Nucleotide and deduced amino acid sequence of the mouse LXA₄R. The mouse LXA₄R has an open reading frame encoding 351 amino acids. Putative transmembrane regions (TM) are indicated with bars, and possible N-glycosylation sites are indicated by an asterisk (*). These sequence data are available from EMBL/GenBank/DDBJ under the accession number U78299.

Alignment of human and mouse LXA₄R. (A) The amino acid sequence of human LXA₄R was aligned to the mouse homologue. Their amino acid sequences were 73% identical. The vertical bars indicate the identical residues. (B) Percentage of identity of each segment is shown.

GTP hydrolysis by the mouse LXA₄R stable transformant. Rates of γ-[³²P]dCTP hydrolysis were determined by calculating the linear regression of ³²Pi release in the initial 3 min after ligand addition (10⁻⁸ M) to electropermeabilized CHO cells that were stably transfected with mouse LXA₄R cDNA or vector alone. Open bars, mock transfected cells. Hatched bars, mouse LXA₄R stable transformant. Data are mean ± SEM, n = 4–5. *P <0.05 to all the other bars.

Competitive displacement of [³H]LXA₄ and [³H]LTD₄ by LXA₄ analogues. (A) Specific binding of [³H]LXA₄ on PMN: comparison of 15(R/S)-methylLXA₄, 16-phenoxy-LXA₄ and 15epi-LXA₄. PMN were suspended in DPBS (5 × 10⁷cells/ml) and binding of [³H]LXA₄ (0.3 nM) was determined at 4°C in the presence or absence of LXA₄ (▪), 15(R/S)-methyl-LXA₄ (○), 15epi-LXA₄ (•), and 16-phenoxyLXA₄ (▵) as described in Materials and Methods. Result is the representative of three independent experiments with duplicate determination. (B) Evaluation of LXA₄ analogues in [³H]LTD₄ displacement assays with HUVEC. Cells were cultured in 12-wells plate (∼3.5 × 10⁵ cells/well), and [³H]LTD₄ (5 nM) binding was assessed at 4°C in the presence or absence of unlabeled LTD₄ (▪), LXA₄(▪), 15(R/S)- methyl-LXA₄ (○), 15-epi-LXA₄ (•), 16-phenoxy-LXA₄ (▵) or SKF-104353 (▾) as described in Materials and Methods. Results are the mean ± SEM of three separate experiments with duplicate determinations.

Ear biopsies: inhibition of LTB₄-induced neutrophil infiltration by 16-phenoxy-LXA₄. (A) Section of ear exposed to vehicle alone (8 h). (B) sections of ears exposed to LTB₄ (5 μg) for 8 h as in Fig. 8, upper panel, low power field; bottom panel, high power. Arrow indicates presence of neutrophils. (C) Ears exposed to 16-phenoxy-LXA₄ and LTB₄ as in Fig. 8; upper panel, low power field and bottom, high power field showing a sharp reduction in neutrophils. Sections were prepared as described in Materials and Methods and stained with hematoxylin and eosin.

Northern hybridization of the mouse and human LXA₄R. (A) Mouse multiple tissue blot: each lane of mouse multiple tissue blot contains 2μg poly(A)⁺ RNA. 10 μg of total RNA was used for mouse neutrophil. Filters were hybridized and washed as described in Materials and Methods. Filters were exposed to x-ray film with intensifier at −80°C for 120 h (multiple tissue blot) or 72 h (neutrophil). (B) Human multiple tissue blot: each lane contains 2μg poly(A)⁺ RNA. Filter was hybridized and washed as described in Materials and Methods. Filter was exposed to x-ray film for 24 h.

Specific binding characteristics of mouse LXA₄R expressed in CHO cells. After transfection with mouse LXA₄R cDNA (48 h), intact CHO cells were resuspended in DPBS²⁺ (10⁶ cells /ml). Cells were incubated with 0.3 nM of [³H]LXA₄ in the presence of indicated concentrations of homoligand for 5 min at 4°C. The incubations were terminated by rapid centrifugation (12,000 g, 60 s) through silicon oil (d = 1.028), and cell-associated radioactivity was determined with 10⁶ cells used per incubation. Data were analyzed with the Ligand program (Biosoft Elsevier). Results are representative of four independent experiments (mean ± SEM, n = 4). Specific binding was not detected in the mock transfected cells with vector (pcDNA3) alone.

Structure of LXA₄ stable analogues. Structures of LXA₄ stable analogues used in these experiments. 15-epi-LXA₄ is an aspirin triggered lipoxin, and carried a C-15 alcohol at the R configuration, opposite to the S configuration in native LXA₄. 16-phenoxy-LXA₄ has phenoxy group at C-16, and 15-epi-16-phenoxy-LXA₄ carried its C-15 alcohol at the R configuration, in addition to the phenoxy group at C-16, and is a stable analogue of 15-epi-LXA₄. In 15(R/S)-methyl-LXA₄, hydrogen at C-15 was replaced by a methyl group as a racemate at C-15. 15(R/S)-16phenoxy-11,12-acetylenic-LXA₄ has a phenoxy group at C-16 and racemic (∼50:50) C-15 alcohol, and also carried an acetylenic bond at C11-12.

Topical application of LXA₄ and 15-epi-LXA₄ analogues inhibit neutrophil infiltration in vivo. (A) Mouse ears were topically treated with equimolar amounts of LTB₄ (1 μg), FMLP (1.3 μg), or platelet-activating factor (1.6 μg) in 20 μl of acetone. After 24 h, punch biopsy samples (6-mm diam) were obtained from each ear, and MPO activity was measured as described in Materials and Methods. MPO activity was further converted into number of neutrophils using the standard curve obtained using peritoneal neutrophils (inset). Neutrophils (2 × 10⁶ cells) gave an absorbance change of 0.25 units per min at 460 nm. Results are mean ± SEM of n = 3–5. (B) Mouse ears were topically treated either with vehicle (acetone), 16-phenoxyLXA₄ (240 nmol), LTB₄ (5 μg), or LTD₄ (5 μg). After 8 h, PMN infiltration was determined as in Fig. 8 A. Results are mean ± SEM of n = 4 (vehicle, LTB₄), n = 3 (LTD₄), or n = 2 (16-phenoxy-LXA₄). (C) Mouse ears were topically treated with either vehicle (○) or 16-phenoxy-LXA₄ (240 nmol) (•) and then exposed to 5 μg LTB₄ (see Materials and Methods). Results are mean ± SEM of n = 4. *P <0.01; #, P <0.05 vs. 16-phenoxy-LXA₄ treatment. (D) Mouse ears were topically treated either with vehicle or indicated amount of 16-phenoxy-LXA₄ or dexamethasone and then stimulated by 5 μg LTB₄ for 8 h. Percent inhibition of PMN infiltration was calculated with vehicle treated ear as 100% after background levels (MPO activity of ear treated with acetone alone) were subtracted. Results are mean ± SEM of n = 3 or 4. (E) Mouse ears were topically treated either with vehicle or 15(R/S)- 16-phenoxy-11,12-acetylenic-LXA₄ (120 nmol), or 16-phenoxy-LXA₄ (240 nmol), or 15-epi-16-phenoxy-LXA₄ (240 nmol), and then exposed to LTB₄ (5 μg) for 8 h. Percent inhibition of PMN infiltration was calculated as in D. (Results for 16-phenoxy-LXA₄ are the same in D.) Results are mean ± SEM of n = 3–6.