Lack of activity of 15-epi-lipoxin A₄ on FPR2/ALX and CysLT1 receptors in interleukin-8-driven human neutrophil function

Abstract

Neutrophil recruitment and survival are important control points in the development and resolution of inflammatory processes. 15-epi-lipoxin (LX)A interaction with formyl peptide receptor 2 (FPR2)/ALX receptor is suggested to enhance anti-inflammatory neutrophil functions and mediate resolution of airway inflammation. However, it has been reported that 15-epi-LXA₄ analogues can also bind to cysteinyl leukotriene receptor 1 (CysLT1) and that the CysLT1 antagonist MK-571 binds to FPR2/ALX, so cross-reactivity between FPR2/ALX and CysLT1 ligands cannot be discarded. It is not well established whether the resolution properties reported for 15-epi-LXA4 are mediated through FPR2/ALX, or if other receptors such as CysLT1 may also be involved. Evaluation of specific FPR2/ALX ligands and CysLT1 antagonists in functional biochemical and cellular assays were performed to establish a role for both receptors in 15-epi-LXA₄-mediated signalling and function. In our study, a FPR2/ALX synthetic peptide (WKYMVm) and a small molecule FPR2/ALX agonist (compound 43) induced FPR2/ALX-mediated signalling, enhancing guanosine triphosphate-gamma (GTPγ) binding and decreasing cyclic adenosine monophosphate (cAMP) levels, whereas 15-epi-LXA₄ was inactive. Furthermore, 15-epi-LXA4 showed neither binding affinity nor signalling towards CysLT1. In neutrophils, 15-epi-LXA₄ showed a moderate reduction of interleukin (IL)-8-mediated neutrophil chemotaxis but no effect on neutrophil survival was observed. In addition, CysLT1 antagonists were inactive in FPR2/ALX signalling or neutrophil assays. In conclusion, 15-epi-LXA₄ is not a functional agonist or an antagonist of FPR2/ALX or CysLT1, shows no effect on IL-8-induced neutrophil survival and produces only moderate inhibition in IL-8-mediated neutrophil migration. Our data do not support an anti-inflammatory role of 15-epi-LXA₄- FPR2/ALX interaction in IL-8-induced neutrophil inflammation.

Keywords: 15-epi-LXA4; FPR2/ALX; apoptosis; interleukin-8; neutrophils.

Fig. 1

Chemical structure of reference compounds. The chemical structures of the FPR2/ALX natural ligand 15-epi-lipoxin A₄ (LXA₄), the FPR2/ALX agonist compound 43, two cysteinyl leukotriene receptor 1 (CysLT1) antagonists montelukast (MK-476) and MK-571, and the synthetic peptide WKYMVm are detailed.

Fig. 2

FPR2/ALX agonists and CysLT1 antagonist behaviour in cyclic adenosine monophosphate (cAMP) and CysLT1 Ca²⁺ flux signalling assays in Chinese hamster ovary (CHO) over-expressing human FPR2/ALX and CysLT1 receptors, respectively. (a) Effect of the reference compounds on cAMP in human FPR2/ALX recombinant cells. The agonist behaviour was measured as the percentage of inhibition after preincubation of the compounds (100 μM–0·1 pM) with human FPR2/ALX recombinant cells for 1 h prior to adding 10 μM forskolin. Fluorescence signal was detected after 1 h incubation with time-resolved fluorescence resonance energy transfer (TR-FRET) dynamic-2 cAMP kit (further information detailed in Material and methods). Compound 43 and the proinflammatory peptide (WKYMVm) inhibited cAMP in recombinant cells, whereas 15-epi-lipoxin (LX)A₄, montelukast (MK-476) or MK-571 did not exert any effect. (b) Effect of the reference compounds on calcium release in human CysLT1 recombinant cells. The antagonist behaviour was measured as the percentage of inhibition after compound preincubation (0·01 pM–100 μM) and agonist addition [leukotriene D4 (LTD₄), 1 nM]. Cytoplasmic calcium concentration was determined by fluorescence signal in the Flexstation (further information detailed in Material and methods). In the CysLT1 calcium flux assay only montelukast and MK-571 inhibited the signal potently.

Fig. 3

Effect of 15-epi-lipoxin (LX)A₄ and compound 43 on interleukin (IL)-8-induced neutrophil chemotaxis. (a) The percentage of inhibition of neutrophil chemotaxis was calculated after preincubation of the compounds (0·1 nM–1 μM) with human peripheral blood isolated neutrophils for 30 min. Briefly, chemotaxis was run for additional 30 min against an IL-8 (1·25 nM) gradient, as described in Material and methods. IL-8-induced neutrophil chemotaxis was dampened by 15-epi-LXA₄ (maximum inhibition 40% at 10 nM) and compound 43 (IC₅₀ = 67 nM). SCH527123, a CXCR2 antagonist, was used as a positive control of inhibition of neutrophil migration (IC₅₀ = 9·3 nM). *P < 0·05; ***P < 0·001. (b) Direct chemotactic effect of the reference compounds on neutrophils was measured by incubating 15-epi-LXA₄, montelukast, MK-571, SCH527123 (0·1 μM) and compound 43 (0·01, 0·1 and 1 μM) alone in the lower chemotactic chamber compartment without IL-8. Neutrophils were added on top of the filter that separated the upper compartment containing cells, but no agonist, from the lower compartment containing the reference compounds. IL-8 (1·25 nM) was used as a positive control of neutrophil migration and controls of inhibition of IL-8-induced chemotaxis were assessed by incubating compound 43 (0·1 μM) and SCH527123 (0·1 μM) with neutrophils prior to placing them in the upper compartment. ***P < 0·001 IL-8 versus vehicle; *P < 0·05 compound 43 (1 μM) versus vehicle; #P < 0·05 compound 43 and SCH527123 versus IL-8.

Fig. 4

Effect of 15-epi-lipoxin (LX)A₄, compound 43, montelukast and MK-571 on interleukin (IL)-8-induced neutrophil apoptosis arrest by measuring caspase 3/7 activity. Human neutrophil survival was induced by incubation with IL-8 (100 nM), and the apoptosis produced by the reference compounds (0·1 nM–1 μM) was measured by the percentage of induction of caspase 3/7 activity after 4 h of IL-8 addition. Caspase inhibitor I (Caspase inh.) at 5 μM was used as a control of apoptosis inhibition. None of the reference compounds reversed IL-8-induced neutrophil apoptosis arrest at the doses tested.

Fig. 5

Measurement of interleukin (IL)-8-induced neutrophil survival by annexin V staining: effect of 15-epi-lipoxin (LX)A₄. Cell apoptosis was measured in unstimulated human neutrophils (a) stimulated with IL-8 (100 nM) for 4 h (b) or preincubated with 15-epi-LXA₄ (100 nM) for 30 min before addition of IL-8 (100 nM) for 4 h (c). Apoptosis induction was measured by annexin V staining. Propidium iodide staining was included for necrotic cell detection. Total neutrophils were gathered (left panel) and the number of viable neutrophils (double-negative cells), apoptotic neutrophils (annexin V-positive cells) and necrotic neutrophils (IP-positive cells) was detected using the proper probes by flow cytometry. Percentage of viable, apoptotic and necrotic cells are shown in the insert table. The percentage of apoptotic neutrophils after IL-8 and IL-8 in addition to 15-epi-LXA4 treatment compared to vehicle is represented in the bottom right graph. 15-epi-LXA₄ did not increase annexin V-positive cells and did not reverse IL-8-induced neutrophil apoptosis arrest. *P < 0·05 IL-8 versus Vehicle.

Fig. 6

Effect of 15-epi-lipoxin (LX)A₄, compound 43, montelukast, MK-571 and SCH527123 alone or in the presence of interleukin (IL)-8 on neutrophil apoptosis by annexin V staining assessment. 15-epi-LXA₄, montelukast and MK-571 did not produce any effect by themselves on neutrophil survival, whereas compound 43 alone arrested the neutrophils to enter apoptosis. Similar to the caspase 3/7 activity results, none of the compounds reversed IL-8 induced neutrophil apoptosis arrest, except for the CXCR2 antagonist SCH527123 that restores the normal levels of apoptosis. *P < 0·05 compound 43 versus Veh; **P < 0·001 IL8 versus Veh; ^#P < 0·001 SCH527123 versus IL-8 (Student's t-test).