PMC

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.

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.

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.

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.

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.