The effect of the cromoglycate-like drugs on the disposition and release of the anti-inflammatory protein Annexin A1 by U937 cells.

Samia Yazid1, Egle Solito2, Nicolas Goulding1, Roderick Flower1
1WHRI, Queen Mary University of London, London, United Kingdom, 2Imperial College School of Medicine, London, United Kingdom

The “cromoglycate-like” anti-allergic drugs inhibit the early and the late phase of the asthmatic reaction (Murphy et al., 1987), an activity attributed to their anti-inflammatory properties (Barnes, 1993). These drugs act as “mast cell stabilisers” apparently preventing the release of mast cell mediators such as histamine and eicosanoids (e.g. Cox, 1967), although no clear mechanism of action has been established.

Annexin A1 (ANXA1) is an anti-inflammatory protein induced in cells by glucocorticoids (GCs). These drugs also promote ANXA1 phosphorylation on Ser27P-ANXA1 (Solito et al., 2006) leading to its release from cells. ANXA1 suppresses the release of histamine from mast cells and reduces antigen-induced pleurisy (Bandeira-Melo et al., 2005). ANXA1 null mice exhibit enhanced sensitivity to mast cell de-granulating agent such as compound 48/80 (Damazo et al., 2005). We have explored the ability of the cromoglycate-like drugs to alter the disposition and release of ANXA1 using a U937 model cell system.

Treatment of differentiated (10ng/ml PMA /24h) U937 cells with dexamethasone or hydrocortisone (0.01-10 nM) for 0-30 min activated PKCα/β and produced a dose-related increase in the content of Ser27P-ANXA1 with no change in the total ANXA1 protein level (n=5). Analysis of the sub-cellular distribution of this protein, by western blotting, indicated that the majority of Ser27P-ANXA1 was found in the cytoplasm although this varied between the two GCs. Administration of sodium cromoglycate or nedocromil alone (0.01-10 nM) produced a small increase in Ser27P-ANXA1 (15%, n=3) but this effect was greatly enhanced (40%, n=3) by the presence of low level (2nM) of either GC. Analysis by ELISA of thromboxane(Tx) B2 generation from the cells indicated that dexamethasone (2nM) inhibited TxB2 released by 15%. In the presence of (0.5nM) nedocromil, this inhibition was raised by 28% (p<0.01, ANOVA, n=3). The effects of these drugs on the cellular distribution of ANXA1 was determined by inverted deconvolved microscopy in U937 clones stably transfected (Darmafect reagent 2) with ANXA1-GFP. GCs caused a translocation of ANXA1 to the plasma membrane with nedocromil releasing the protein from this location.

We conclude that cromoglycate-like drugs increase the external release of ANXA1, especially when this is “primed” by low doses of GCs. This explains how these drugs can modulate eicosanoid release from U937 cells and possibly points the way towards an understanding of their mast cell inhibitory activities.

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Support from the Research Advisory Board of St Bart’s and the London is acknowledged.