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Aspirin-triggered 15-epi-lipoxin A4 (LXA4) and LXA4 stable analogues are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors.

Takano T, Fiore S, Maddox JF, Brady HR, Petasis NA, Serhan CN - J. Exp. Med. (1997)

Bottom Line: Topical application of LXA4 analogues and novel aspirin-triggered 15-epi-LXA4 stable analogues to mouse ears markedly inhibited neutrophil infiltration in vivo as assessed by both light microscopy and reduced myeloperoxidase activity in skin biopsies.The 15(R)-16-phenoxy-17,18, 19,20-tetranor-LXA4 methyl ester (15-epi-16-phenoxy-LXA4), an analogue of aspirin triggered 15-epi-LXA4, and 15(S)-16-phenoxy-17,18,19,20-tetranor-LXA4 methyl ester (16-phenoxy-LXA4) were each as potent as equimolar applications of the anti-inflammatory, dexamethasone.These findings provide direct in vivo evidence for an anti-inflammatory action for both aspirin-triggered LXA4 and LXA4 stable analogues and their site of action in vivo.

View Article: PubMed Central - PubMed

Affiliation: Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesia, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Lipoxins are bioactive eicosanoids that are immunomodulators. In human myeloid cells, lipoxin (LX) A4 actions are mediated by interaction with a G protein-coupled receptor. To explore functions of LXA4 and aspirin-triggered 5(S),6(R),15(R)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (15-epi-LXA4) in vivo, we cloned and characterized a mouse LXA4 receptor (LXA4R). When expressed in Chinese hamster ovary cells, the mouse LXA4R showed specific binding to [3H]LXA4 (K(d) approximately 1.5 nM), and with LXA4 activated GTP hydrolysis. Mouse LXA4R mRNA was most abundant in neutrophils. In addition to LXA4 and 15-epi-LXA4, bioactive LX stable analogues competed with both [3H]LXA4 and [3H]leukotriene D4 (LTD4)-specific binding in vitro to neutrophils and endothelial cells, respectively. Topical application of LXA4 analogues and novel aspirin-triggered 15-epi-LXA4 stable analogues to mouse ears markedly inhibited neutrophil infiltration in vivo as assessed by both light microscopy and reduced myeloperoxidase activity in skin biopsies. The 15(R)-16-phenoxy-17,18, 19,20-tetranor-LXA4 methyl ester (15-epi-16-phenoxy-LXA4), an analogue of aspirin triggered 15-epi-LXA4, and 15(S)-16-phenoxy-17,18,19,20-tetranor-LXA4 methyl ester (16-phenoxy-LXA4) were each as potent as equimolar applications of the anti-inflammatory, dexamethasone. Thus, we identified murine LXA4R, which is highly expressed on murine neutrophils, and showed that both LXA4 and 15-epi-LXA4 stable analogues inhibit neutrophil infiltration in the mouse ear model of inflammation. These findings provide direct in vivo evidence for an anti-inflammatory action for both aspirin-triggered LXA4 and LXA4 stable analogues and their site of action in vivo.

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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.
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Figure 1: 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.

Mentions: A mouse spleen cDNA library was purchased from Clontech (Palo Alto, CA), and 6 × 105 clones were screened with the EcoRI fragment from the human LXA4R cDNA employing high stringency. A positive clone (designated 15-2) was isolated. Phage DNA was amplified and purified, and the insert cDNA was excised by EcoRI digestion and subcloned into the EcoRI site of pBluescript II KS(+) (obtained from Stratagene, La Jolla, CA). Sequence analysis showed that this clone 15-2 was a partial clone lacking the amino terminal region (nucleotide 87, of full-length clone; see Fig. 1). To obtain the missing amino-terminal region, we used the rapid amplification of cDNA end or rapid amplification of cDNA end (RACE) technique. The 5′-RACE-Ready cDNA™ from spleen was purchased from Clontech (Palo Alto, CA), and RACE was performed according to the manufacturer's instructions. The first round of PCR was done between the anchor primer provided by the manufacturer and synthetic primer 5′-GCCATTTCAACAAGAAGGAATGGTAGAG-3′ (antisense of nucleotide 229–257) for 30 cycles (94°C for 30 s, 60°C for 45 s, 72°C for 2 min). The first PCR product was diluted to 1:50, and a second round of PCR was carried out between the anchor primer and a synthetic primer 5′-GCTGTGAAAGAGAAGTCAGCCAATGCTA-3′ (antisense of nucleotide 199–227) using the same condition for 35 cycles. A PCR product of ∼300 bp was obtained and subcloned into pBluescript II KS(+) for sequencing. Overlapping regions of RACE product and clone 15-2 (nucleotide 87–198) were found to be identical. The RACE product was subcloned to the 5′ end of clone 15-2 to construct a full-length clone, using a SpeI site at nucleotide 136. Hydrophobicity analysis of amino acid sequence and homology comparison were performed using Lasergene (DNASTAR Inc., Madison WI).


Aspirin-triggered 15-epi-lipoxin A4 (LXA4) and LXA4 stable analogues are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors.

Takano T, Fiore S, Maddox JF, Brady HR, Petasis NA, Serhan CN - J. Exp. Med. (1997)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196289&req=5

Figure 1: 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.
Mentions: A mouse spleen cDNA library was purchased from Clontech (Palo Alto, CA), and 6 × 105 clones were screened with the EcoRI fragment from the human LXA4R cDNA employing high stringency. A positive clone (designated 15-2) was isolated. Phage DNA was amplified and purified, and the insert cDNA was excised by EcoRI digestion and subcloned into the EcoRI site of pBluescript II KS(+) (obtained from Stratagene, La Jolla, CA). Sequence analysis showed that this clone 15-2 was a partial clone lacking the amino terminal region (nucleotide 87, of full-length clone; see Fig. 1). To obtain the missing amino-terminal region, we used the rapid amplification of cDNA end or rapid amplification of cDNA end (RACE) technique. The 5′-RACE-Ready cDNA™ from spleen was purchased from Clontech (Palo Alto, CA), and RACE was performed according to the manufacturer's instructions. The first round of PCR was done between the anchor primer provided by the manufacturer and synthetic primer 5′-GCCATTTCAACAAGAAGGAATGGTAGAG-3′ (antisense of nucleotide 229–257) for 30 cycles (94°C for 30 s, 60°C for 45 s, 72°C for 2 min). The first PCR product was diluted to 1:50, and a second round of PCR was carried out between the anchor primer and a synthetic primer 5′-GCTGTGAAAGAGAAGTCAGCCAATGCTA-3′ (antisense of nucleotide 199–227) using the same condition for 35 cycles. A PCR product of ∼300 bp was obtained and subcloned into pBluescript II KS(+) for sequencing. Overlapping regions of RACE product and clone 15-2 (nucleotide 87–198) were found to be identical. The RACE product was subcloned to the 5′ end of clone 15-2 to construct a full-length clone, using a SpeI site at nucleotide 136. Hydrophobicity analysis of amino acid sequence and homology comparison were performed using Lasergene (DNASTAR Inc., Madison WI).

Bottom Line: Topical application of LXA4 analogues and novel aspirin-triggered 15-epi-LXA4 stable analogues to mouse ears markedly inhibited neutrophil infiltration in vivo as assessed by both light microscopy and reduced myeloperoxidase activity in skin biopsies.The 15(R)-16-phenoxy-17,18, 19,20-tetranor-LXA4 methyl ester (15-epi-16-phenoxy-LXA4), an analogue of aspirin triggered 15-epi-LXA4, and 15(S)-16-phenoxy-17,18,19,20-tetranor-LXA4 methyl ester (16-phenoxy-LXA4) were each as potent as equimolar applications of the anti-inflammatory, dexamethasone.These findings provide direct in vivo evidence for an anti-inflammatory action for both aspirin-triggered LXA4 and LXA4 stable analogues and their site of action in vivo.

View Article: PubMed Central - PubMed

Affiliation: Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesia, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Lipoxins are bioactive eicosanoids that are immunomodulators. In human myeloid cells, lipoxin (LX) A4 actions are mediated by interaction with a G protein-coupled receptor. To explore functions of LXA4 and aspirin-triggered 5(S),6(R),15(R)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (15-epi-LXA4) in vivo, we cloned and characterized a mouse LXA4 receptor (LXA4R). When expressed in Chinese hamster ovary cells, the mouse LXA4R showed specific binding to [3H]LXA4 (K(d) approximately 1.5 nM), and with LXA4 activated GTP hydrolysis. Mouse LXA4R mRNA was most abundant in neutrophils. In addition to LXA4 and 15-epi-LXA4, bioactive LX stable analogues competed with both [3H]LXA4 and [3H]leukotriene D4 (LTD4)-specific binding in vitro to neutrophils and endothelial cells, respectively. Topical application of LXA4 analogues and novel aspirin-triggered 15-epi-LXA4 stable analogues to mouse ears markedly inhibited neutrophil infiltration in vivo as assessed by both light microscopy and reduced myeloperoxidase activity in skin biopsies. The 15(R)-16-phenoxy-17,18, 19,20-tetranor-LXA4 methyl ester (15-epi-16-phenoxy-LXA4), an analogue of aspirin triggered 15-epi-LXA4, and 15(S)-16-phenoxy-17,18,19,20-tetranor-LXA4 methyl ester (16-phenoxy-LXA4) were each as potent as equimolar applications of the anti-inflammatory, dexamethasone. Thus, we identified murine LXA4R, which is highly expressed on murine neutrophils, and showed that both LXA4 and 15-epi-LXA4 stable analogues inhibit neutrophil infiltration in the mouse ear model of inflammation. These findings provide direct in vivo evidence for an anti-inflammatory action for both aspirin-triggered LXA4 and LXA4 stable analogues and their site of action in vivo.

Show MeSH