Serhan, C. N. (1987) Leukotrienes and lipoxins: structures, biosynthesis, and biological effects. Science 237, 1171–1176[Abstract/Free Full Text]
3. Clària, J., and Serhan, C. N. (1995) Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interactions. Proc. Natl. Acad. Sci. USA 92, 9475–9479[Abstract/Free Full Text]
4. Serhan, C. N., Haeggstrom, J. Z., and Leslie, C. C. (1996) Lipid mediator networks in cell signaling: update and impact of cytokines. FASEB J. 10, 1147–1158[Abstract]
5. Chavis, C., Vachier, I., Chanez, P., Bousquet, J., and Godard, P. (1996) 5(S),15(S)-dihydroxyeicosatetraenoic acid and lipoxin generation in human polymorphonuclear cells: dual specificity of 5-lipoxygenase towards endogenous and exogenous precursors. J. Exp. Med. 183, 1633–1643[Abstract/Free Full Text]
6. Thomas, E., Leroux, J. L., Blotman, F., and Chavis, C. (1995) Conversion of endogenous arachidonic acid to 5,15-diHETE and lipoxins by polymorphonuclear cells from patients with rheumatoid arthritis. Inflamm. Res. 44, 121–124[Medline]
7. Popov, G. K., Nekrasov, A. S., Khshivo, A. L., Pochinskii, A. G., and Lankin, V. Z. (1989) Effect of lipoxin B on colony-forming capacity of human peripheral blood mononuclear cells in diffusion chambers. Biull. Eksp. Biol. Med. 107, 80–83[Medline]
8. Beckman, B. S., Despinasse, B. P., and Spriggs, L. (1992) Actions of lipoxins A₄ and B₄ on signal transduction events in Friend erythroleukemia cells. Proc. Soc. Exp. Biol. Med. 201, 169–173[Medline]
9. Kantha, S. S., Matsumura, H., Kubo, E., Kawase, K., Takahata, R., Serhan, C. N., and Hayaishi, O. (1994) Effect of prostaglandin D₂, lipoxins and leukotrienes on sleep and brain temperature of rats. Prostaglandins, Leukotrienes, and Essential Fatty Acids 51, 87–93[Medline]
10. Maddox, J. F., and Serhan, C. N. (1996) Lipoxin A₄ and B₄ are potent stimuli for human monocyte migration and adhesion: selective inactivation by dehydrogenation and reduction. J. Exp. Med. 183, 137–146[Abstract/Free Full Text]
11. Stenke, L., Mansour, M., Edenius, C., Reizenstein, P., and Lindgren, J. Å. (1991) Formation and proliferative effects of lipoxins in human bone marrow. Biochem. Biophys. Res. Commun. 180, 255–261[Medline]
12. Papayianni, A., Serhan, C. N., and Brady, H. R. (1996) Lipoxin A₄ and B₄ inhibit leukotriene-stimulated interactions of human neutrophils and endothelial cells. J. Immunol. 156, 2264–2272[Abstract]
13. Serhan, C. N. (1997) Lipoxins and novel aspirin-triggered 15-epi-lipoxins: a jungle of cell–cell interactions or a therapeutic opportunity? Prostaglandins 53, 107–137[Medline]
14. Serhan, C. N., Maddox, J. F., Petasis, N. A., Akritopoulou-Zanze, I., Papayianni, A., Brady, H. R., Colgan, S. P., and Madara, J. L. (1995) Design of lipoxin A₄ stable analogs that block transmigration and adhesion of human neutrophils. Biochemistry 34, 14609–14615[Medline]
15. Maddox, J. F., Hachicha, M., Takano, T., Petasis, N. A., Fokin, V. V., and Serhan, C. N. (1997) Lipoxin A₄ stable analogs are potent mimetics that stimulate human monocytes and THP-1 cells via a G-protein linked lipoxin A₄ receptor. J. Biol. Chem. 272, 6972–6978[Abstract/Free Full Text]
16. Takano, T., Fiore, S., Maddox, J. F., Brady, H. R., Petasis, N. A., and Serhan, C. N. (1997) Aspirin-triggered 15-epi-lipoxin A₄ (LXA₄) and LXA₄ stable analogues are potent inhibitors of acute inflammation: Evidence for anti-inflammatory receptors. J. Exp. Med. 185, 1693–1704[Abstract/Free Full Text]
17. Braithwaite, S. S., and Jarabak, J. (1975) Studies on a 15-hydroxy-prostaglandin dehydrogenase from human placenta. Purification and partial characterization. J. Biol. Chem. 250, 2315–2318[Abstract/Free Full Text]
18. Böyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 77–89
19. Parkos, C. A., Delp, C., Arnaout, M. A., and Madara, J. L. (1991) Neutrophil migration across a cultured intestinal epithelium. Dependence on a CD11b/CD18-mediated event and enhanced efficiency in physiological direction. J. Clin. Invest. 88, 1605–1612
20. Ensor, C. M., and Tai, H.-H. (1994) Bacterial expression and site-directed mutagenesis of two critical residues (tyrosine-151 and lysine-155) of human placental NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase. Biochim. Biophys. Acta 1208, 151–156[Medline]
21. Griffiths, W. J., Yang, Y., Sjovall, J., and Lindgren, J. Å. (1996) Electrospray/collision-induced dissociation mass spectrometry of mono-, di- and tri-hydroxylated lipoxygenase products, including leukotrienes of the B-series and lipoxins. Rapid Commun. Mass Spectrom. 10, 183–196[Medline]
22. Clària, J., Lee, M. H., and Serhan, C. N. (1996) Aspirin-triggered lipoxins (15-epi-LX) are generated by the human lung adenocarcinoma cell line (A549)-neutrophil interactions and are potent inhibitors of cell proliferation. Mol. Med. 2, 583–596[Medline]
23. Liu, Y., Yoden, K., Shen, R. F., and Tai, H. H. (1985) 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) is an excellent substrate for NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase. Biochem. Biophys. Res. Commun. 129, 268–274[Medline]
24. Fiore, S., Maddox, J. F., Perez, H. D., and Serhan, C. N. (1994) Identification of a human cDNA encoding a functional high affinity lipoxin A₄ receptor. J. Exp. Med. 180, 253–260[Abstract/Free Full Text]
25. Thierauch, K.-H., Dinter, H., and Stock, G. (1994) Prostaglandins and their receptors: II. Receptor structure and signal transduction. J. Hypertens. 12, 1–5[Medline]
26. Takano, T., Clish, C. B., Gronert, K., Petasis, N., and Serhan, C. N. (1998) Neutrophil-mediated changes in vascular permeability are inhibited by topical application of aspirin-triggered 15-epi-lipoxin A₄ and novel lipoxin B₄ stable analogues. J. Clin. Invest. In Press
27. Martin, P. (1997) Wound healing—aiming for perfect skin regeneration. Science 276, 75–81[Abstract/Free Full Text]