Endogenous lipid- and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin A4 receptor

Abstract

Aspirin (ASA) and dexamethasone (DEX) are widely used anti-inflammatory agents yet their mechanism(s) for blocking polymorphonuclear neutrophil (PMN) accumulation at sites of inflammation remains unclear. Here, we report that inhibition of PMN infiltration by ASA and DEX is a property shared by aspirin-triggered lipoxins (ATL) and the glucocorticoid-induced annexin 1 (ANXA1)-derived peptides that are both generated in vivo and act at the lipoxin A(4) receptor (ALXR/FPRL1) to halt PMN diapedesis. These structurally diverse ligands specifically interact directly with recombinant human ALXR demonstrated by specific radioligand binding and function as well as immunoprecipitation of PMN receptors. In addition, the combination of both ATL and ANXA1-derived peptides limited PMN infiltration and reduced production of inflammatory mediators (that is, prostaglandins and chemokines) in vivo. Together, these results indicate functional redundancies in endogenous lipid and peptide anti-inflammatory circuits that are spatially and temporally separate, where both ATL and specific ANXA1-derived peptides act in concert at ALXR to downregulate PMN recruitment to inflammatory loci.

Fig. 1

Additive actions with DEX and ASA on PMN extravasation. DEX or ASA were injected into murine air pouches 15 min before Zymosan A (1 mg) injection. PMN accumulation was measured 4 h later by light microscopy. Results are the mean ± s.e.m of n = 8 per group. *, P < 0.05; **, P < 0.01 versus Zymosan A alone. □, Zymosan A alone; , +DEX; , +ASA; ■, +DEX & ASA.

Fig. 2

Direct interaction of ANXA1 with ALXR: competitive [¹²⁵I-Tyr]Ac2-26 and [³H]LXA₄ binding. a and b, Human ALXR-transfected HEK293 cells (0.5 × 10⁶) were incubated with [³H]LXA₄ (a) or [¹²⁵I-Tyr]Ac2-26 (b) in the presence of an increasing concentration of unlabeled compounds: LXA₄ (◆), Ac2-26 (■), Ac2-12 (△), ANXA1 (○) or scrambled Ac2-6 (□). c and d, Suspension (c) or adherent human PMNs (d) were incubated with [¹²⁵I-Tyr]Ac2-26 in the presence of increasing concentration of unlabeled compounds: LXA₄ (◆), Ac2-26 (■) or SAA (□). Insets in b and c, Scatchard plots of specific [¹²⁵I-Tyr]Ac2-26 binding. Results represent the mean ± s.e.m. from duplicates of n =3.

Fig. 3

ANXA1 peptides directly interact with recombinant as well as endogenous PMN ALXR. a, CHO-Gqo-ALXR cells were added to the upper compartment of a microchamber (5 × 10⁴ cells per well). Chemotaxis was initiated by addition of Ac2-26 peptide (100 μM; ) or aspirin-triggered lipoxin A₄ analog 1 (ATLa1; 100 nM; ■) to the lower compartment. □, vehicle. b, CHO-Gqo-ALXR cells were pretreated with Ac2-26 peptide for 30 min at 37 °C and added to the upper compartment of a microchamber (5 × 10⁴ cells per well). Chemotaxis was initiated by addition of ATLa1 (100 nM) to the lower compartment. c, Human PMNs were added to the upper compartment of a microchamber (5 × 10⁴ cells per well). Chemotaxis was initiated by addition of Ac2-26 peptide (1–10 μM; ■) to the lower compartment. In some cases, cells were treated with 10 () or 100 nM (□) ATLa1 for 30 min at 37 °C. #, P = 0.01, versus vehicle; *, P < 0.01, versus Ac2-26 alone (a and c) or % inhibition of ATLa1- evoked chemotaxis (*, P = 0.02; **, P < 0.01, versus ATLa1 alone in b). Data represent the mean ± s.e.m. from n = 3 experiments. d, Fura2-AM-loaded human PMNs (5 × 10⁶ cells/incubation) were incubated with the different stimuli, and rapid changes in intracellular [Ca²⁺] measured by fluorimetry. Additions of agonists are denoted by arrows. hrANXA1: human recombinant annexin 1. Traces are representative of 3 independent experiments.

Fig. 4

Both ATL and ANXA1 are generated in vivo and interact with ALXR. a, Adherent human PMNs (5 × 10⁶) incubated (in 6-well plates) in presence of 0.1 μM PMA or 0.1 μM fMLP for 30 min at 37 °C. b, Murine blood-borne PMNs (BL) or air pouch (AP) cells (>95% PMNs) collected 4 h after injection of 1 mg Zymosan A were immunoprecipitated (IP) with either the monoclonal antibody against ANXA1 (top) or polyclonal antibody against ALXR peptide (bottom). Immunoprecipitated proteins were probed with anti-ALXR (top) or anti-ANXA1 (bottom) by western blotting (WB). Immunoblots represent 3 independent experiments. DEX or ASA were injected into murine air pouches with the indicated doses 15 min before Zymosan A (1 mg) injection. c–e, After 4 or 16 h, PMN accumulation (c), 15-epi-LXA₄ (d) and ANXA1 generation (e) were determined. Insets, Representative western blot exudates at 4 and 16 h. Results are the mean ± s.e.m of n = 8 per group. *, P < 0.05 versus Zymosan A alone. For c–e: □, Zymosan alone; , + 3 μg DEX; , + 300 μg ASA; ■, + 3 μg DEX + 300 μg ASA.

Fig. 5

Synergism with ANXA1-derived peptides and ATLa in vivo. a, ANXA1-derived peptides were administered at the reported doses via the tail vein 15 min prior to injection of 1 mg Zymosan A into 6-d-old air pouches. PMN accumulation was measured 4 h later. The number of migrated PMNs in the control group (vehicle-treated mice) was 7.5 ± 0.5 × 10⁶ PMNs per mouse. Data are mean ± s.e.m of 6–12 mice; *, P < 0.05 versus control migration as calculated on original values. □, Ac2-26; ●, Ac2-12; ■, Ac2-6; ○, Ac2-6 scramble. b, Animals were treated i.v. with the reported doses of ATLa (■; 0.5–5 μg, 1.2–12 nmol; chemical structure shown, R = para-fluoro-phenoxy), Ac2-26 (□; 5–100 μg, corresponding to ~1.5–33 nmol) or both ATLa and Ac2-26 () 15 min before local injection of Zymosan A. PMN accumulation was measured after 4 h. The number of migrated PMNs in the control group (mice treated with Zymosan A alone) was 6.5 ± 0.55 × 10⁶ PMNs per mouse. Data are mean ± s.e.m of n = 6–8 mice per group; *, P < 0.05 versus control migration as calculated on original values. c, Air-pouch biopsies. Sections from the top dose groups (that is, 5 μg of ATLa and 100 μg of Ac2-26; see b) were prepared and were stained with H&E. Magnifications, ×10 for the top panel and ×40 for the inset and lower panels.