Activation of lipoxin A(4) receptors by aspirin-triggered lipoxins and select peptides evokes ligand-specific responses in inflammation

Abstract

Lipoxin (LX) A(4) and aspirin-triggered LX (ATL) are endogenous lipids that regulate leukocyte trafficking via specific LXA(4) receptors (ALXRs) and mediate antiinflammation and resolution. ATL analogues dramatically inhibited human neutrophil (polymorphonuclear leukocyte [PMN]) responses evoked by a potent necrotactic peptide derived from mitochondria as well as a rogue synthetic chemotactic peptide. These bioactive lipid analogues and small peptides each selectively competed for specific (3)H-LXA(4) binding with recombinant human ALXR, and its N-glycosylation proved essential for peptide but not LXA(4) recognition. Chimeric receptors constructed from receptors with opposing functions, namely ALXR and leukotriene B(4) receptors (BLTs), revealed that the seventh transmembrane segment and adjacent regions of ALXR are essential for LXA(4) recognition, and additional regions of ALXR are required for high affinity binding of the peptide ligands. Together, these findings are the first to indicate that a single seven-transmembrane receptor can switch recognition as well as function with certain chemotactic peptides to inhibitory with ATL and LX (lipid ligands). Moreover, they suggest that ALXR activation by LX or ATL can protect the host from potentially deleterious PMN responses associated with innate immunity as well as direct effector responses in tissue injury by recognition of peptide fragments.

Figure 1

Structures of inhibitory lipid mediators and stimulatory peptides: LXA₄, ATLa₁, ATLa₂, MHC binding peptide, and MMK-1 surrogate peptide.

Figure 2

ATLa inhibits PMN chemotaxis induced by MHC binding peptide and surrogate MMK-1 peptide. (A) Freshly isolated human PMNs were added to the upper compartment of a microchamber (5 × 10⁴/well). Chemotaxis was initiated by addition of MHC binding peptide (▪), MMK-1 (•), or ATLa₁ (○). (B) PMNs were incubated with vehicle or ATLa₁ (10 nM) for 30 min at 37°C and added to the upper compartment of a microchamber (5 × 10⁴/well). Chemotaxis was initiated by addition of MHC peptide (black bar) or MMK-1 (gray bar) (1 nM) to the lower compartment. Data represent the mean ± SEM from three separate healthy donors.

Figure 3

ATLa inhibits MHC binding peptide– and surrogate MMK-1 peptide–induced extracellular acidification in human PMNs. Human PMNs (∼2 × 10⁵/chamber) were embedded in agar and placed in a microphysiometer. Extracellular acidification rate was normalized to base line at t = 0. PMNs were perfused with 1 μM of MHC binding peptide (black bar) or MMK-1 (gray bar) in the presence or absence of ATLa₂ (1 μM). Data are expressed as percent inhibition of extracellular acidification rate by ATL (percent change of extracellular acidification rate in the absence of ATL is considered as 100%). Data represent n = 3. Inset, Data represent on-line microphysiometer tracings. Challenge of PMNs with ATLa₂ (○), MHC binding peptide (▾), or MMK-1 (•) is indicated by an arrow. Data are expressed as percent change of extracellular acidification rate from n = 3.

Figure 4

Surrogate peptide MMK-1 and ATLa₁ display distinct actions in vivo. 6-d murine dorsal air pouches were raised and injected locally with (A) TNF-α (20 ng) plus MMK-1 or ATLa₁, or TNF-α alone; or (B) MMK-1 alone. After 4 h, pouches were lavaged and leukocytes were enumerated. Data represent the mean ± SEM from n = 3 (*P = 0.04, **P = 0.03).

Figure 5

LXs and peptides specifically compete for ³H-LXA₄ on HEK293 cells. ALXR-transfected HEK293 cells (5 × 10⁶/ml) were incubated with ³H-LXA₄ for 30 min at 4°C in the presence of an increasing concentration of unlabeled LXA₄ (▪) or (A) ATLa₁ (♦), ATLa₂ (•), 15-deoxy-LXA₄ (⋄), (B) MMK-1 (▵), MHC binding peptide (○), or SAA (♦). Bound and unbound radioligands were separated by filtration and specific binding was determined. Data represent the mean ± SEM from duplicates of n = 3.

Figure 5

LXs and peptides specifically compete for ³H-LXA₄ on HEK293 cells. ALXR-transfected HEK293 cells (5 × 10⁶/ml) were incubated with ³H-LXA₄ for 30 min at 4°C in the presence of an increasing concentration of unlabeled LXA₄ (▪) or (A) ATLa₁ (♦), ATLa₂ (•), 15-deoxy-LXA₄ (⋄), (B) MMK-1 (▵), MHC binding peptide (○), or SAA (♦). Bound and unbound radioligands were separated by filtration and specific binding was determined. Data represent the mean ± SEM from duplicates of n = 3.

Figure 6

ATLa₁ as well as surrogate MMK-1 peptide evoke chemotaxis via ALXR. CHO-FPR or CHO-Gqo-ALXR cells were pretreated with vehicle alone (white, hatched, and black bars, respectively) or ATLa₁ (10 nM, gray bar) for 30 min at 37°C and added to the upper compartment of a microchamber (5 × 10⁴/well). Chemotaxis was initiated by addition of FMLP (10 nM, white bar), ATLa₁ (100 nM, hatched bar), or MMK-1 (1 nM, black and gray bars) to the lower compartment. Data were expressed as percent chemotaxis above vehicle control. FMLP-evoked chemotaxis in CHO-FPR is considered as 100%. Data represent the mean ± SEM from n = 3 (*P < 0.01).

Figure 7

Deglycosylation of human ALXR attenuates ligand recognition for peptides but not LXA₄. Human ALXR-transfected HEK293 cells (5 × 10⁵/ml) were pretreated with glycosidase F (1 U/ml) for 24 h at 37°C and then incubated with ³H-LXA₄ for 30 min at 4°C in the presence of an increasing concentration of unlabeled LXA₄ (▪), MMK-1 (▵), or MHC binding peptide (○). Bound and unbound radioligands were separated by filtration and specific binding was determined. Data represent the mean ± SEM from n = 3.

Figure 8

The seventh transmembrane segment and third extracellular loop of human ALXR are essential for LXA₄ and peptide recognition: BLT/ALXR chimeras. (A) Sequences of human ALXR (bold type), BLT (regular type), and B/A₂₅₄ chimera at sixth transmembrane, third extracellular loop, and seventh transmembrane. Inset shows e1–e3, representing the putative extracellular loops; TMI–VII, the transmembrane segments;and i1–i3, intracellular loops for ALXR (bold line) or BLT (regular line). (B) ALXR-, BLT-, or B/A₂₅₄-transfected HEK293 cells (5 × 10⁵/ml) were incubated with ³H-LXA₄ (1 nM, black bar) or ³H-LTB₄ (1 nM, white bar) in the absence or presence of 100 nM of unlabeled homoligand. (C) B/A₂₅₄-transfected HEK293 cells were incubated with ³H-LXA₄ (1 nM) in the presence of an increasing concentration of unlabeled LXA₄ (▪), MMK-1 (▵), or MHC binding peptide (○) for 30 min at 4°C. Bound and unbound radioligands were separated by filtration and specific binding was determined. Data represent the mean ± SEM from n = 3.

Figure 8

The seventh transmembrane segment and third extracellular loop of human ALXR are essential for LXA₄ and peptide recognition: BLT/ALXR chimeras. (A) Sequences of human ALXR (bold type), BLT (regular type), and B/A₂₅₄ chimera at sixth transmembrane, third extracellular loop, and seventh transmembrane. Inset shows e1–e3, representing the putative extracellular loops; TMI–VII, the transmembrane segments;and i1–i3, intracellular loops for ALXR (bold line) or BLT (regular line). (B) ALXR-, BLT-, or B/A₂₅₄-transfected HEK293 cells (5 × 10⁵/ml) were incubated with ³H-LXA₄ (1 nM, black bar) or ³H-LTB₄ (1 nM, white bar) in the absence or presence of 100 nM of unlabeled homoligand. (C) B/A₂₅₄-transfected HEK293 cells were incubated with ³H-LXA₄ (1 nM) in the presence of an increasing concentration of unlabeled LXA₄ (▪), MMK-1 (▵), or MHC binding peptide (○) for 30 min at 4°C. Bound and unbound radioligands were separated by filtration and specific binding was determined. Data represent the mean ± SEM from n = 3.