Objective-To determine whether the inhibitory action of the antiallergic cromone "mast cell stabilizing" drugs on polymorphonuclear leukocyte (PMN) trafficking is mediated through an annexin-A1 (Anx-A1) dependent mechanism. Methods and Results-Intravital microscopy was used to monitor the actions of cromones in the inflamed microcirculation. Reperfusion injury provoked a dramatic increase in adherent and emigrated leukocytes in the mesenteric vascular bed, associated with augmented tissue levels of myeloperoxidase. Nedocromil, 2 to 20 mg/kg, significantly (PϽ0.05) inhibited cell adhesion and emigration, as well as myeloperoxidase release, in wild-type but not Anx-A1 Ϫ/Ϫ mice. Short pretreatment of human PMNs with nedocromil, 10 nmol/L, inhibited cell adhesion (PϽ0.05) in the flow chamber assay, and this effect was reversed by specific anti-AnxA1 or a combination of antiformyl peptide receptors 1 and 2, but not irrelevant control, antibodies. Western blotting experiments revealed that cromones stimulate protein kinase C-dependent phosphorylation and release Anx-A1 in human PMNs. Conclusion-We propose a novel mechanism to explain the antiinflammatory actions of cromones on PMN trafficking, an effect that has long puzzled investigators. Key Words: nedocromil cromoglycate PKC inflammation Ⅲ FPR receptors Ⅲ reperfusion injury Ⅲ vascular biology C romones are a group of antiallergic drugs; sodium cromoglycate and sodium nedocromil are exemplars. Early studies on the mechanism of action of these mast cell stabilizers suggested that they blocked mast cell degranulation and, thus, mediator release, when this was triggered by various stimuli, including IgE. 1 However, these drugs can affect other facets of the inflammatory process, including inhibition of eicosanoid 2 and cytokine release. 3 Leukocyte emigration 4 is also inhibited by these drugs eg, cromoglycate and ketotifen exhibit a protective role in neutrophil-dependent pathological features, including the intestinal 5 and pulmonary 6 ischemia-reperfusion (IR) model. Mast cells, located in close apposition to the vessels, are an important source of proinflammatory mediators released at the site of inflammation. 7 Mast-cell derived mediators activate adhesion molecules from the local environment, leading to leukocyte recruitment, 7 using a well-characterized multistep mechanism. 8 Clearly, inhibition of the release of mediators from these mast cells could explain the effect of the cromones on polymorphonuclear leukocyte (PMN) trafficking. However, other research 9 points to a separate and direct effect of these compounds on PMNs.Annexin-A1 (Anx-A1) is a 37-kDa glucocorticoid (GC)-regulated protein that mimics the effects of proteins in several in vivo and in vitro model systems. 10 -12 GCs induce the Anx-A1 gene in many cells and also increase secretion of the protein from existing intracellular pools by stimulating protein kinase C (PKC) activity. 13 Once secreted, the protein acts in a paracrine/autocrine fashion, using formyl peptide receptors (FPRs) 14,15 to pro...

Background and PurposeAlthough the ‘cromones’ (di-sodium cromoglycate and sodium nedocromil) are used to treat allergy and asthma, their ‘mast cell stabilising’ mechanism of pharmacological action has never been convincingly explained. Here, we investigate the hypothesis that these drugs act by stimulating the release of the anti-inflammatory protein Annexin-A1 (Anx-A1) from mast cells.Experimental approachWe used biochemical and immuno-neutralisation techniques to investigate the mechanism by which cromones suppress histamine and eicosanoid release from cord-derived human mast cells (CDMCs) or murine bone marrow-derived mast cells (BMDMCs) from wild type and Anx-A1 null mice.Key resultsCDMCs activated by IgE-FcRε1 crosslinking, released histamine and prostaglandin (PG) D2, which were inhibited (30–65%) by 5 min pre-treatment with cromoglycate (10 nM) or nedocromil (10 nM), as well as dexamethasone (2 nM) and human recombinant Anx-A1 (1–10 nM). In CDMCs cromones potentiated (2–5 fold) protein kinase C (PKC) phosphorylation and Anx-A1 phosphorylation and secretion (3–5 fold). Incubation of CDMCs with a neutralising anti-Anx-A1 monoclonal antibody reversed the cromone inhibitory effect.Nedocromil (10 nM) also inhibited (40–60%) the release of mediators from murine bone marrow derived-mast cells from wild type mice activated by compound 48/80 and IgE-FcRε1 cross-linking, but were inactive in such cells when these were prepared from Anx-A1 null mice or when the neutralising anti-Anx-A1 antibody was present.Conclusions and ImplicationsWe conclude that stimulation of phosphorylation and secretion of Anx-A1 is an important component of inhibitory cromone actions on mast cells, which could explain their acute pharmacological actions in allergy. These findings also highlight a new pathway for reducing mediator release from these cells.

Graphical abstract

Background and purpose: Annexin-A1 (ANXA1), a glucocorticoid-regulated protein, mediates several of the antiinflammatory actions of the glucocorticoids. Previous studies demonstrated that ANXA1 is involved in pain modulation. The current study, using ANXA1 knockout mice (ANXA1 À/À ), is aimed at addressing the site and mechanism of the modulatory action of ANXA1 as well as possible involvement of ANXA1 in mediating the analgesic action of glucocorticoids. Experimental approach: The acetic acid-induced writhing response was performed in ANXA1À/À and wild-type (ANXA1 þ / þ ) mice with spinal and brain levels of prostaglandin E 2 (PGE 2 ) examined in both genotypes. The effect of the ANXA1 peptomimetic Ac2-26 as well as methylprednisolone on the writhing response and on spinal cord PGE 2 of ANXA1 þ / þ and ANXA1 À/À was compared. The expression of proteins involved in PGE 2 synthesis, cytosolic phospholipase A 2 (cPLA 2 ) and cyclooxygenases (COXs), in the spinal cord of ANXA1 þ / þ and ANXA1 À/À was also compared. Key results: ANXA1 À/À mice exhibited a significantly greater writhing response and increased spinal cord levels of PGE 2 compared with ANXA1 þ / þ mice. Ac2-26 produced analgesia and reduced spinal PGE 2 levels in ANXA1 þ / þ and ANXA1 À/À mice, whereas methylprednisolone reduced the writhing response and spinal PGE 2 levels in ANXA1mice. The expression of cPLA 2 , COX-1, COX-2 and COX-3 in spinal cord tissues was upregulated in ANXA1 À/À compared with ANXA1 þ / þ . Conclusions and implications: We conclude that ANXA1 protein modulates nociceptive processing at the spinal level, by reducing synthesis of PGE 2 by modulating cPLA 2 and/or COX activity. The analgesic activity of methylprednisolone is mediated by spinal ANXA1.