The role of Annexin A1 in the actions of nedocromil in human and murine mast cells.

A Sinniah1, S Yazid2, M Perretti1, R.J Flower1. 1William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London, UK, 2Division of Molecular Therapy, Institute of Ophthalmology, London, UK

The synthesis and secretion of the protein Annexin (Anx) A1 is regulated by glucocorticoids (GCs) in many cell types. In addition, ‘mast cell stabilising’ cromones such as nedocromil also mobilise Anx-A1 by promoting its phosphorylation by PKC, an essential step for its secretion (1). Extracellular Anx-A1 produces its anti-inflammatory actions through FPR receptor ligation. Since Anx-A1 peptides inhibit histamine release in allergic pleuritis model (2) and cytokine release in murine model of asthma (3), we have here investigated the role of Anx-A1 in the actions of nedocromil in human and murine mast cells.

Cultured (4) cord blood derived mast cells (CDMCs) and bone marrow derived murine mast cells (BMDMCs) from wild type or fpr2/3-/- mice were pre-treated with nedocromil (0.5-10nM) for 5 min prior to 10 min stimulation with compound 48/80 (10μg/ml) to induce degranulation. Mediator release was assayed using ELISA kits. Tryptase release from these cells was determined by Western blots and immunofluorescence techniques. In some experiments, anti-Anx-A1 neutralising antibody (20μg/ml; or isotype matched control) was used. Data are given as mean±SEM and analysis was performed using one-way ANOVA, followed by a Bonferonni post-hoc test.

PKC activation is crucial for Anx-A1 export in CDMCs because the PKC inhibitor (10μM) blocked (43.5±11.9%; p<0.05) nedocromil-induced Anx-A1 phosphorylation. Confocal analysis showed that nedocromil (10nM) prolonged the duration of PKC activation produced by dexamethasone (2nM) and thus promoted release of Anx-A1 from CDMC. Stimulation with compound 48/80 provoked a release of 40.0% of net CDMC histamine release and 1331±237.8 pg ml-1 of PGD2. Pre-treatment with nedocromil significantly (p<0.05) inhibited the release of histamine (10.7±0.8% net histamine release) and PGD2 (657.7±38.2 pg ml-1) but was completely inactive in the presence of anti-Anx-A1 neutralising antibody. Western blot and immunofluorescence analysis indicated that nedocromil was similarly able to inhibit (30%) the release of intracellular tryptase and β-hexosaminidase from stimulated CDMCs, but was again inactive in the presence of anti-Anx-A1 neutralising antibody.

The possibility that FPR2 might be involved in the acute actions of nedocromil was tested using the FPR2 antagonist (10μM) or BMDMCs from fpr2/3-/- mice. Nedocromil failed to block PGD2 release in either model, but its action on histamine release does not seem to depend solely upon FPR2 and may involve another member of the FPR family. Limited testing indicated that Anx-A1 differentially regulates the activation of p38 and JNK treated with nedocromil in CDMCs, suggesting that this cromone is able to yield specific signalling profiles in these cells.

These findings indicate a novel paradigm by which Anx-A1 mediates the pharmacological actions of nedocromil and thus has an important role in preventing mast cell degranulation.

References

(1) Yazid S et al. (2013). PLoS One 8(3): e58963.

(2) Bandero-Melo C et al. 2005. J Pharmacol Exp Ther 313: 1416-1422.

(3) Lee SH et al.2012.Biochem Biophys Res Commun 417: 1024-1029, 2012

(4) Dahl C et al. 2002. J Immunol Methods 262(1-2): 137-143.