On the Mechanism of Action of the Anti-allergic Cromones

Ajantha Sinniah, Samia Yazid, Rod Flower. William Harvey Research Institute, London, UK

Background

Annexin A1 (Anx-A1) is a 37kDa protein secreted by some cells in response to glucocorticoids (GCs) and which mediates several of their acute anti-inflammatory effects. Anti-allergic ‘mast cell stabilising’ cromones such as nedocromil also mobilise Anx-A1 by promoting its phosphorylation by protein kinase C triggering its secretion. This may explain their acute efficacy as anti-allergic/anti-inflammatory agents (Yazid et al., 2009). Some other anti-allergic drugs have also been reported to have mast cell stabilising effects and one purpose of this study was to test for a correlation between this activity and Anx-A1 release. Using mouse bone marrow-derived mast cells (BMDMC) we confirm here that the acute ability of nedocromil to prevent histamine release is dependent upon Anx-A1 mobilisation. We further compared the ability of 9 other anti-allergic drugs to promote Anx-A1 phosphorylation and release in a U937 cells, including 3 histamine H1 antagonists (Group A), 3 mast cell stabilisers (Group B) and 3 drugs reported (Cook et al., 2002) to have both anti-histamine and mast cell stabilising activity (Group C).

Methods

Cultured (Fukuda et al, 2009) BMDMCs from C57/BL6 Anx-A1+/+ or Anx-A1-/- mice were pre-treated with nedocromil (0.5-10nM) for 10 min prior to 15 min stimulation with compound 48/80 (10μg/ml) to induce degranulation. Histamine release (% net release) was assayed using ELISA kits (SPI Bio, Strasbourg, France). In some experiments, a specific anti-Anx-A1 neutralizing antibody was used and an irrelevant IgG1 isotype was used as a negative control. Western blotting was used to assess Anx-A1 phosphorylation (at Ser27) and release from U937 cells treated with anti-allergic drugs (2–500nM) .

Results

Nedocromil produced a concentration-dependent inhibition of histamine release (EC50 ˜ 3nM; n=3) from Anx-A1+/+ cells, but not in the Anx-A1-/- cells (< 15% inhibition at 10 nM; n=3). However, exogenous Anx-A1 (20nM) significantly (p<0.05; ANOVA) inhibited (>75%) the release of histamine (3.43±0.50%; control 14.18±2.055%; n=3).

In immuno-neutralisation experiments using BMDMCs (Anx-A1+/+), nedocromil (10nM) significantly (p<0.001; ANOVA) decreased histamine release (14.31±2.36%; control 33.64±4.30%; n=3). However, it was without effect on histamine release (41.60±3.98%) in cells pre-treated with 10μg/ml Anx-A1 neutralising antibody.

In the U937 cells, densitometry analysis of Ser27 Anx-A1 revealed that Group A drugs were inactive or only weakly (< 1.5 fold; n=3) stimulated phosphorylation at concentrations >100nM (promethazine = pheniramine > antazoline). Group B drugs moderately (<2.5 fold; n=3) stimulated Anx-A1 phosphorylation (nedocromil>cromoglycate = pemirolast) and Group C drugs strongly (>3.5 fold; n=3) strongly stimulated phosphorylation (olopatidine>ketotifen>epinastine).

Conclusion

Anx-A1 release is crucial to the acute anti-allergic of nedocromil. Other anti-allergic drugs with dual H1 antagonist and mast cell stabilising properties also promote Anx-A1 phosphorylation suggesting that they too may act in this manner to prevent histamine release.

Cook E et al. Curr Drug Targets Inflamm Allergy 2002; 1: 167-180

Fukuda K et al. J Allergy Clin Immunol 2009; 124: 827-833

Yazid S et al. Biochem Pharmacol 2009; 77; 1814-1826

We thank the Government of Malaysia and the Wellcome Trust for their support.