Selectivity of recombinant human leukotriene D(4), leukotriene B(4), and lipoxin A(4) receptors with aspirin-triggered 15-epi-LXA(4) and regulation of vascular and inflammatory responses

Abstract

Aspirin-triggered lipoxin A(4) (ATL, 15-epi-LXA(4)) and leukotriene D(4) (LTD(4)) possess opposing vascular actions mediated via receptors distinct from the LXA(4) receptor (ALX) that is involved in leukocyte trafficking. Here, we identified these receptors by nucleotide sequencing and demonstrate that LTD(4) receptor (CysLT(1)) is induced in human vascular endothelia by interleukin-1beta. Recombinant CysLT(1) receptor gave stereospecific binding with both [(3)H]-LTD(4) and a novel labeled mimetic of ATL ([(3)H]-ATLa) that was displaced with LTD(4) and ATLa ( approximately IC(50) 0.2 to 0.9 nmol/L), but not with a bioinactive ATL isomer. The clinically used CysLT(1) receptor antagonist, Singulair, showed a lower rank order for competition with [(3)H]-ATLa (IC(50) approximately 8.3 nmol/L). In contrast, LTD(4) was an ineffective competitive ligand for recombinant ALX receptor with [(3)H]-ATLa, and ATLa did not compete for [(3)H]-LTB(4) binding with recombinant LTB(4) receptor. Endogenous murine CysLT(1) receptors also gave specific [(3)H]-ATLa binding that was displaced with essentially equal affinity by LTD(4) or ATLa. Systemic ATLa proved to be a potent inhibitor (>50%) of CysLT(1)-mediated vascular leakage in murine skin (200 microg/kg) in addition to its ability to block polymorphonuclear leukocyte recruitment to dorsal air pouch (4 microg/kg). These results indicate that ATL and LTD(4) bind and compete with equal affinity at CysLT(1), providing a molecular basis for aspirin-triggered LXs serving as a local damper of both vascular CysLT(1) signals as well as ALX receptor-regulated polymorphonuclear leukocyte traffic.

Figure 1.

Identification of human vascular endothelial-derived CysLT₁ receptor. A: CysLT₁ receptor RNA expression in mucosal epithelial cells and IL-1β induction in HUVECs. RT-PCR results (see Methods) are from two separate T84 cell RNA samples in parallel lanes (n = 4), a representative for HUVECs exposed to IL-1β (6 or 24 hours) or media alone (n = 3), and PCR control (without RNA, molecular size of products is indicated by arrow). B: Competition for specific [³H]-LTD₄ (1 nmol/L) binding with increasing concentrations of LTD₄ or ATLa in isolated membrane preparations from CysLT₁ receptor stable transfectants (COS-7 cells, n = 3 with duplicates). Structures of the CysLT₁ receptor competitors are shown in inset. C: Competition for [11,12-³H]-ATLa (1 nmol/L)-specific binding with increasing concentrations of ATLa, the specific CysLT₁ receptor antagonist montelukast or the bioinactive 6S-LXA₄ (inset) in isolated membrane preparations from CysLT₁ receptor stable transfectants (COS-7 cells, n = 3 with duplicates).

Figure 2.

Selectivity of recombinant human LXA₄ and LTB₄ receptor. A: Competition for [11,12-³H]-ATLa-specific binding (1 nmol/L) with increasing concentrations of ATLa, LXA₄, or the specific CysLT₁ receptor antagonist MK571 in intact cells of ALX receptor stable transfectants (HEK293 cells, n = 3 with duplicates). B: Competition for [³H]-LTB₄ (1 nmol/L)-specific binding with ATLa or LTB₄ (inset) in intact cells of BLT receptor stable transfectants (HEK293 cells, n = 3 with duplicates).

Figure 3.

ATLa competes for specific binding at murine CysLT₁ receptor as well as inhibits both vascular leakage and PMN trafficking. A: Competition for [11,12-³H]-ATLa (1 nmol/L)-specific binding with increasing concentrations of LTD₄ or ATLa in isolated membrane preparations from rat lungs (n = 3 with duplicates). B: Inhibition of vascular leakage and leukocyte trafficking. Mice were injected intravenously (left tail vein) with LTD₄ (0.5 μg), ATLa (5 μg), or a single combined injection of LTD₄ (0.5 μg) and ATLa (5 μg) and/or a CysLT₁ antagonist (MK571, 5 μg). A second injection of Evans Blue was given 90 seconds after treatment (see Methods). Results represent the mean (n = 3 to 8 with duplicates) above vehicle. #, P < 0.02 compared to LTD₄ treatment alone; **, P < 0.01 compared to vehicle alone. Top inset: LTD₄ (0.5 μg) stimulated vascular leakage (10 minutes after injection) and bottom inset: Inhibition with a combined dose of antagonists (ie, ATLa and MK571, each at 5 μg). Right ordinate: TNF-α (20 ng) was injected locally into 6-day dorsal air pouch and ATLa (100 ng) or vehicle injected intravenously (left tail vein) and infiltrated leukocytes quantitated at 4 hours after treatment (n = 3).