[Show abstract][Hide abstract] 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.
Full-text · Article · Mar 2016 · International immunopharmacology
[Show abstract][Hide abstract] ABSTRACT: Endogenous glucocorticoids are pro-resolving mediators, an example of which is the endogenous glucocorticoid-regulated protein Annexin A1 (AnxA1). Since silicosis is an occupational lung disease characterized by unabated inflammation and fibrosis, in this study we tested the therapeutic properties of the N-terminal AnxA1-derived peptide Ac2-26 on experimental silicosis.
Swiss-Webster mice received silica particles intranasally and were subsequently treated with intranasal peptide Ac2-26 (200 μg.mouse(-1) ) or dexamethasone (25 μg.mouse(-1) ) for 7 days, starting 6 h post-challenge. Peptide Ac2-26 abolished leukocyte infiltration, collagen deposition, granuloma formation and the generation of pro-inflammatory cytokines following silica provocation, readouts only partially inhibited by dexamethasone.
A clear exacerbation of these pathological changes was observed in AnxA1 knockout mice as compared to the wild-type littermate controls. Lung fibroblasts from WT mice, but not from formyl peptide receptor (Fpr) type 1 knockout, had the IL-13 or TGFβ-induced production of CCL2/MCP-1 and collagen reduced after incubation with peptide Ac2-26 in vitro. This compound also inhibited the production of CCL2/MCP-1 from fibroblasts of Fpr2 knockout mice.
Collectively, our findings unveil novel protective properties of the AnxA1 derivative peptide Ac2-26 on the inflammatory and fibrotic responses promoted by silica, and suggest that AnxA1 mimetic agents might be a promising strategy in innovative anti-fibrotic approaches for treatment of silicosis.
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Full-text · Article · Feb 2015 · British Journal of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Ever since Stone Age men discovered that knapping flint produced sharp stone edges that could be used in combat as well as for cooking and hunting, technological advances of all kinds have been adapted and adopted by the military. The opportunities provided by modern neuroscience are proving no exception, but their application in a military context is accompanied by complex practical and ethical considerations.
No preview · Article · Dec 2014 · Nature reviews Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Ever since Stone Age men discovered that knapping flint produced sharp stone edges that could be used in combat as well as for cooking and hunting, technological advances of all kinds have been adapted and adopted by the military.The opportunities provided by modern neuroscience are proving no exception, but their application in a military context is accompanied by complex practical and ethical considerations.
No preview · Article · Nov 2014 · Nature reviews Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Although 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.
We 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.
CDMCs 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.
We 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.
[Show abstract][Hide abstract] ABSTRACT: The action of anti-inflammatory and anti-allergic drugs on the eicosanoid system is briefly reviewed. In addition to the aspirin-like drugs, which directly inhibit the cyclo-oxygenase enzymes, other drugs such as the glucocorticoids and the cromones also inhibit the formation of eicosanoids. In the latter cases this is bought about through the release of a protein factor that acts through formyl peptide receptors on the target cell surface. Of growing interest, is the observation that this receptor is also a target for other eicosanoids, such as lipoxins and resolvins that modulate host defence systems.
No preview · Article · Nov 2011 · Prostaglandins & other lipid mediators
[Show abstract][Hide abstract] ABSTRACT: Glucocorticoids (GCs) exert diverse actions in the body, which are required for the maintenance of homeostasis. Their complex effects are mediated mainly by mechanisms involving the induction or repression of key target genes. Lipocortin 1 is the product of one such gene; it fulfills a prominent role in effecting the regulatory actions of the steroids in the host defense and neuroendocrine systems.
[Show abstract][Hide abstract] ABSTRACT: Autoimmune diseases, like multiple sclerosis, are triggered by uncontrolled activation of cells of the immune system against self-antigen present, for instance, in the central nervous system. We have reported novel biological functions for Annexin A1, an effector of endogenous anti-inflammation, to produce positive actions on the adaptive immune system by reducing the threshold of T cell activation. In this study, we investigated the potential modulatory role of Annexin A1 in the development of experimental autoimmune encephalomyelitis, a model of multiple sclerosis.
Male control C57/BL6 and AnxA1 null mice were immunized subcutaneously with an emulsion consisting of 300 microg of MOG35-55 in PBS combined with an equal volume of CFA. Lymph node cells obtained from mice immunized with MOG33-55 for 14 days were re-stimulated in vitro with MOG33-55 (100 microg/ml) for 4 days and the Th1/Th17 cytokine profile measured by ELISA. Spinal cords were processed either to isolate the infiltrated T cells or fixed and stained with haematoxylin and eosin. Statistical analyses were performed using two-tailed, unpaired Student's t tests or ANOVA.
Our results show a direct correlation between Annexin A1 expression and severity of EAE. Analysis of MOG35-55-induced EAE development in Annexin A1 null mice showed decreased signs of the disease compared to wild type mice. This defect was significant at the peak of the disease and accompanied by reduced infiltration of T cells in the spinal cord. Finally, analysis of the T cell recall response in vitro following stimulation with MOG35-55 showed a decrease proliferation of Annexin A1 null T cells, with a significantly reduced Th1/Th17 phenotype, compared to wild type cells.
Together these findings suggest that Annexin A1 null mice have an impaired capacity to develop EAE. Furthermore strategies aiming at reducing Annexin A1 functions or expression in T cells might represent a novel therapeutic approach for multiple sclerosis.
Full-text · Article · Nov 2009 · Journal of Neuroinflammation
[Show abstract][Hide abstract] ABSTRACT: Objective and design: A dual-purpose targeting/reporter vector, incorporating GFP, was utilised to monitor promoter activity in transgenic alx/fpr-rs2 null mice. Materials and Methods:
alx/fpr-rs2 null mice were generated by homologous recombination in embryonic stem (ES) cells. A GFP construct was fused inframe within the promoter region allowing representative promoter activity to be monitored by flow cytometry. TNF-α secretion was measured by ELISA. Results: Germline transmission of the alx/fpr-rs2 was confirmed by southern blotting. PCR primer pairs were designed to recognise GFP allowing genotyping. Inducible GFP expression was observed during bone marrow derived macrophage (BMM) differentiation (P<0.01) and following subsequent treatment with LPS (P<0.05), TNF-a secretion mirrored GFP induction. Furthermore GFP positive cell populations were observed in vivo within nai’ve and inflammatory environments. Conclusions: A dual targeting/reporter strategy utilising GFP is a highly effective way of identifying germline transmission in a transgenic colony. In addition the insertion into the promoter region allows a quantitative measure of alx/fpr-rs2 promoter activity and acts as a phenotypic marker in vivo.
No preview · Article · Jul 2009 · Inflammation Research
[Show abstract][Hide abstract] ABSTRACT: The glucocorticoids are the most potent anti-inflammatory drugs that we possess and are effective in a wide variety of diseases. Although their action is known to involve receptor mediated changes in gene transcription, the exact mechanisms whereby these bring about their pleiotropic action in inflammation are yet to be totally understood. Whilst many different genes are regulated by the glucocorticoids, we have identified one particular protein-annexin A1 (Anx-A1)-whose synthesis and release is strongly regulated by the glucocorticoids in many cell types. The biology of this protein, as revealed by studies using transgenic animals, peptide mimetics and neutralizing antibodies, speaks to its role as a key modulator of both of the innate and adaptive immune systems. The mechanism whereby this protein exerts its effects is likely to be through the FPR receptor family-a hitherto rather enigmatic family of G protein coupled receptors, which are increasingly implicated in the regulation of many inflammatory processes. Here we review some of the key findings that have led up to the elucidation of this key pathway in inflammatory resolution.
Full-text · Article · Sep 2008 · British Journal of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Annexin-A1 (ANXA1), a glucocorticoid-regulated protein, mediates several of the anti-inflammatory actions of the glucocorticoids. Previous studies demonstrated that ANXA1 is involved in pain modulation. The current study, using ANXA1 knockout mice (ANXA1-/-), 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.
The acetic acid-induced writhing response was performed in ANXA1-/- and wild-type (ANXA1+/+) mice with spinal and brain levels of prostaglandin E2 (PGE2) examined in both genotypes. The effect of the ANXA1 peptomimetic Ac2-26 as well as methylprednisolone on the writhing response and on spinal cord PGE2 of ANXA1+/+ and ANXA1-/- was compared. The expression of proteins involved in PGE2 synthesis, cytosolic phospholipase A2 (cPLA2) and cyclooxygenases (COXs), in the spinal cord of ANXA1+/+ and ANXA1-/- was also compared.Key results:ANXA1-/- mice exhibited a significantly greater writhing response and increased spinal cord levels of PGE2 compared with ANXA1+/+ mice. Ac2-26 produced analgesia and reduced spinal PGE2 levels in ANXA1+/+ and ANXA1-/- mice, whereas methylprednisolone reduced the writhing response and spinal PGE2 levels in ANXA1+/+, but not in ANXA1-/- mice. The expression of cPLA2, COX-1, COX-2 and COX-3 in spinal cord tissues was upregulated in ANXA1-/-compared with ANXA1+/+.
We conclude that ANXA1 protein modulates nociceptive processing at the spinal level, by reducing synthesis of PGE2 by modulating cPLA2 and/or COX activity. The analgesic activity of methylprednisolone is mediated by spinal ANXA1.
Full-text · Article · Aug 2008 · British Journal of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Annexin-1 (Anx-A1) has been recently shown to play a key role in T-cell activation and to be highly expressed in T cells from RA patients. Here, we investigated the effects of glucocorticoids (GCs) on Anx-A1 expression in T cells in vitro and in vivo.
To evaluate the effects of dexamethasone (Dex) on Anx-A1 expression, human peripheral blood T cells were incubated with Dex and then analysed by real-time PCR and western blotting. Similar experiments were carried out in vivo by measuring Anx-A1 levels in T cells from patients with RA before and after administration of steroids.
Incubation of T cells with Dex decreased Anx-A1 levels in a time-dependent fashion and almost abolished its expression after 12 h. Stimulation of T cells pre-incubated with Dex for 12 h with anti-CD3/CD28 led to significant reduction of IL-2 production. Addition of human recombinant Anx-A1 to Dex-treated cells reversed the inhibitory effects of the steroids on anti-CD3/CD28-induced IL-2 production. Treatment of RA patients with steroid decreased Anx-A1 expression in T cells.
GCs suppress Anx-A1 expression in T cells in vitro and in vivo. These results provide evidence for a novel pathway by which steroids regulate the adaptive immune response and suggest that Anx-A1 may represent a target for the treatment of autoimmune diseases.
Full-text · Article · Jun 2008 · Rheumatology (Oxford, England)
[Show abstract][Hide abstract] ABSTRACT: We have previously reported a role for annexin-A1 in liver proliferation and tumorogenicity as well as its action as an acute phase protein in a model of endotoxemia in interleukin-6 null mice.
In this study, we have investigated the analysis of the gene and protein expression in annexin-A1 null mice and the wild type livers during foetal and adult life, and in the presence of a proinflammatory stimulus.
The data indicate a link between the expression of the annexin-A1 as serine-phosphorylated-protein during early events of the inflammatory response and as tyrosine-phosphorylated-form at later time-points, during the resolution of inflammation.
The study of annexin-A1 post-translation modification may promote a new annexin-A1 peptide discovery programme to treat specific pathologies.
No preview · Article · Apr 2008 · Inflammation Research
[Show abstract][Hide abstract] ABSTRACT: The N-formyl peptide receptors (FPRs) are a family of G-protein coupled receptors that respond to proinflammatory N-formylated bacterial peptides (e.g., formyl-Met-Leu-Phe, fMLF) and, thus, contribute to the host response to bacterial infection. Paradoxically, a growing body of evidence suggests that some members of this receptor family may also be targets for certain anti-inflammatory molecules, including annexin A1 (ANXA1), which is an important mediator of glucocorticoid (GC) action. To explore further the potential role of FPRs in mediating ANXA1 actions, we have focused on the pituitary gland, where ANXA1 has a well-defined role as a cell-cell mediator of the inhibitory effects of GCs on the secretion of corticotrophin (ACTH), and used molecular, genetic, and pharmacological approaches to address the question in well-established rodent models. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis identified mRNAs for four FPR family members in the mouse anterior pituitary gland, Fpr-rs1, Fpr-rs2, Fpr-rs6, and Fpr-rs7. Functional studies confirmed that, like dexamethasone, ANXA1 and two ANXA1-derived peptides (ANXA1(1-188) and ANXA1(Ac2-26)) inhibit the evoked release of ACTH from rodent anterior pituitary tissue in vitro. Fpr1 gene deletion failed to modify the pituitary responses to dexamethasone or ANXA1(Ac2-26). However, lipoxin A4 (LXA4, 0.02-2 microM, a lipid mediator with high affinity for Fpr-rs1) mimicked the inhibitory effects of ANXA1 on ACTH release as also did fMLF in high (1-100 microM) but not lower (10-100 nM) concentrations. Additionally, a nonselective FPR antagonist (Boc1, 100 microM) overcame the effects of dexamethasone, ANXA1(1-188), ANXA1(Ac2-26), fMLF, and LXA4 on ACTH release, although at a lower concentration (50 microM), it was without effect. Together, the results suggest that the actions of ANXA1 in the pituitary gland are independent of Fpr1 but may involve other FPR family members, in particular, Fpr-rs1 or a closely related receptor. They thus provide the first evidence for a role of the FPR family in the regulation of neuroendocrine function.
Full-text · Article · May 2007 · The FASEB Journal
[Show abstract][Hide abstract] ABSTRACT: There is accumulating and convincing evidence indicating a role for glutamate in the pathogenesis of the human demyelinating disease multiple sclerosis (MS). Studies in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, demonstrate that pharmacological inhibition of specific glutamate receptors suppresses neurological symptoms and prevents blood-brain barrier (BBB) breakdown. The mechanisms through which glutamate influences BBB function during EAE remain unclear. Glutamate triggers the production of nitric oxide and superoxide, which can lead to the formation of peroxynitrite (ONOO(-)). Recent studies have implicated ONOO(-) in the loss of neurovascular integrity during EAE. We propose that glutamate contributes to BBB breakdown via the actions of ONOO(-). The present investigation examined glutamate-induced ONOO(-) formation in the b.End3 brain-derived endothelial cell line. b.End3 cells were incubated with a concentration range of glutamate and ONOO(-) production was assessed over time. Results showed a concentration- and time-dependent increase in ONOO(-) levels in glutamate-treated cells that were suppressed by selective and non-selective inhibitors of ONOO(-)-mediated reactions. Specific activation of b.End3-associated NMDA receptors also resulted in a concentration-dependent increase in ONOO(-) production. The ability of b.End3 cells to respond to the presence of glutamate was confirmed through the detection of NMDA receptor immnuoreactivity in cell extracts. In addition, the use of the NMDA receptor antagonists MK-801 and memantine reduced glutamate-mediated ONOO(-) generation from b.End3 cells. The data reinforce the important relationship between glutamate and the NMDA receptor, positioned at neurovascular sites, which may be of particular relevance to the pathogenesis of demyelinating disease.
Full-text · Article · Feb 2007 · Biochemical Pharmacology