Fulvio D'Acquisto

Annexin A1 and glucocorticoids as effectors of the resolution of inflammation

Glucocorticoids are widely used for the management of inflammatory diseases. Their clinical application stems from our understanding of the inhibitory effect of the corticosteroid hormone cortisol on several components of the immune system. Endogenous and exogenous glucocorticoids mediate their multiple anti-inflammatory effects through many effector molecules. In this Opinion article, we focus on the role of one such effector molecule, annexin A1, and summarize the recent studies that provide insight into its molecular and pharmacological functions in immune responses. In addition, we propose a model in which glucocorticoids regulate the expression and function of annexin A1 in opposing ways in innate and adaptive immune cells to mediate the resolution of inflammation.

Involvement of NF‐κB in the regulation of cyclooxygenase‐2 protein expression in LPS‐stimulated J774 macrophages

We investigated the involvement of NF‐κB in the regulation of COX‐2 protein expression and prostaglandin production in LPS‐stimulated J774 macrophages. Incubation of J774 cells with LPS (1 μg/ml) for 24 h caused an increase of COX‐2 protein expression and accumulation of both PGE2 and 6‐keto‐PGF1α in the cell culture medium. Ammonium pyrrolidinedithiocarbamate (APDC, 0.1, 1, 10 μM) and N‐α‐p‐tosyl‐l‐lysine chloromethylketone (TLCK, 1, 10, 100 μM), two inhibitors of NF‐κB activation, suppressed in a concentration‐dependent manner both LPS‐induced COX‐2 protein expression and prostanoid generation. Moreover, APDC and TLCK both inhibited the LPS‐induced increase of NF‐κB DNA binding activity and prevented IκB‐α degradation. Our results show for the first time that NF‐κB is involved in COX‐2 protein expression in LPS‐stimulated J774 macrophages and suggest that inhibitors of NF‐κB activation may represent a useful tool for the pharmacological control of inflammation.

Anti-Inflammatory Role of the Murine Formyl-Peptide Receptor 2: Ligand-Specific Effects on Leukocyte Responses and Experimental Inflammation

The human formyl-peptide receptor (FPR)-2 is a G protein-coupled receptor that transduces signals from lipoxin A4, annexin A1, and serum amyloid A (SAA) to regulate inflammation. In this study, we report the creation of a novel mouse colony in which the murine FprL1 FPR2 homologue, Fpr2, has been deleted and describe its use to explore the biology of this receptor. Deletion of murine fpr2 was verified by Southern blot analysis and PCR, and the functional absence of the G protein-coupled receptor was confirmed by radioligand binding assays. In vitro, Fpr2−/− macrophages had a diminished response to formyl-Met-Leu-Phe itself and did not respond to SAA-induced chemotaxis. ERK phosphorylation triggered by SAA was unchanged, but that induced by the annexin A1-derived peptide Ac2–26 or other Fpr2 ligands, such as W-peptide and compound 43, was attenuated markedly. In vivo, the antimigratory properties of compound 43, lipoxin A4, annexin A1, and dexamethasone were reduced notably in Fpr2−/− mice compared with those in wild-type littermates. In contrast, SAA stimulated neutrophil recruitment, but the promigratory effect was lost following Fpr2 deletion. Inflammation was more marked in Fpr2−/− mice, with a pronounced increase in cell adherence and emigration in the mesenteric microcirculation after an ischemia–reperfusion insult and an augmented acute response to carrageenan-induced paw edema, compared with that in wild-type controls. Finally, Fpr2−/− mice exhibited higher sensitivity to arthrogenic serum and were completely unable to resolve this chronic pathology. We conclude that Fpr2 is an anti-inflammatory receptor that serves varied regulatory functions during the host defense response. These data support the development of Fpr2 agonists as novel anti-inflammatory therapeutics.

Annexin‐A1: a pivotal regulator of the innate and adaptive immune systems

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.

Critical Protective Role for Annexin 1 Gene Expression in the Endotoxemic Murine Microcirculation

The inflammatory response is a protective process of the body to counteract xenobiotic penetration and injury, although in disease this response can become deregulated. There are endogenous biochemical pathways that operate in the host to keep inflammation under control. Here we demonstrate that the counterregulator annexin 1 (AnxA1) is critical for controlling experimental endotoxemia. Lipopolysaccharide (LPS) markedly activated the AnxA1 gene in epithelial cells, neutrophils, and peritoneal, mesenteric, and alveolar macrophages--cell types known to function in experimental endotoxemia. Administration of LPS to AnxA1-deficient mice produced a toxic response characterized by organ injury and lethality within 48 hours, a phenotype rescued by exogenous application of low doses of the protein. In the absence of AnxA1, LPS generated a deregulated cellular and cytokine response with a marked degree of leukocyte adhesion in the microcirculation. Analysis of LPS receptor expression in AnxA1-null macrophages indicated an aberrant expression of Toll-like receptor 4. In conclusion, this study has detailed cellular and biochemical alterations associated with AnxA1 gene deletion and highlighted the impact of this protective circuit for the correct functioning of the homeostatic response to sublethal doses of LPS.

Prostaglandins prevent inducible nitric oxide synthase protein expression by inhibiting nuclear factor-κB activation in J774 macrophages

We investigated the effect of PGE2 and iloprost (a prostacyclin analogue) on inducible nitric oxide synthase (iNOS) protein expression and nuclear factor‐κB (NF‐κB) activation in lipopolysaccharide (LPS)‐stimulated J774 macrophages. Incubation of J774 cells with LPS (10 μg/ml) caused an increase of iNOS protein expression which was prevented in a concentration‐dependent fashion by PGE2 (0.1, 1, 10 μM) and iloprost (0.01, 0.1, 1 μM). Electrophoretic mobility shift assay indicated that both prostanoids blocked the activation of NF‐κB, a transcription factor necessary for NO synthase induction. PGE2 and iloprost also blocked disappearance of IκB‐α from cytosolic fraction and nuclear translocation of NF‐κB subunits p50 and p65. These results show for the first time that PGE2 and iloprost down‐regulate iNOS protein expression by inhibiting NF‐κB activation and suggest a negative feed‐back mechanism that may be important for limiting excessive or prolonged NO production in pathological events.

Annexin 1 cleavage in activated neutrophils: A pivotal role for proteinase 3

Annexin 1 is an anti-inflammatory protein that plays a key role in innate immunity by modulating the activation of several types of cells, including neutrophils. Here we have developed a cleavage assay using tagged annexin 1 and observed marked activity in the membrane fraction of activated neutrophils. A combination of inhibitors, transfected cells, and proteomic analyses allowed us to identify proteinase 3 as the main enzyme responsible for this cleavage in the N terminus region of the protein, at least in the context of neutrophil activation. Because annexin 1 is an important endogenous anti-inflammatory mediator, blocking its cleavage by proteinase 3 would augment its homeostatic pro-resolving actions and could represent an opportunity for innovative anti-inflammatory drug discovery.

Reduced inflammatory and neuropathic pain and decreased spinal microglial response in fractalkine receptor (CX3CR1) knockout mice

J. Neurochem. (2010) 114, 1143–1157.

Impaired T cell activation and increased Th2 lineage commitment in Annexin-1-deficient T cells

Annexin‐1 is a well‐known endogenous anti‐inflammatory protein that modulates the activation of cells of the innate immune system such as neutrophils and macrophages. We have recently reported a positive role for the exogenous protein on T cell differentiation, however, whether such a role holds true for the endogenous protein has yet to be determined. This aspect has been investigated here finding that Annexin‐1‐deficient T cells display an impaired activation and proliferation in response to anti‐CD3 plus anti‐CD28 stimulation. Furthermore, differentiation of T cells from Annexin‐1‐deficient mice in Th0/Th1/Th2 or Th17 skewing conditions demonstrated an increased Th2 phenotype compared to cells from control littermates. Similar results were obtained when we analyzed the Th1/Th2 profile of lymph node cells obtained from mice immunized with keyhole limpet hemocyanin or the inflammatory infiltrate in mouse model of allergic inflammation. These results demonstrate a novel modulatory role of endogenous Annexin‐1 in TCR signaling and T cell differentiation and suggest this protein might play a dual and complementary role in the innate and adaptive immune response.

Design and characterization of a cleavage-resistant Annexin A1 mutant to control inflammation in the microvasculature

Human polymorphonuclear leukocytes adhesion to endothelial cells during the early stage of inflammation leads to cell surface externalization of Annexin A1 (AnxA1), an effector of endogenous anti-inflammation. The antiadhesive properties of AnxA1 become operative to finely tune polymorphonuclear leukocytes transmigration to the site of inflammation. Membrane bound proteinase 3 (PR3) plays a key role in this microenvironment by cleaving the N terminus bioactive domain of AnxA1. In the present study, we generated a PR3-resistant human recombinant AnxA1-named superAnxA1 (SAnxA1)-and tested its in vitro and in vivo properties in comparison to the parental protein. SAnxA1 bound and activated formyl peptide receptor 2 in a similar way as the parental protein, while showing a resistance to cleavage by recombinant PR3. SAnxA1 retained anti-inflammatory activities in the murine inflamed microcirculation (leukocyte adhesion being the readout) and in skin trafficking model. When longer-lasting models of inflammation were applied, SAnxA1 displayed stronger anti-inflammatory effect over time compared with the parental protein. Together these results indicate that AnxA1 cleavage is an important process during neutrophilic inflammation and that controlling the balance between AnxA1/PR3 activities might represent a promising avenue for the discovery of novel therapeutic approaches.