Resolvin D1 limits polymorphonuclear leukocyte recruitment to inflammatory loci: receptor-dependent actions

Abstract

Objective: Resolvin D1 (RvD1) limits neutrophil recruitment during acute inflammation and is derived from omega-3 docosahexaenoic acid to promote catabasis. The contribution of its specific receptors, the lipoxin A(4)/Annexin-A1 receptor formyl-peptide receptor 2 (FPR2/ALX) and the orphan receptor G-protein-coupled receptor 32 (GPR32) are of considerable interest.

Methods and results: RvD1 reduced human polymorphonuclear leukocytes recruitment to endothelial cells under shear conditions as quantified using a flow chamber system. Receptor-specific antibodies blocked these anti-inflammatory actions of RvD1, with low (1 nmol/L) concentrations sensitive to GPR32 blockade, while the higher (10 nmol/L) concentration appeared FPR2/ALX-specific. Interestingly, polymorphonuclear leukocytes surface expression of FPR2/ALX but not GPR32 increased following activation with pro-inflammatory stimuli, corresponding with secretory vesicle mobilization. Lipid mediator metabololipidomics carried out with 24-hour exudates revealed that RvD1 in vivo gave a significant reduction in the levels of a number of pro-inflammatory mediators including prostaglandins and leukotriene B(4). These actions of RvD1 were abolished in fpr2 null mice.

Conclusions: Pro-resolving lipid mediators and their receptors, such as RvD1 and the 2 G-protein-coupled receptors, studied here regulate resolution and may provide new therapeutic strategies for diseases with a vascular inflammatory component.

Figure 1. RvD1 potently reduces neutrophil-endothelial interactions under flow

Human PMN were incubated with vehicle (0.1% EtOH) or RvD1 (0.01 to 100 nM) for 10 min at 37°C. Cells were then flowed over TNF-stimulated endothelial monolayers at 1 dyne/cm² for 8 min, and the extent of cell capture (A), rolling (B) and adhesion (C) were quantified from 6 frames per treatment using Image Pro-plus software analysis. Results are mean ± SEM, n=7 donors, *P<0.05 vs. Vehicle (0.1% EtOH), one-way ANOVA followed by Dunnett’s post-hoc test.

Figure 2. RvD1 reduces PMN recruitment: GPCR dependent actions

Human PMN were pre-incubated with anti-FPR2/ALX or anti-GPR32 (10 μg/ml, 10 min, 37°C) prior to incubation for 10 min at 37°C with vehicle or RvD1 at 1 nM (A) or 10 nM (B) final concentration. Then, PMN were flowed over TNF-stimulated endothelial monolayers at 1 dyne/cm² for 8 min, and neutrophil capture, rolling and adhesion were quantified. Results are mean ± SEM, n=3-5 donors, *P<0.05 vs. Vehicle (0.1% EtOH), #P<0.05 vs. RvD1 treatment, one-way ANOVA followed by Bonferroni’s post-hoc test.

Figure 3. Neutrophil activation mobilizes FPR2/ALX, but not GPR32, to the cell surface

Freshly isolated human PMN were stimulated for 15 min with various pro-inflammatory or chemoattractive substances and FPR2/ALX (A) and GPR32 (B) cell surface expression was assessed by flow cytometry. Neutrophil activation was monitored with antibodies recognizing adhesion molecules CD11b (C) and CD62L (D). PMN granule mobilization was assessed using specific surface markers for secretory vesicles; CD35 (E), specific granules; CD66b (F) and azurophilic granules; CD63 (G). Results are mean ± SEM, n=6 donors, *P<0.05 vs. Vehicle (0.1% EtOH), one-way ANOVA followed by Dunnett’s post-hoc test.

Figure 4. FPR2/ALX mediates the RvD1 anti-inflammatory actions in vivo

Total leukocyte and neutrophil infiltration to the peritoneum in wild-type (WT) and Fpr2 null mice was assessed 4 h (A, B) and 24 h (C, D) after injection of zymosan (0.2 mg, i.p.). Mice were treated with vehicle (0.1% EtOH) or RvD1 (1 or 10 ng, i.v.) immediately prior to zymosan administration. Results are mean ± SEM, n=3-6 mice per group. *P<0.05 vs. Vehicle (0.1% EtOH), one-way ANOVA followed by Dunnett’s post-hoc test.

Figure 5. RvD1 regulates exudate lipid mediators via fpr2

Mice were treated with vehicle (0.1% EtOH) or RvD1 (10 ng, i.v.) immediately prior to zymosan administration and exudates collected after 24 h. Lipid mediators in peritoneal in cell free exudates were assessed using LC-MS/MS techniques following solid phase extraction. (A) Representative schedule reaction monitoring chromatograms for selected ion pairs for arachidonic acid derived lipoxins and eicosanoids, Compound I = 12-epi-6-trans-LTB₄ and compound II = 6-trans-LTB₄ (B) Representative MS/MS spectra detailing diagnostic ions employed for the identification of LXB₄ and 5,15-diHETE based on published criteria . Quantification of peritoneal lipid mediators (C-I). Results are mean ± SEM, 4 mice per group. *P<0.05 vs. WT + Vehicle (0.1% EtOH), one-way ANOVA followed by Dunnett’s post-hoc test.

Figure 6. Pro-resolving actions of RvD1 are FPR2/ALX dependent

Bio-gel elicited macrophages were harvested from WT or Fpr2 null mice and pretreated with vehicle or RvD1 (0.01-100 nM, 30 min) prior to addition of fluorescent zymosan (1:20 ratio, 20 min). Zymosan phagocytosis was evaluated following vigorous washing, using a fluorescent plate reader (NOVOstar). Results are mean ± SEM, n=6 experiments, **P<0.01 vs. Veh (0.1% EtOH), one-way ANOVA followed by Dunnett’s post-hoc test.