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. 2016 Mar:32:87-95.
doi: 10.1016/j.intimp.2016.01.003. Epub 2016 Jan 21.

The role of the Annexin-A1/FPR2 system in the regulation of mast cell degranulation provoked by compound 48/80 and in the inhibitory action of nedocromil

Ajantha Sinniah  1 , Samia Yazid  2 , M Perretti  1 , Egle Solito  3 , R J Flower  4
Affiliations

The role of the Annexin-A1/FPR2 system in the regulation of mast cell degranulation provoked by compound 48/80 and in the inhibitory action of nedocromil

Ajantha Sinniah et al. Int Immunopharmacol. 2016 Mar.

Abstract

1.We investigated the role of Annexin (ANX)-A1 and its receptor, ALX/FPR2, in the regulation of mast cell degranulation produced by compound 48/80. 2.Both human cord-blood derived mast cells (CBDMCs) and murine bone marrow derived mast cells (BMDMCs) release phosphorylated ANX-A1 during treatment with glucocorticoids or the mast cell 'stabilising' drugs ketotifen and nedocromil. 3.Compound 48/80 also stimulated ANX-A1 phosphorylation and release and this was also potentiated by nedocromil. Anti-ANX-A1 neutralising monoclonal antibodies (Mabs) enhanced the release of pro-inflammatory mediators in response to compound 48/80. 4.Nedocromil and ketotifen potently inhibited the release of histamine, PGD2, tryptase and β-hexosaminidase from mast cells challenged with compound 48/80. Anti-ANX-A1 neutralising Mabs prevented the inhibitory effect of these drugs. 5.BMDMCs derived from Anx-A1−/− mice were insensitive to the inhibitory effects of nedocromil or ketotifen but cells retained their sensitivity to the inhibitory action of hu-r-ANX-A1. 6.The fpr2/3 antagonist WRW4 blocked the action of nedocromil on PGD2, but not histamine, release. BMDMCs derived from fpr2/3−/− mice were insensitive to the inhibitory effects of nedocromil on PGD2, but not histamine release. 7.Compound 48/80 stimulated both p38 and JNK phosphorylation in CBDMCs and this was inhibited by nedocromil. Inhibition of p38 phosphorylation was ANX-A1 dependent. 8.We conclude that ANX-A1 is an important regulator of mast cell reactivity to compound 48/80 exerting a negative feedback effect through a mechanism that depends at least partly on the FPR receptor.

Keywords: ANX-A1; Compound 48/80; Cromones; Mast cell; PKC; PP2A.

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Figures

Fig. 1
Fig. 1
Nedocromil prolongs the dexamethasone stimulation of PKC phosphorylation and increases the dwell time of PKC at the CBDMC plasma membrane. Panel A: Western blot analysis of PKC phosphorylation at different time points in CBDMCs treated with nedocromil (10 nM; LH block), dexamethasone (2 nM; centre) or both in combination (RH block). Increased PKC phosphorylation is detectable at 5 min and peaks at 10 min when CBDMCs were treated with nedocromil and at 5 min following treatment with dexamethasone. In the presence of both dexamethasone and nedocromil, phosphorylation of PKC is potentiated by 4 fold at 40 min. Blots are a representative of 3 independent experiments. Panel B: The confocal images show a complementary experiment to that in panel A. The three columns represent the information from the two colour channels and the merged channel. Cells were stained for total PKC (red; Alexa Fluor 546; anti-rabbit) and total ANX-A1 1B (green; Alexa Fluor 488; anti-mouse). The micrographs of untreated CBDMCs (left hand block) show similar cytoplasmic distribution of total PKC and ANX-A1 across various time points. There is some localization of ANX-A1, but not PKC, at the membrane. The effect of nedocromil alone on the co-localisation of ANX-A1 and PKC was undetectable and indistinguishable from untreated cells. Cells treated with dexamethasone (middle block) for 5 min shows that there is some co-localisation of PKC and ANX-A1 at membrane (white arrow). However, this had disappeared by 10 min. Cells treated with both dexamethasone and nedocromil for 5 min (right hand block) shows that there is a striking degree of co-localisation at the membrane level between total PKC and total ANX-A1 (white arrows) and this persisted for 20 min, suggesting that nedocromil prolonged the activity of PKC at the membrane level probably by inhibiting the PP2A enzyme. The images were taken at 63 × oil magnification. Scale bars: 10 μm. This composite figure is representative of three independent experiments.
Fig. 2
Fig. 2
Compound 48/80 itself stimulates histamine release, PKC and Anx-A1 phosphorylation in CBDMCs in a concentration-dependent manner and this is potentiated by nedocromil. Panel A. Exposure of CBDMCs to compound 48/80 (0.01–0.1 μg/ml) caused a concentration-dependent release of histamine reaching a maximum (~ 55% total cellular histamine) at a concentration of 10 μg/ml. Data are expressed as mean ± SEM of n = 3 experiment (***p < 0.001). Panel B. Exposure of CBDMCs to compound 48/80 (0.01–0.1 μg/ml) is accompanied by a concentration-dependent increase in Ser-27 phospho ANX-A1 and release of the protein from the cell into the surrounding medium (representative example from 3 blots). Panel C. When assessed by Western blotting techniques, the phosphorylation of ANX-A1 induced by nedocromil (10 nM) or compound 48/80 (0.05 μg/ml) alone decreases after 10 min, however when the two are combined, the effect of compound 48/80 persists for 30 min (representative example from 3 blots). Panel D. A graphical representation of the densitometry analysis of panel C illustrating this effect. The values are expressed as mean ± SEM (n = 3). *p < 0.05 and **p < 0.01 vs untreated samples; ♯♯ p < 0.01 and ♯♯♯ p < 0.001 vs compound 48/80 values; §§§p < 0.001 vs compound 48/80 + nedocromil values).
Fig. 3
Fig. 3
The inhibition by nedocromil and ketotifen of mediator release from CBDMCs stimulated with compound 48/80 is dependent upon Anx-A1. CBDMCs were plated at a density of 2 × 105 cells per well and the stipulated groups were treated with 20 μg/ml ANX-A1 neutralising antibody or an irrelevant isotype control. Subsequently, the cells were pre-treated with nedocromil (10 nM) for 5 min followed by compound 48/80 (10 μg/ml) stimulation for 10 min. To assess the effects of ANX-A1 removal, the cells were incubated with the ANX-A1 neutralising antibody (or an irrelevant isotype control) only. The supernatants were collected from the samples and assessed for histamine (top panel) and PGD2 (lower panel). Nedocromil produced consistent inhibition of histamine and PGD2 release, but this was abrogated in the presence of the immuno-neutralising mAb, but not the control reagent. Data are expressed as mean ± SEM from n = 3 experiment and were analysed using one-way analysis of variance (ANOVA), followed by a Bonferroni post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001 vs unstimulated).
Fig. 4
Fig. 4
The inhibitory action of nedocromil and ketotifen is dependent upon the presence of mast cell Anx-A1. BMDMCs from both Anx-A1+/+ (panels A and B) and Anx-A1−/− (panels C and D) mice were cultured and prepared as described. The cells were incubated with either nedocromil (10 nM) or ketotifen (10 nM) and, in the case of the Anx-A1−/− cells, 10 nM hu-r-Anx-A1. From panels A and B it can be seen that both nedocromil and ketotifen inhibit histamine (43.6 and 30% respectively) as well as PGD2 (61.04% and 58.1% respectively). These drugs do not produce significant inhibitory actions in the Anx-A1−/− cells (panels C and D) although hu-r-Anx-A1 retains its potent activity demonstrating that this is not caused by a failure of the cells to respond. *0.05 < p; **0.01; ***0.001 vs stimulated release; n = 5.
Fig. 5
Fig. 5
The role of FPR receptors in the inhibitory action of nedocromil in CBDMCs and BMDMCs. Nedocromil (10 nM; panel A) inhibited the release of histamine from CBDMCs stimulated with compound 48/80 (10 μg/ml). The FPR2 antagonist WRW4 (10 μM) alone partially inhibited the release of histamine but had little effect on the inhibitory action of nedocromil. In the case of PGD2 however (panel B), WRW4 substantially reversed the inhibitory effect of nedocromil while having no significant effect itself on the release of PGD2 (*0.05 < p; **0.01; ***0.001 vs stimulated release; n = 6). In the case of BMDMCs, nedocromil was found to exert a concentration-dependent inhibition of histamine release in both WT and fpr2/3 null mice (panel C) whereas nedocromil was without effect on the release of PGD2 from cells derived from the fpr2/3 null mice whilst producing the expected inhibition in WT cells (panel D). Data are expressed as mean ± SEM from n = 3 experiment and were analysed using one-way analysis of variance (ANOVA), followed by a Bonferroni post-hoc test, *p < 0.05, **/++ p < 0.01, ***p < 0.001 vs control.
Fig. 6
Fig. 6
Downstream signalling in CBDMCs is differentially altered by nedocromil. Compound 48/80 (10 μg/ml) stimulation of CBDMCs increased p-38 and JNK phosphorylation, and this was inhibited by nedocromil (10 nM). The presence of ANX-A1 neutralising antibody reversed the phospho-p38 inhibitory actions of nedocromil but did not alter its inhibitory actions on JNK phosphorylation. Data are expressed as mean ± SEM (n = 3 independent experiments; ***p < 0.001 vs unstimulated).
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