Resolvin D1 binds human phagocytes with evidence for proresolving receptors

Abstract

Endogenous mechanisms that act in the resolution of acute inflammation are essential for host defense and the return to homeostasis. Resolvin D1 (RvD1), biosynthesized during resolution, displays potent and stereoselective anti-inflammatory actions, such as limiting neutrophil infiltration and proresolving actions. Here, we demonstrate that RvD1 actions on human polymorphonuclear leukocytes (PMNs) are pertussis toxin sensitive, decrease actin polymerization, and block LTB(4)-regulated adhesion molecules (beta2 integrins). Synthetic [(3)H]-RvD1 was prepared, which revealed specific RvD1 recognition sites on human leukocytes. Screening systems to identify receptors for RvD1 gave two candidates--ALX, a lipoxin A(4) receptor, and GPR32, an orphan--that were confirmed using a beta-arrestin-based ligand receptor system. Nuclear receptors including retinoid X receptor-alpha and peroxisome proliferator-activated receptor-alpha, -delta, -gamma were not activated by either resolvin E1 or RvD1 at bioactive nanomolar concentrations. RvD1 enhanced macrophage phagocytosis of zymosan and apoptotic PMNs, which increased with overexpression of human ALX and GPR32 and decreased with selective knockdown of these G-protein-coupled receptors. Also, ALX and GPR32 surface expression in human monocytes was up-regulated by zymosan and granulocyte-monocyte-colony-stimulating factor. These results indicate that RvD1 specifically interacts with both ALX and GPR32 on phagocytes and suggest that each plays a role in resolving acute inflammation.

Fig. 1.

RvD1 counter-regulates LTB₄ actions with human leukocytes. (A) Actin polymerization in PMNs incubated with RvD1 (10 nM) alone (15 min at 37 °C) or following treatment with PTX or cholera toxin for 2 h. (B) Actin polymerization in PMNs incubated with LTB₄ (10 nM, 10 sec) alone or with RvD1 (10 nM) followed by LTB₄ (10 nM). (*, P < 0.05 compared to LTB₄). (C) CD11b surface expression with PMNs incubated with LTB₄ (10 nM, 15 min) alone or with RvD1 (10 nM) followed by LTB₄ (10 nM) (*, P < 0.05 vs. vehicle alone; #, P < 0.05 vs. LTB₄; MFI, mean fluorescence intensity). (D) RvD1 (10 nM) does not mobilize intracellular Ca²⁺ in PMNs compared to LTB₄ (10 nM). Arrow denotes time intervals (RFU, relative fluorescence intensity). Results in A and D are representative of three healthy subjects. (B and C) Means ± SEMs from three healthy subjects.

Fig. 2.

[³H]-RvD1 specific binding to human leukocytes. (A) Saturation curve and Scatchard plot (inset) obtained with PMNs incubated with indicated concentrations of [³H]-RvD1 in the presence or absence of 3 log orders excess of unlabeled RvD1. (B) Competition binding of [³H]-RvD1 with homoligand RvD1 and heteroligands LXA₄ or Annexin (Ac2-12) peptide. Results are the percentage displacement of [³H]-RvD1-specific binding. Results in A are representative of n = 4 and in B are the mean of triplicate determinations with healthy subjects.

Fig. 3.

Screening of RvD1 GPCR candidates. (Left) Inhibition of NF-κB activity in HeLa cells overexpressing GPCRs (see text for accession numbers) treated with RvD1 (10 nM, 30 min) followed by TNF-α (1 ng) for 6 h. Values were subtracted from those obtained with mock transfected cells (n = 6; *, P < 0.05 compared with BLT2, assigned as negative control). (Inset) The screening system (see text for details). (Right) Radial phylogenetic tree for GPCR protein sequences. GPCRs screened as putative RvD1 receptors are in boldface type, and the cluster generated with ClustalW2 (www.ebi.ac.uk/Tools/clustalw2) is based on deduced amino acid sequences in the NCBI database and constructed with TreeIllustrator software (www.genohm.com). ATL, aspirin-triggered lipoxin; 12-HHT, 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid (33).

Fig. 4.

RvD1 activates GPR32 and ALX receptors. (A) The β-arrestin system used to monitor receptor–ligand interaction (see text for details). (B–D) Dose responses obtained with RvD1, LXA₄, and compound (Compd) 43 with β-arrestin cells stably expressing GPR32. Results are mean ± SEM (n = 4–8). (E) RvE1 comparison with RvD1 with β-arrestin cells stably expressing the ChemR23 receptor; mean ± SEM (n = 4). (F–H) Dose responses obtained with RvD1, LXA₄, and compound 43 on β-arrestin cells stably expressing ALX; mean ± SEM (n = 4–7). (Insets in F and G) Concentration-dependent inhibition of ALX activation by RvD1 (F) and LXA₄ (G) in the presence of escalating concentrations of antagonist t-Boc-Met-Leu-Phe; means from triplicates of two separate experiments. (RLU, relative luminescence unit).

Fig. 5.

RvD1 enhances human MΦ phagocytosis regulated by GPR32 and ALX. (A) RvD1 enhances MΦ phagocytosis of FITC–STZ and apoptotic human PMNs. Increases in phagocytosis were determined by monitoring total fluorescence from ingested FITC–STZ particles or apoptotic PMNs labeled with carboxyfluorescein diacetate-succinimidyl ester; mean ± SEM, *P < 0.05 vs. vehicle, n = 5 healthy subjects. (B and C) Overexpression of GPR32 (B) and ALX (C) in human MΦ increases RvD1-stimulated phagocytosis. Results are the percentage increase in phagocytosis and are representative of three healthy subjects in quadruplicates. (Insets in B and C) Expression of GPR32 (B) and ALX (C) mRNAs from MΦ transfected with cDNAs for the respective GPCRs. (D–F) shRNA knockdown of ALX, GPR32, or both reduces RvD1-enhanced phagocytosis. (D) Surface expression levels of ALX (Upper) and GPR32 (Lower) in single and double knockdown of GPCRs compared to sh scramble-transfected MΦ; representative of three healthy subjects. (E) Representative histograms from three healthy subjects showing ALX (Upper) and GPR32 (Lower) staining in shRNA-transfected MΦ. (F) Percentage RvD1-enhanced phagocytosis; comparison between sh scramble, shALX, shGPR32, and double knockdown MΦ; mean ± SEM of quadruplicate determinations from six healthy subjects. (Inset) Percentage of RvD1-enhanced phagocytosis in sh scramble-transfected MΦ.