Formation of Endogenous "Antiinflammatory" Lipid Mediators by Transcellular Biosynthesis
Lipoxins and Aspirin-triggered Lipoxins Inhibit Neutrophil Recruitment and Vascular Permeability

CHARLES N. SERHAN, TOMOKO TAKANO, NAN CHIANG, KARSTEN GRONERT, and CLARY B. CLISH

Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts

	INTRODUCTION

TOP INTRODUCTION TRANSCELLULAR GENERATION OF LX... METHODS RESULTS DISCUSSION REFERENCES

Neutrophil-dependent vascular injury gives rise to increased vascular permeability, edema, and further release of chemoattractants. Leukotriene B₄ (LTB₄) is among the most potent neutrophil stimuli and thus participates in tissue injury via recruiting neutrophils (PMNs, polymorphonuclear leukocytes) in pathophysiologic scenarios (1). Lipoxins are trihydroxytetraene-containing eicosanoids that are, among other in vivo sites, also generated within vascular lumen primarily by platelet-leukocyte interactions by pathways (Figure 1) that are activated during multicellular responses such as inflammation, atherosclerosis, and thrombosis (for a review, see Reference 2). In addition, aspirin has been found to trigger the transcellular biosynthesis of a new group of compounds termed 15-epi-lipoxins (15-epi-LXs) or aspirin-triggered lipoxins, which are likely to contribute to some of the beneficial actions ascribed to aspirin by serving as local endogenous antiinflammatory lipid mediators (2). Thus, these LX branches illustrated in Figure 1 involve cell-cell interactions that appear to produce "endogenous stop signals" or "antiinflammatory compounds," while the 5-lipoxygenase pathway generates leukotrienes that evoke cellular events that are considered to be proinflammatory.

View larger version (18K): [in this window] [in a new window]   Figure 1.   Transcellular lipoxin biosynthesis. Illustration of the three main transcellular routes to generate lipoxins in mammalian tissues. Each of these is an independent route initiated by selective addition of molecular oxygen to arachidonic acid in the donor cell type of origin.

	TRANSCELLULAR GENERATION OF LX AND 15-EPI-LX

TOP INTRODUCTION TRANSCELLULAR GENERATION OF LX... METHODS RESULTS DISCUSSION REFERENCES

Platelet-leukocyte interactions and/or platelet-leukocyte microaggregates promote the formation of LX by transcellular conversion of the leukocyte 5-lipoxygenase (5-LO) product LTA₄. Once thought to be solely an intracellular intermediate in leukotriene production, it is now clear that LTA₄ released by activated leukocytes is available for enzymatic conversion by neighboring cell types. When platelets are adherent, their 12-LO converts LTA₄ to lipoxin A₄ and B₄ (Figure 1). For further mechanistic details, see Reference 2 for a review. Hence, human platelets, which do not produce LX on their own, become a major source of lipoxins, given their abundance in vivo and their highly active 12-LO.

15-LO-initiated LX production is best illustrated by airway epithelial cells, monocytes or eosinophils, which upregulate their 15-LO when exposed to cytokines such as interleukin 4 (IL-4) or IL-13. When these cell types are activated, they generate and release 15S-hydroxyeicosatetraenoic acid (15S-HETE), which is rapidly taken up and converted by neutrophils to lipoxins via the action of their 5-LO (Figure 1). This event not only leads to LX biosynthesis, but also "turns off" leukotriene formation. 5-LO conversion of 15R-HETE also results in inhibition of leukotriene biosynthesis. Since 15R-HETE is a product of cyclooxygenase II (COX-II) after acetylation by aspirin, it is possible that aspirin can regulate the in vivo production of leukotrienes by 15R-HETE conversion to 15-epi-LX, and that 15-epi-LX can in turn also regulate the cellular actions of leukotrienes.

We sought evidence for alternative explanations for the therapeutic actions of aspirin because many beneficial new actions have been documented in clinical studies. These new potential therapeutic indicators for aspirin (ASA, acetylsalicylic acid) include decreasing incidence of lung, colon, and breast cancer [reviewed by Levy (3)], and prevention of cardiovascular diseases (4). Inhibition of cyclooxygenase and biosynthesis of prostaglandins can account for many of the therapeutic properties of ASA (5); however, its ability to regulate neutrophil-mediated inflammation or cell proliferation remains of interest. Along these lines, we uncovered a new action of aspirin that involves COX-II-bearing cells such as vascular endothelial cells or epithelial cells and their coactivation with neutrophils (Figure 1). Hence, inflammatory stimuli (i.e., tumor necrosis factor  [TNF-], lipopolysaccharide [LPS], etc.) induce COX-II to generate 15R-HETE when ASA is administered. This intermediate carries a carbon-15 alcohol in the R configuration that is rapidly converted by activated neutrophils to 15-epimeric lipoxins, or lipoxins that carry their 15-position alcohol in the R configuration (2) rather than 15S native LX, which results from LO-LO interaction (Figure 1).

The native LXs regulate human PMN responses that are relevant to inflammation and reperfusion injury. These include (1) inhibition of formyl-methionyl-leucyl-phenylalanine (fMLP) and LTB₄-induced chemotaxis (6), (2) adhesion and transmigration with endothelial cell (7), and (3) transmigration through epithelial cells (8). These actions of LXA₄ were also demonstrated in an acute murine inflammation model: in this model, (1) PMN infiltration was dramatically inhibited by stable analogs of both LXA₄ and aspirin-triggered 15-epi-LXA₄; (2) 15-epi-LXA₄ and LXA₄ were found to interact with a common receptor on human PMNs; and (3) bioactive LXA₄ analogs compete with [³H]LXA₄ binding to LXA₄ receptors (9). Thus, these inhibitory actions of LX analogs are likely to be mediated by specific LXA₄ receptors on mouse and human PMNs.

LXB₄ is a positional isomer of LXA₄, carrying alcohol groups at Carbon 5S, -14R, and -15S positions, instead of the C-5S, -6R, and -15S positions present in LXA₄. Aspirin-triggered LXB₄ carries a 15R alcohol, hence 15-epi-LXB₄ (see Figure 1). Although LXA₄ and LXB₄ show similar bioactivities in some systems (7), in many others they each show distinct actions (10) (see Reference 2 for a review). For example, 15-epi-LXB₄ is an inhibitor of cell proliferation (2). Here, we review findings indicating that 15-epi-LXA₄ is generated in inflammatory exudates in an aspirin-dependent manner and that aspirin-triggered LXA₄ and novel fluorinated LXA₄ as well as LXB₄ stable analogs are potent, topically active inhibitors of PMN-directed actions in vivo.

	METHODS

TOP INTRODUCTION TRANSCELLULAR GENERATION OF LX... METHODS RESULTS DISCUSSION REFERENCES

Materials

BALB/c mice (6-8 wk old), 5-LO^+/+ mice (129/SV IMR strains, JR2448), and 5-LO^/ mice (129/SV-ALOX5^ltm1Fun, JR2485) were from Jackson Laboratory (Bar Harbor, ME). Evans blue, fMLP and C5a were from Sigma (St. Louis, MO). Synthetic LX analogs, 15(R/S)-methyl-LXA₄ [5(S),6(R),15(R/S)-trihydroxy-15-methyl-7,9,13-trans-11-cis-eicosatetraenoic acid methyl ester], 16-phenoxy-LXA₄ [15(R)-16-phenoxy- 17,18,19,20-tetranol-LXA₄ methyl ester], 16-(para-fluoro)-phenoxy-LXA₄, 15-epi-16-(para-fluoro)-phenoxy-LXA₄, 5(S)-methyl-LXB₄ [5(S), 14(R),15(S)-trihydroxy-5(R)-methyl-6,8,12-trans-10-cis-eicosatetraenoic acid], and 5(R)-methyl-LXA₄ [5(R),14(R),15(S)-trihydroxy-5(S)- methyl-6,8,12-trans-10-cis eicosatetraenoic acid] (used as methyl ester unless indicated) were designed from knowledge of both 15-epi-LXA₄ [5(S),6(R),15(R)-trihydroxy-7,9,13-trans-11-cis eicosatetraenoic acid] and LXB₄ [5(S),14(R),15(S)-trihydroxy-6,8,12-trans-10-cis-eicosatetraenoic acid] structure and bioactivities, respectively (2). Total organic synthesis of the LX analogs was performed by N. Petasis and V. Fokin (Department of Chemistry, University of Southern California) as part of our ongoing collaborative efforts. Each analog was isolated by reversed-phase high-performance liquid chromatography (RP-HPLC), and their identities were confirmed by nuclear magnetic resonance as in Serhan and coworkers (11) in the case of LXA₄ analogs, and Maddox and coworkers (12) for the LXB₄ analogs. The LTB₄ antagonist U-75302 was from BioMol Research Laboratories (Plymouth Meeting, PA).

Mouse Ear Inflammation

BALB/c mice (6-8 wk old) were used as in Takano and coworkers (13). The inner side of the right ear was treated with acetone (i.e., vehicle control), and the inner side of the left ear was topically treated with the compounds to be tested prepared in acetone. After 5 to 7 min, LTB₄ (1 µg in acetone) was applied to the inner sides of both ears. At 24 h, punch biopsy samples (6 mm in diameter) (Acu-Punch; Acuderm, Ft. Lauderdale, FL) were obtained, and myeloperoxidase (MPO) activity and PMN infiltration were quantified (9). PMN infiltration was calculated after background levels of MPO activity present in mouse ear skin were subtracted. To quantify vascular permeability, 0.2 ml of Evans blue (0.5% in PBS² without Ca²⁺ or Mg²⁺) was intravenously injected immediately after topical application of the compounds to be evaluated. After 24 h, punch biopsies (4 mm in diameter; Acu-Punch) were obtained and Evans blue was extracted in formamide (55° C for 1 h) and quantified by measuring absorbances at 610 nm with subtraction of reference absorbance at 450 nm as in Takano and coworkers (13).

Liquid Chromatography-Tandem Mass Spectrometry Analysis: Lipoxin Analogs within Murine Ear Tissue

The right ear was biopsied as soon as the ear surface appeared dry, and placed in ice-cold methanol (400 µl containing 1 µg of prostaglandin B₂ [PGB₂] for internal standard), and kept at 20° C until extracted. The left ear was processed in the same fashion at 24 h. Each sample was gently homogenized with a glass homogenizer, washed twice with methanol (200 µl), once with ethanol (200 µl), and extracted with Extract-Clean solid-phase extraction cartridges (500 mg C₁₈; Alltech Associates, Deerfield, IL). Materials that eluted in the methyl formate fractions were concentrated under a stream of N₂ and injected into a liquid chromatography-tandem mass spectrometer (LC/MS/MS) (LCQ; Finnigan MAT, San Jose, CA) using settings reported in reference 12. Each compound was quantified with PGB₂ as an internal standard.

	RESULTS

TOP INTRODUCTION TRANSCELLULAR GENERATION OF LX... METHODS RESULTS DISCUSSION REFERENCES

ASA-dependent Generation of 15-epi-LXA₄ by Mouse Peritoneal Exudates

To determine whether 15-epi-LXA₄ (Figure 1) could be detected in animal models, experiments were carried out with a mouse peritonitis model (14). In this model, COX-II protein levels were found to be upregulated by intraperitoneal injection of LPS. Peritonitis was induced by intraperitoneal injection of casein. In these experiments, upregulation of COX-II was also demonstrated by Western blot analysis and the immunoreactive bands were observed at ~ 70 kD in peritoneal lavage samples from LPS-treated mice.

Four hours after leukocyte infiltration was initiated, approximately 25 × 10⁶ cells were obtained by peritoneal lavage of each mouse. The exudate leukocyte populations were ~ 73% PMNs and 10% monocytes (and/or macrophages), respectively, as determined by hematoxylin and eosin (H&E) staining and enumeration by light microscopy. To test if ASA treatment of the mice results in the generation of 15-epi-LXA₄ during an inflammatory event, ASA was administrated by intraperitoneal injection (see experimental timeline, Figure 2, top). 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 using a newly developed specific enzyme-linked immunosorbent assay (ELISA) (14). Without administration of ASA, approximately 0.5 ng of 15-epi-LXA₄ per 5 ml of lavage was associated with peritoneal exudates from each mouse. Given one or two doses of ASA (Figure 2), the mean values for 15-epi-LXA₄ production were ~ 1.5 and 1.8 ng per 5 ml of peritoneal lavage per mouse. Naive animals (without any treatment) gave low levels (< 0.2 ng/ 5 ml of peritoneal lavage per mouse) of 15-epi-LXA₄. The physiological relevance of this value obtained in the absence of experimental challenge is currently not known. Because LPS upregulates COX-II (3) and can induce neutrophil recruitment, we tested the possibility that animals treated with LPS could give rise to 15-epi-LXA₄ in the absence of casein. The results obtained showed a low level of 15-epi-LXA₄ generation in these LPS-treated animals in the presence or absence of ASA (0.25 and 0.30 ng per 5 ml of peritoneal lavage per mouse, respectively), suggesting that LPS alone was not sufficient to elicit neutrophil infiltration into peritoneal lavage. Casein-induced neutrophil infiltration and ASA are required in this scenario to generate significant levels of 15-epi-LXA₄. Thus, these results demonstrate that ASA administration in a murine model of peritonitis gives inflammatory exudates that, when stimulated, generate 15-epi-LXA₄ in appreciable levels from endogenous substrate with inflammatory cells.

View larger version (20K): [in this window] [in a new window]   Figure 2.   Peritoneal inflammatory exudates from ASA-treated mice generate 15-epi-LXA4. Mice were injected with LPS (1.25 mg/kg body weight) to induce COX-II and 16 h later they were treated with vehicle or (a) one bolus dose of ASA (0.125 g/kg body weight) by intraperitoneal injection for 30 min before casein-initiated induction of neutrophil infiltration for 4 h. (b) Consecutive doses of ASA were given in a separate group of animals that received ASA (0.125 g/kg body weight) by intraperitoneal injection 30 min before casein injection and before killing the mice. The lavage exudates were collected and stimulated with 5 µM A23187 (30 min, 37° C). The values are expressed as the mean ± SEM from three independent experiments. The values from both treatment groups were significantly different (*p = 0.05 for group a, **p = 0.03 for group b) from those obtained with animals that did not receive ASA (vehicle). The samples were extracted as reported in Chiang and coworkers (14). BW = body weight; IP = intraperitoneal injection.

PMN Infiltration in 5-Lipoxygenase Knockout Mice

To assess the role of 5-LO products in recruitment of PMNs to murine skin, we evaluated the chemotactic capacity for LTB₄ in 5-LO knock out mice. Topical application of either 1 or 5 µg of LTB₄ induced significant PMN infiltration to mouse ear that reached maximum at 24 h (13). Both 5-LO^/ and 5-LO^+/+ mice showed essentially equivalent levels of PMN infiltration into the ears, indicating that LTB₄ receptor signaling was intact in 5-LO^/mice and suggesting that inhibitors of 5-lipoxygenase might be of limited utility in this tissue. After a 24-h exposure to topical LTB₄, approximately 5 × 10⁵ PMNs infiltrated per 6-mm punch biopsy, which was equivalent to the levels induced by five times more LTB₄ within 8 h. Thus, topically applied LTB₄ at 1 µg/ear was selected for further experiments.

LXA₄ Stable Analogs Inhibit Both PMN Infiltration and Vascular Permeability Changes

To evaluate other approaches, we studied LX stable analogs that we designed as mimetics of the inhibitory actions noted for LXA₄ (11) and more recently for LXB₄ (12) in vitro. We examined several LXA₄ stable analogs and tested their ability to inhibit PMN infiltration and changes in vascular permeability. 15(R/S)-methyl-LXA₄ having a methyl group at C-15 position (racemate 15R/S), is an analog of both the aspirin-triggered 15-epi-LXA₄ and native LXA₄; and 16-phenoxy-LXA₄, which has a phenoxy group at the C-16 position, is an analog of native LXA₄ that prevents enzymatic inactivation with recombinant 15-prostaglandin dehydrogenase in vitro (11) (and see structures in Figure 3). Both analogs act at LXA₄ receptors (9).

View larger version (17K): [in this window] [in a new window]   Figure 3.   Structures of several lipoxin stable analogs. For further details, see References 11 and 12.

Applied topically to mouse ears, these LX stable analogs inhibited both PMN infiltration and vascular permeability changes in a concentration-dependent fashion (Figures 4A and 4B). At 130 nmol per ear, the degree of inhibition of PMN infiltration was more than 90% for both analogs, with apparent median inhibitory concentrations (IC₅₀S) noted at a range of ~ 13-26 nmol per ear for each analog. In the same concentration range, these two LXA₄ stable analogs also inhibited vascular permeability, namely, extravasation of Evans blue. At 130 nmol per ear, the inhibition of vascular permeability change was > 98% for 15(R/S)-methyl-LXA₄, and ~ 87% for 16-phenoxy-LXA₄, respectively, and their impact was visible (see Figure 4C). The inhibition of vascular permeability changes paralleled inhibition of PMN infiltration with both analogs.

View larger version (14K): [in this window] [in a new window]   View larger version (45K): [in this window] [in a new window]   Figure 4.   LXA4 analogs inhibit vascular permeability change and PMN infiltration in the mouse ear. Mouse ears were topically treated with vehicle (acetone) or the indicated amounts of LXA4 analogs, and then inflammation was induced by topical application of LTB4 (1 µg). Evans blue (0.5%; 200 µl) was intravenously injected into the tail vein immediately after treatment of the ears. Punch biopsies were obtained after 24 h, and MPO activity (A) and Evans blue (B) were quantified as in Takano and coworkers (13). Black bars, 15(R/S)-methyl-LXA4; white bars, 16-phenoxy-LXA4. Results are means ± SEM of n = 3-5 experiments, except for 15(R/S)-methyl-LXA4 at 130 nmol per ear in (B) (n = 2). (C ) Photographs of mouse ears showing LTB4-stimulated vascular permeability and inhibition by topical application of LX analog. Right ear was treated with acetone, and left ear was treated simultaneously with 15(R/S)-methyl-LXA4 (130 nmol). LTB4 was applied to both ears.

Direct Comparison of A Series and B Series LX Stable Analogs

We also compared the actions of three LXA₄ analogs with those of native LXA₄ and the LTB₄ receptor antagonist U-75302. In addition, we evaluated the impact of LXB₄ analogs that resist enzymatic inactivation in vitro (12) (for structures, see Figure 3). When applied topically at 26 nmol per ear, the stable analogs were three to four times more potent than native LXA₄. Of the LX stable analogs tested, 15(R/S)-methyl-LXA₄ was the most potent (> 70% inhibition), and its inhibitory actions on PMN infiltration and vascular permeability changes were significantly greater than those of topically applied native LXA₄ (p < 0.05), indicating that these analogs also increase bioavailability as topical agents because LXA₄ and these analogs are within a similar potency range in in vitro assays of leukocyte responses (11, 12). A 16-para-fluoro derivative of 16-phenoxy-LXA₄ was prepared for these experiments to assess whether fluorination of the phenoxy ring could enhance potency. Results in Figures 5A and 5B indicate that 16- (para-fluoro)-phenoxy-LXA₄ was also potent and retained the activity at levels comparable to 16-phenoxy-LXA₄. Both of the two LXB₄ analogs inhibited PMN infiltration and vascular permeability. The S enantiomer 5(S)-methyl-LXB₄, was significantly more potent than 5(R)-methyl-LXB₄ indicating a preferred stereoselectivity for inhibition. The rank order of inhibitory potency was 15(R/S)-methyl-LXA₄ > 16-para-fluoro-phenoxy-LXA₄  5(S)-methyl-LXB₄ > 16-phenoxy-LXA₄ > 5(R)-methyl-LXA₄ for both PMN infiltration and vascular permeability changes.

View larger version (24K): [in this window] [in a new window]   Figure 5.   LXA4 and LXB4 stable analogs inhibit PMN infiltration and vascular permeability change: the rank order of potency. Experiments were carried out as in Figure 4, using 26 nmol of LX analog per ear, and compared with the LTB4 receptor antagonist U-75302. (A) Percent inhibition of PMN infiltration; (B) percent inhibition of vascular permeability change (Evans blue extravasation). Results are means ± SEM of n = 3 experiments.

Clearance of Topical Lipoxin Analogs

LX are rapidly inactivated by conversion to oxo- and dihydro-containing products (reviewed in Reference 2). Each biologically active LX analog tested also resisted rapid conversion by recombinant dehydrogenase in vitro (11, 12). To determine whether these analogs were cleared and/or present in an extractable form within ear skin tissues after topical application, biopsied ear tissues were taken for LC/MS/MS analysis at the time of application and after a time interval (i.e., 24 h), when high-level inhibition was found for several LX analogs (Table 1 and Figure 4). The eicosanoids recovered after extraction of ear biopsies (i.e., immediately after application and at 24 h) were identified and quantitated by LC/MS/MS. LTB₄ was monitored for purposes of direct comparison, and selected ion chromatograms of PGB₂ (internal standard) and LTB₄ were obtained after extraction from biopsies. Two of the more active LXA₄ analogs and an LXB₄ analog were examined. After initial applications to the ears, only ~ 10-20% of the applied compounds were present within the target area of the biopsies (Table 1). At 24 h after application, this value was reduced to < 10%, indicating that > 95% of the added LX analogs were not recoverable in extractable forms and thus were likely cleared by the ear tissue.

Do LX Analogs Block Other Inflammatory Stimuli?

A panel of known inflammatory mediators was examined to test the specificity and/or generality of the actions of LX analogs (Table 2). PGE₂ dramatically augmented the LTB₄- induced PMN infiltration and vascular permeability change, although the effects of this prostaglandin by itself were minimal. Phorbol myristate acetate (PMA), a tumor promotor and topical irritant that bypasses cell surface receptors, caused concentration-dependent changes (not shown) in both PMN infiltration and vascular permeability (see Table 1), and 100 ng of PMA per ear was chosen for further evaluation. Several potent agents such as fMLP, C5a, IL-8, platelet-activating factor (PAF), or LTD₄ did not give significant changes in these parameters when applied in amounts as high as 1 to 25 µg (data not shown) compared with LTB₄ (Table 2), suggesting that they are not topically active, perhaps because they do not gain access in intact skin tissues to locations that would establish a chemotactic gradient.

We evaluated the impact of 15-epi-LXA₄ with a stable analog, 15-epi-16-(para-fluoro)-phenoxy-LXA₄ (see Figure 3). This LX analog not only inhibited LTB₄ but also inhibited PGE₂-enhanced inflammation (Table 2). Of interest, it also inhibited PMA-induced PMN influx, with little impact at 24 h on PMA-induced vascular leakage. Thus, LXA₄ stable analogs clearly inhibit native mediators as topically active agents and partially blocked PMA-evoked actions that were restricted to inhibition of PMN influx. The observations with PMA indicate that a major component of PMA-induced vascular leakage is not mediated by PMN-dependent mechanisms and likely involves direct vascular sites of actions for PMA that are not blocked by LX analogs.

	DISCUSSION

TOP INTRODUCTION TRANSCELLULAR GENERATION OF LX... METHODS RESULTS DISCUSSION REFERENCES

To briefly summarize, 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 selectively interact with 15-epi-LXA₄ (14). Utilizing this ELISA, we established that 15-epi-LXA₄ generation proceeds via transcellular biosynthesis during heterotypic leukocyte-leukocyte interactions and provided the first evidence of ASA-dependent 15-epi-LXA₄ generation by inflammatory exudate cells from a murine model with LPS and casein-induced peritonitis (Figure 2).

The stable LX analogs reviewed here and found to be potent inhibitors of both PMN infiltration and PMN-mediated vascular permeability changes (Figures 3-5) were designed to resist rapid inactivation and proved able to resist conversion by cells and isolated recombinant enzymes in vitro (11, 12). Therefore, it was of interest to determine whether the LX analogs remained within the ear tissues after topical application or if they were effectively cleared. These results indicate that at 24 h after topical application greater than ~ 95% of the LX analogs were not present in an extractable or recoverable forms from the biopsies, which suggests that they were either cleared from the biopsied areas or were present within these tissues in forms that were not extractable by the current methodology. We also did not find evidence, using LC/MS/MS workstation-based analyses, of anticipated local metabolites of these LX analogs persisting within the biopsies. It is noteworthy that, in addition to local clearance, an alternative explanation may be that LX analogs could have been subject to local metabolism and/or covalent modification that results in their binding to tissue matrix components. Whether such matrix forms of LX analogs exist and whether they are in an inactive or bioactive configuration is of interest. Nevertheless, it is clear from the present results that LX analogs are not retained in their native form within the local microenvironment (i.e., ear biopsies), and this may be another useful property of these LX analogs.

Taken together, our findings reviewed here indicate that aspirin initiates the formation of 15-epi-LXA₄ in murine inflammatory exudates and that stable analogs of aspirin-triggered 15-epi-LXA₄, LXA₄, and LXB₄ are potent, topically active agents that inhibit both PMN recruitment to murine skin and PMN-mediated changes in vascular permeability. Moreover, they provide additional new tools for investigating the actions of LX and evidence that the cell-cell interactions that generate LX do so to limit further PMN recruitment.

	Footnotes

Correspondence and requests for reprints should be addressed to Charles N. Serhan, Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesia, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115.

Acknowledgments: The authors thank Mary Halm Small for expert assistance in the preparation of this manuscript.

Supported in part by National Institutes of Health Grants GM38765 and DK50305 and by a grant from Schering AG (to C. N. Serhan).

	References

TOP INTRODUCTION TRANSCELLULAR GENERATION OF LX... METHODS RESULTS DISCUSSION REFERENCES

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