Egle Solito

Endogenous lipid- and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin A4 receptor.

Aspirin (ASA) and dexamethasone (DEX) are widely used anti-inflammatory agents yet their mechanism(s) for blocking polymorphonuclear neutrophil (PMN) accumulation at sites of inflammation remains unclear. Here, we report that inhibition of PMN infiltration by ASA and DEX is a property shared by aspirin-triggered lipoxins (ATL) and the glucocorticoid-induced annexin 1 (ANXA1)-derived peptides that are both generated in vivo and act at the lipoxin A4 receptor (ALXR/FPRL1) to halt PMN diapedesis. These structurally diverse ligands specifically interact directly with recombinant human ALXR demonstrated by specific radioligand binding and function as well as immunoprecipitation of PMN receptors. In addition, the combination of both ATL and ANXA1-derived peptides limited PMN infiltration and reduced production of inflammatory mediators (that is, prostaglandins and chemokines) in vivo. Together, these results indicate functional redundancies in endogenous lipid and peptide anti-inflammatory circuits that are spatially and temporally separate, where both ATL and specific ANXA1-derived peptides act in concert at ALXR to downregulate PMN recruitment to inflammatory loci.

Annexin 1: more than an anti-phospholipase protein.

Annexin 1 (ANXA1) is the first characterized member of the annexin family of proteins able to bind (i.e. to annex) to cellular membranes in a calcium-dependent manner. ANXA1 may be induced by glucocorticoids in inflammatory cells and shares with these drugs many anti-inflammatory effects. Originally described as a phospholipase A2 (PLA2)-inhibitory protein, ANXA1 can affect many components of the inflammatory reaction besides the metabolism of arachidonic acid. Recent data have shown that ANXA1 may specifically target cytosolic PLA2 by both direct enzyme inhibition and suppression of cytokine-induced activation of the enzyme. ANXA1 inhibits the expression and/or activity of other inflammatory enzymes like inducible nitric oxide synthase (iNOS) in macrophages and inducible cyclooxygenase (COX-2) in activated microglia. The inhibition of iNOS expression may be caused by the stimulation of IL-10 release induced by ANXA1 in macrophages. Like glucocorticoids, ANXA1 exerts profound inhibitory effects on both neutrophil and monocyte migration in inflammation. Several mechanisms may contribute to the protein effect on cell migration, namely the activation of receptors like the formyl peptide receptor (FPR) and the lipoxin A4 receptor (ALXR), the shedding of L-selectin, the binding to α4β1 integrin and carboxylated N-glycans. Furthermore, again mimicking the action of glucocorticoids, ANXA1 promotes inflammatory cell apoptosis associated with transient rise in intracellular calcium and caspase-3 activation. Finally, ANXA1 has been recently identified as one of the ‘eat-me’ signals on apoptotic cells to be recognised and ingested by phagocytes. Thus, ANXA1 may contribute to the anti-inflammatory signalling that allows safe post-apoptotic clearance of dead cells.

A novel calcium-dependent proapoptotic effect of annexin 1 on human neutrophils

The glucocorticoid‐inducible protein annexin (ANXA) 1 is an anti‐inflammatory mediator that down‐regulates the host response. Endogenously, ANXA1 is released in large amounts from adherent polymorphonuclear neutrophils (PMN) and binds to their cell surface to inhibit their extravasation into inflamed tissues. The present study determined the effects of exogenous ANXA1 on several functions of human PMN in vitro. Addition of 0.1–1 µM human recombinant ANXA1 to the PMN provoked rapid and transient changes in intracellular Ca2+ concentrations that were blocked by the Ca2+ channel inhibitor SKF‐96365. Although ANXA1 did not affect oxidant production and only minimally affected PMN chemotactic properties, the ANXA1‐promoted Ca2+ influx was associated with two important functional effects: shedding of L‐selectin and acceleration of PMN apoptosis. The latter effect was confirmed using three distinct technical procedures, namely, cell cycle, Hoechst staining, and ANXA5 binding assay. ANXA1‐induced PMN apoptosis was insensitive to inhibitors of L‐selectin shedding, whereas it appeared to be associated with dephosphorylation of the proapoptotic intracellular mediator BAD. In conclusion, exogenous ANXA1 displayed selective actions on human PMN. We propose that the new proapoptotic effect reported here may be part of the spectrum of ANXA1‐mediated events involved in the resolution of acute inflammation.

Involvement of the Receptor for Formylated Peptides in the in Vivo Anti-Migratory Actions of Annexin 1 and its Mimetics

An innovative avenue for anti-inflammatory therapy is inhibition of neutrophil extravasation by potentiating the action of endogenous anti-inflammatory mediators. The glucocorticoid-inducible protein annexin 1 and derived peptides are effective in inhibiting neutrophil extravasation. Here we tested the hypothesis that an interaction with the receptor for formylated peptide (FPR), so far reported only in vitro, could be the mechanism for this in vivo action. In a model of mouse peritonitis, FPR antagonists abrogated the anti-migratory effects of peptides Ac2-26 and Ac2-12, with a partial reduction in annexin 1 effects. A similar result was obtained in FPR (knock-out) KO mice. Binding of annexin 1 to circulating leukocytes was reduced (>50%) in FPR KO mice. In vitro, annexin binding to peritoneal macrophages was also markedly reduced in FPR KO mice. Finally, evidence of direct annexin 1 binding to murine FPR was obtained with HEK-293 cells transfected with the receptor. Overall, these results indicate a functional role for FPR in the anti-migratory effect of annexin 1 and derived peptides.

Transfection of annexin 1 in monocytic cells produces a high degree of spontaneous and stimulated apoptosis associated with caspase-3 activation

Transfection of the pre‐monomyelocytic U937 cell line with a plasmid coding for full‐length annexin 1 (ANX1, 347 amino acid) leads to cell death by promoting apoptosis. In addition, over‐expression of the N‐terminal and the first domain of the protein (144 amino acids, clone ANX1‐S), which does not contain the Ca2+ binding sites, gives susceptibility to cell apoptosis following activation by either 5 ng ml−1 tumour necrosis factor (TNF)‐α or 1 – 40 μg ml−1 etoposide. This was demonstrated by using the fluorescent labelled annexin V, cell cycle and nuclear staining analyses. Transfection with an empty plasmid (clone CMV) or with a plasmid carrying the cDNA antisense for ANX1 (clone ANX1‐AS) did not alter U937 cells to the degree of apoptosis promoted by either stimulant. Treatment of CMV U937 cells with TNF‐α increased ANX1 mRNA and protein expression in a time‐dependent manner, with maximal increases at 3 and 6 h, respectively. Clone ANX1‐S showed higher constitutive (more than 2 fold) and activated caspase‐3 activity, associated with higher phospholipase A2 (PLA2) activity (in the region of +50 – 100%), whereas expression of cytosolic PLA2 Bax and Bcl‐2 were similar in all cell clones, as determined by Western blotting. In conclusion, this study demonstrates a complex regulatory role of cell apoptosis for ANX1, at least with regards to cells of the myelo‐monocytic lineage.

Dexamethasone induces the expression of the mRNA of lipocortin 1 and 2 and the release of lipocortin 1 and 5 in differentiated, but not undifferentiated U‐937 cells

The effect of dexamethasone on mRNA and protein synthesis of lipocortins (LCT) 1, 2 and 5 has been investigated in U‐937 cells. A constitutive expression of both mRNAs and proteins was detected in undifferentiated U‐937 cells. This constitutive level was increased time‐ and dose‐ dependently by incubation with phorbol myristate acetate (PMA). In U‐937 cells differentiated by 24 h incubation with 6 ng/ml PMA, dexamethasone (DEX) (1 μM for 16 h) caused an increased synthesis of the mRNA level of LCT‐1 and 2, but not of LCT‐5, over the level induced by PMA. DEX had no effect in undifferentiated cells, Moreover, DEX stimulated the extracellular release of LCT‐1 and 5, but not of LCT‐2, and inhibited the release of PGE2 and TXB2 only in the differentiated U‐937 cells. These results suggest that the responsiveness of these cells to glucocorticoids is dependent on the phase of cell differentiation. The selective release of lipocortins by differentiated U‐937 cells may explain, at least in part, the inhibition by DEX of the prostanoid release.

Annexin A1: A Central Player in the Anti-Inflammatory and Neuroprotective Role of Microglia

The brain microenvironment is continuously monitored by microglia with the detection of apoptotic cells or pathogens being rapidly followed by their phagocytosis to prevent inflammatory responses. The protein annexin A1 (ANXA1) is key to the phagocytosis of apoptotic leukocytes during peripheral inflammatory resolution, but the pathophysiological significance of its expression in the CNS that is restricted almost exclusively to microglia is unclear. In this study, we test the hypothesis that ANXA1 is important in the microglial clearance of apoptotic neurons in both noninflammatory and inflammatory conditions. We have identified ANXA1 to be sparingly expressed in microglia of normally aged human brains and to be more strongly expressed in Alzheimer’s disease. Using an in vitro model comprising microglial and neuronal cell lines, as well as primary microglia from wild-type and ANXA1 null mice, we have identified two distinct roles for microglial ANXA1: 1) controlling the noninflammatory phagocytosis of apoptotic neurons and 2) promoting resolution of inflammatory microglial activation. In particular, we showed that microglial-derived ANXA1 targets apoptotic neurons, serving as both an “eat me” signal and a bridge between phosphatidylserine on the dying cell and formyl peptide receptor 2 on the phagocytosing microglia. Moreover, inflammatory activation of microglia impairs their ability to discriminate between apoptotic and nonapoptotic cells, an ability restored by exogenous ANXA1. We thus show that ANXA1 is fundamental for brain homeostasis, and we suggest that ANXA1 and its peptidomimetics can be novel therapeutic targets in neuroinflammation.

Identification of an essential endogenous regulator of blood-brain barrier integrity, and its pathological and therapeutic implications.

The blood–brain barrier (BBB), a critical guardian of communication between the periphery and the brain, is frequently compromised in neurological diseases such as multiple sclerosis (MS), resulting in the inappropriate passage of molecules and leukocytes into the brain. Here we show that the glucocorticoid anti-inflammatory messenger annexin A1 (ANXA1) is expressed in brain microvascular endothelial cells, where it regulates BBB integrity. In particular, ANXA1−/− mice exhibit significantly increased BBB permeability as a result of disrupted interendothelial cell tight junctions, essentially related to changes in the actin cytoskeleton, which stabilizes tight and adherens junctions. This situation is reminiscent of early MS pathology, a relationship confirmed by our detection of a selective loss of ANXA1 in the plasma and cerebrovascular endothelium of patients with MS. Importantly, this loss is swiftly restored by i.v. administration of human recombinant ANXA1. Analysis in vitro confirms that treatment of cerebrovascular endothelial cells with recombinant ANXA1 restores cell polarity, cytoskeleton integrity, and paracellular permeability through inhibition of the small G protein RhoA. We thus propose ANXA1 as a critical physiological regulator of BBB integrity and suggest it may have utility in the treatment of MS, correcting BBB function and hence ameliorating disease.

Post-translational modification plays an essential role in the translocation of annexin A1 from the cytoplasm to the cell surface

Annexin A1 (ANXA1) has an important role in cell‐cell communication in the host defense and neuroendocrine systems. In both systems, its actions are exerted extracellularly via membrane‐bound receptors on adjacent sites after translocation of the protein from the cytoplasm to the cell surface of adjacent cells. This study used molecular, microscopic, and pharmacological approaches to explore the mechanisms underlying the cellular exportation of ANXA1 in TtT/GF (pituitary folliculo‐stellate) cells. LPS caused serine‐phosphorylation of ANXA1 (ANXA1‐S27‐PO4) and translocation of the phosphorylated protein to the cell membrane. The fundamental requirement of phosphorylation for membrane translocation was confirmed by immunofluorescence microscopy on cells transfected with wild‐type or mutated (S27/A) ANXA1 constructs tagged with enhanced green fluorescence protein. The trafficking of ANXA1‐S27‐PO4 to the cell surface was dependent on PI3‐kinase and MAP‐kinase. It also required HMG‐coenzyme A and myristoylation. The effects of HMG‐coenzyme A blockade were overcome by mevalonic acid (the product of HMG‐coen‐zyme A) and farnesyl‐pyrophosphate but not by geranyl‐geranylpyrophosphate or cholesterol. Together, these results suggest that serine‐27 phosphorylation is essential for the translocation of ANXA1 across the cell membrane and also identify a role for isoprenyl lipids. Such lipids could target consensus sequences in ANXA1. Alternatively, they may target other proteins in the signal transduction cascade (e.g., transporters).—Solito, E., Christian, H. C., Festa, M., Mulla, A., Tierney, T., Flower, R. J., Buckingham, J. C. Post‐transla‐tional modification plays an essential role in the translocation of annexin A1 from the cytoplasm to the cell surface. FASEB J. 20, E677–E687 (2006)

Lipocortin 1 reduces myocardial ischemia-reperfusion injury by affecting local leukocyte recruitment

We assessed here the effect of the glucocorticoid‐regulated protein lipocortin 1 (LC1) in a model of rat myocardial ischemia reperfusion. Treatment of animals with human recombinant LC1 at the end of a 25‐min ischemic period significantly reduced the extent of infarct size in the area at risk as measured 2 h later, with ~50% inhibition at the highest dose tested of 50 per rat (equivalent to 5.4 nmol/kg). The protective effect of LC1 was abolished by protein denaturation and not mimicked by the structurally related protein annexin V. A combination of electron and light microscopy techniques demonstrated the occurrence of the myocardial damage at the end of the reperfusion period, with loss of fiber organization. LC1 provided a partial and visible protection. The dose‐dependent protection afforded by LC1 was paralleled by lower values of myeloperoxidase activity, tumor necrosis factor α, and macrophage inflammatory protein‐1α. The functional link between migrated leukocytes and the myocardial damage was confirmed by electron and light microscopy, and a significantly lower number of extravasated leukocytes was counted in the group of rats treated with LC1 (50 μg). In conclusion, we demonstrate for the first time that LC1 reduces the leukocyte‐dependent myocardial damage associated with an ischemia‐reperfusion procedure.