55. Sharpless, N. E. & DePinho, R. A. How stem cells age and why this makes us grow old. Nature Rev. Mol. Cell Biol. 8, 703–713 (2007).

56. Krishnamurthy, J. et al. Ink4a/Arf expression is a biomarker of aging. J. Clin. Invest. 114, 1299–1307 (2004).

57. Campisi, J. Cancer and ageing: rival demons? Nature Rev. Cancer 3, 339–349 (2003).

58. Signer, R. A. J., Montecino‑Rodriguez, E., Witte, O. N. & Dorshkind, K. Aging and cancer resistance in lymphoid progenitors are linked processes conferred by p16Ink4a and Arf. Genes Dev. 22, 3115–3120 (2008).

59. Rossi, D. J. et al. Cell intrinsic alterations underlie hematopoietic stem cell aging. Proc. Natl Acad. Sci. USA 102, 9194–9199 (2005).

60. Montecino‑Rodriguez, E., Clark, R. & Dorshkind, K. Effects of insulin‑like growth factor administration and bone marrow transplantation on thymopoiesis in aged mice. Endocrinology 139, 4120–4126 (1998).

AcknowledgementsWork from our laboratory was supported by grant AG21450 from the National Institutes of Health, USA.

DAtABAsEsentrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genecD28 | CDKN2A | FGFr2 | growth hormone | iGF1 | iL-6 | iL-7 | KGF

FURtHER inFoRMAtionKenneth Dorshkind’s homepage: http://faculty.uclaaccess.ucla.edu/institution/personnel?personnel_id=45615Us census Bureau: http://www.cdc.gov/nchs/data/hus/hus07.pdf#fig01World Health Organization: http://www.who.int/en/seNieUr Protocol: http://www.medizin.uni-tuebingen.de/eucambis/home/senieur.html

All lInks ArE AcTIvE In ThE onlInE pdf

Inflammation is a primordial response that functions to protect the host against invasion by pathogens or exposure to xenobiotics. However, overly aggressive or prolonged inflammatory responses can be detrimental to the host. Therefore, higher organisms have evolved mechanisms to ensure that the inflammatory response is limited in time and space. Many endogenous anti-inflammatory and pro-resolving mediators function to counteract the properties of pro-inflammatory factors and to ensure a prompt resolution of inflammation. The concept that inflammation is resolved in a time- and space-specific manner is emerging, such that different outcomes can be produced accord-ing to the stage or site at which a given pathway or mediator becomes operative1,2. The action of anti-inflammatory and pro-resolving mediators and pathways on several

components of the inflammatory response is crucial for restoring tissue structure and homeostasis. Elucidation of their effects on immune cells could lead to the identification of the molecular target (or targets) of a given mediator, thereby prompting innovative drug discovery for the treatment of chronic inflammatory conditions2,3.

Glucocorticoids are the first class of endogenous anti-inflammatory mediators that have been successfully used for thera-peutic purposes; budesonide and beclo-methasone are widely used for the treatment of asthma, prednisolone is used for rheu-ma toid arthritis and other auto immune diseases, and mometasone and hydrocorti-sone are used for eczema and psoriasis. In healthy individuals, the circadian release of glucocorticoids (such as cortisol and corticosterone) from the adrenal glands

facilitates the control of several homeostatic processes that are crucial for health. During inflammation, increased levels of cortisol dampen local and systemic inflammatory events, thereby favouring proper resolution of the inflammatory response4,5. These fundamental pathophysiological functions of glucocorticoids are achieved through many molecular mechanisms, which can be broadly divided into genomic mechanisms (involving transactivation or transrepres-sion of gene transcription) and non-genomic mechanisms (that are rapid and independent of de novo protein synthesis) (BOX 1). In this Opinion article, we focus on one downstream mediator of glucocorticoids, the 37 kDa protein annexin A1 (also known as lipocortin 1; encoded by ANXA1). We review the recent evidence indicating that glucocorti coids regulate the synthesis and function of annexin A1 (reFs 4,6), possibly through a combination of both genomic and non-genomic processes, depending on the cell type and the time of induction. In addition, we describe the emerging data showing that glucocorticoids can differentially affect the annexin A1 path-way in cells of the innate and adaptive immune system, in which this pathway can have opposing effects. We propose that the annexin A1 pathway is an important mediator of the anti-inflammatory effects of glucocorticoids.

Effects in innate immunityAnnexin A1 is a member of a superfamily of annexin proteins that bind acidic phospho-lipids with high affinity in the presence of Ca2+ (reF. 7). There are 13 mammalian annexin proteins, each of which has specific biological functions. Similarly to other annexin proteins, annexin A1 is expressed in resting cells and binds to acidic phospho-lipids in the presence of Ca2+; original studies of annexin A1 therefore focused on its role in granule fusion and exocytosis, in which it was shown to promote the fusion of vesicle membranes with plasma membranes in reconstituted systems7.

The actions of annexin A1. In resting con-ditions, human and mouse neutrophils, monocytes and macrophages constitutively contain high levels of annexin A1 in their cytoplasm8–10. Following cell activation (for example, by neutrophil adhesion to endothelial-cell monolayers), annexin A1 is promptly mobilized to the cell surface and secreted11. The molecular mechanisms that are responsible for this rapid secretion are cell specific. In macrophages, the

o P i n i o n

Annexin A1 and glucocorticoids as effectors of the resolution of inflammationMauro Perretti and Fulvio D’Acquisto

Abstract | 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.

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ATP-binding cassette (ABC) transporter system has been shown to be responsible for the secretion of annexin A1 (reF. 12), whereas in pituitary cells the phosphorylation of annexin A1 at the amino-terminal serine 27 residue is required13. In human neutrophils, a high proportion (>60%) of cytoplasmic annexin A1 is stored in gelatinase granules9,14 and can be rapidly mobilized following exposure of the cells to weak activating sig-nals, such as low concentrations of chemo-attractants or adhesion to endothelial-cell monolayers11,15. Cell activation generally leads to the relocation of annexin A1 to the outside leaflet of the plasma membrane, to which it is tethered in a calcium-dependent manner. In addition, extracellular concentra-tions of Ca2+ of ≥1 mM lead to a conforma-tion change in the protein, such that the n-terminal region that is otherwise buried becomes exposed and the active form of annexin A1 is generated7,16 (FIG. 1).

Several studies using experimental models of inflammation have shown that administration of annexin A1, or of the bio-active peptides that comprise its n-terminal region, to mice results in potent inhibition of neutrophil trafficking17,18. In one study19, annexin A1 administration to mice during

an inflammatory response had an important anti-inflammatory effect, as revealed by analysis of the mesenteric microcircula-tion. This effect was associated with the detachment of adherent neutrophils from the vascular wall, which resulted in a reduc-tion in the number of cells that migrated into the subendothelial-matrix tissue19 (FIG. 2). These in vivo effects are supported by in vitro analyses showing that annexin A1 or annexin A1-derived peptides inhibit neutrophil adhesion to and transmigra-tion through endo thelial-cell monolay-ers20,21. Quantification of the interaction of neutrophils with human umbilical-vein endothelial-cell monolayers under flow has recently confirmed these observations, showing that low concentrations of annexin A1 (10 nM) significantly inhibit neutrophil adhesion22. The observed inhibitory activi-ties of recombinant annexin A1 or annexin A1-derived peptides are consistent with the phenotype of Anxa1–/– neutrophils, which show increased transmigration in the cre-master microcirculation of Anxa1–/– mice23. This phenotype is probably neutrophil-intrinsic because Anxa1–/– neutro phils show higher chemotaxis and CD11b upregula-tion in response to chemoattractants than

wild-type neutrophils23. These data are in agreement with an earlier study in which glucocorticoid treatment increased the levels of annexin A1 in circulating leukocytes and inhibition of annexin A1 prolonged leuko-cyte migration time in the post-capillary venules of the hamster cheek pouch24.

Further investigation of Anxa1–/– mice has shown that these mice develop more-severe joint disease (characterized by increased destruction of the cartilage and a thicker pannus at day seven) than wild-type mice in a model of monoarticular antigen-induced arthritis25,26. Increased levels of mRnA encoding interleukin-1 (Il-1) and Il-6 were also detected in the joints of these mice26, which is consistent with the known require-ment for Il-6 in this model of arthritis27. In addition, Anxa1–/– lung fibroblasts showed higher Il-1-induced Il-6 production, an effect that was shown to be an indirect conse-quence of decreased levels of dual-specificity protein phosphatase 1 (DUSP1; also known as MKP1) in these cells28. The potential involvement of DUSP1 in the mechanism of action of annexin A1 is reminiscent of what has been observed following glucocorticoid treatment, in which the induction of DUSP1 expression leads to the inhibition of mitogen-activated protein kinase signalling and thereby to a reduction in cytokine signalling and cell activation (BOX 1). Overactivation of intracellular signalling pathways that induce the release of effector molecules might also contribute to the observed lethality from endotoxic shock in Anxa1–/– mice following the administration of an otherwise sub-lethal dose of lipopolysaccharide (lPS)29. Compared with wild-type mice, lPS-induced endotoxic shock in Anxa1–/– mice was asso-ciated with a delayed and more prolonged increase in the levels of tumour-necrosis factor, Il-1 and Il-6 in the blood, as well as with increased production of these cytokines by peritoneal macrophages.

In summary, exogenous and endogenous annexin A1 counter-regulate the activities of innate immune cells, in particular extra vasation and the generation of pro- inflammatory mediators, and this ensures that a sufficient level of activation is reached but not exceeded.

ALXR: the annexin A1 receptor. Annexin A1 signals through a seven-membrane-spanning G-protein-coupled receptor (GPCR)30 known as formyl peptide recep-tor 2 (FPR2; also known as AlXR in humans), which is also the receptor for the anti-inflammatory molecule lipoxin A4 (BOX 2). Here, we use the term AlXR, to

Box 1 | the molecular mechanisms by which glucocorticoids affect gene expression

genomic effectsGlucocorticoids have numerous effects on gene expression, such that it has been proposed that ~1% of the genome might be modulated by these drugs4. The glucocorticoid receptor is retained in an inactive state by several chaperone proteins, including heat-shock protein 70 (HSP70) and HSP90, and following ligand binding, the glucocorticoid–receptor complex translocates to the nucleus, where it modulates gene expression through transrepression or transactivation.

Transrepression occurs when the glucocorticoid–receptor complex interacts with transcription factors: it can sequester them in the cytoplasm, promote their degradation or inhibit them through other mechanisms. Through transrepression, the glucocorticoid–receptor complex prevents the transcription of the target genes, including pro-inflammatory cytokines, growth factors, adhesion molecules, nitric oxide, prostanoids and other autacoids50. In addition, glucocorticoids transrepress the transcription of nuclear factor-κB (NF-κB)-induced pro-inflammatory genes by inducing the activation of histone deacetylase 2, which reduces the access of NF-κB to the promoter regions of its target genes70.

Transactivation involves the direct binding of the glucocorticoid–receptor complex to specific nucleotide sequences (termed glucocorticoid-response elements) to promote the transcription of genes, such as interleukin-10, β-adrenergic receptor, interleukin-1-receptor antagonist and dual-specificity protein phosphatase 1 (DUSP1), many of which have anti-inflammatory functions41. The anti-inflammatory effects of glucocorticoid-mediated transactivation can also be reinforced indirectly; for example, DUSP1 downregulates mitogen-activated-protein-kinase signalling and thereby inhibits cytokine synthesis. Finally, it is possible that annexin A1 (encoded by ANXA1) functions as a mediator of some of these glucocorticoid effects, as suggested by decreased levels of DUSP1 expression in Anxa1–/– lung fibroblasts28 (see the main text for more details).

non-genomic effectsGlucocorticoids can also have rapid effects, which occur within a few minutes, by modulating the degree of activation and responsiveness of target cells. These actions do not require de novo protein synthesis and are referred to as non-genomic effects71. Initially reported in monocytes, the non-genomic actions of glucocorticoids have been recently described in T cells72 and platelets73. However, it is unclear how non-genomic effects contribute to the overall therapeutic efficacy of glucocorticoids in controlling vascular inflammatory pathologies.

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reflect the biological function of this receptor instead of its structural homology with FPR1. Human AlXR belongs to a small family of receptors consisting of three members (FPR1, AlXR and FPR3) that are coupled to Gi pro-teins and are expressed by several cell types, including human neutrophils, monocytes, macrophages, endothelial cells and epithelial cells31,32. Annexin A1 and peptides derived from its n-terminal region compete with lipoxin A4 and with another AlXR ligand, serum amyloid protein A30,33, for binding to AlXR. Intriguingly, peptides derived from the annexin A1 n-terminal region have been shown in vitro to activate all three receptors of the FPR family34. However, the biological significance of this finding is not clear, as the generation of short and bioactive annexin A1 fragments in vivo has not been proven.

Downstream signalling following annexin A1-induced AlXR activation involves transient phosphorylation of extracellular-regulated kinase 1 (ERK1; also known as MAPK3) and ERK2 (also known as MAPK1)22,33, which is associated with a

rapid increase in the concentration of intra-cellular calcium32,35. Blocking of AlXR with a monoclonal antibody prevented annexin A1-induced inhibition of human neutrophil transmigration32 and adhesion to endothelial-cell monolayers under flow22.

As AlXR-deficient mice have yet to be generated and FPR1 is structurally related to AlXR, Fpr1–/– mice and pharmacological pan-receptor antagonists (that can block members of the FPR family in mice) have been used to study the effects of annexin A1 in the absence of signalling through FPRs. In a model of peritonitis, the inhibition of leukocyte migration by exogenous annexin A1 was par-tially reduced in Fpr1–/– mice37. By contrast, the increased neutrophil detachment that was observed in the mesenteric microcircula-tion following the administration of annexin A1-derived peptides or lipoxin A4 remained intact in Fpr1–/– mice, but was abrogated by the administration of the pan-receptor antagonist38. This suggests that mouse AlXR is probably the main receptor for annexin A1; the same is likely to apply in humans.

In addition to sharing a receptor, a com-mon function for annexin A1 and lipoxin A4 has been proposed recently to explain why germ-free mice show inflammatory hypo-responsiveness39. Compared with wild-type mice, germ-free mice showed increased levels of endogenous annexin A1 and lipoxin A4, and this was associated with increased levels of the anti-inflammatory cytokine Il-10 in the gut; the increased Il-10 levels were responsible for the reduced inflammation observed in these mice. A role for endo genous Il-10 in the anti-inflammatory effects of annexin A1 is also consistent with the observation that exposure of macrophages to annexin A1-derived bioactive peptides in vitro induces Il-10 secretion40. Moreover, the finding that gluco corticoids also mediate some of their anti-inflammatory effects through the induction of expression of Il-10 (reF. 41) and DUSP1 (as discussed above) supports the idea that annexin A1 and gluco corticoids have overlapping activities in the regulation of inflammatory responses. However, the contribution made by Il-10 and DUSP1 to the range of biological actions that are elicited by annexin A1 and glucocorticoids remains to be determined.

Glucocorticoids induce annexin A1 and ALXR expression. Administration of gluco-corticoids to healthy human volunteers leads to an increase in the levels of annexin A1 expression by circulating monocytes and neutrophils42. The levels of annexin A1 are also regulated by glucocorticoids during disease. More specifically, leukocytes from patients with Cushing’s disease (which is associated with high levels of cortisol) have higher intracellular levels of annexin A1, and leukocytes from patients with Addison’s disease (which is associated with low levels of cortisol) have lower intracellular levels of annexin A1 than healthy controls10. However, the mechanism by which glucocorticoids regulate annexin A1 has not been defined. The ANXA1 promoter does not seem to contain a canonical glucocorticoid-response element, but it does contain a partial consen-sus-binding site that mediates responsiveness to Il-6 (reF. 43), which suggests that gluco-corticoids indirectly regulate the expres-sion of annexin A1. However, more work is needed to determine the mechanisms that are involved in the cell-specific induction of annexin A1 expression by glucocorticoids, as it remains possible that glucocorticoid-response elements might be present in other regions of the ANXA1 promoter that are yet to be explored.

Nature Reviews | Immunology

Glucocorticoidinduction ofANXA1 expression

Glucocorticoidinduction ofALXR expression

Juxtacrineactivation

Paracrineactivation

Monocyte

Neutrophil

ALXRAnnexin A1

ABCtransporter

ab

cGranule

PAutocrineactivation

Figure 1 | mobilization of annexin A1 in activated cells and its potential mode of action. Following cell activation, such as through adhesion to endothelial-cell monolayers, intracellular annexin A1 is mobilized to the plasma membrane. Depending on the cell type, annexin A1 is then externalized and/or secreted through one of three mechanisms: through the activation of the ATP-binding cassette (ABc) transporter12 (a); through phosphorylation of its amino-terminal serine 27 residue followed by membrane localization to specific lipid domains13 before it moves to the outer leaflet of the plasma membrane and is secreted (b); or through fusion of annexin A1-containing granules with the plasma membrane, followed by release of annexin A1 (c). In the presence of ≥1 mM Ca2+, extra-cellular annexin A1 undergoes a conformational change that leads to exposure of the N-terminal region and binding to its receptor ALXr (also known as FPr2). Annexin A1 can function in an autocrine, para-crine and juxtacrine (involving cell–cell contact) manner to activate ALXr signalling. The juxtacrine interaction between annexin A1 that is tethered on the surface of the producing cell and ALXr on the target cell might be the most plausible mechanism of action in inflammatory conditions, as ‘activated’ annexin A1 could bind to acidic phospholipids at plasma ca2+ concentration of ≥1 mM and could there-fore be presented to target cells that express ALXr. The annexin A1–ALXr pathway can be manipulated by glucocorticoids, which induce the expression of the gene encoding annexin A1, ANXA1, as well as ALXR, by innate immune cells, thereby increasing the effects of this anti-inflammatory circuit.

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Despite the probable involvement of many transcription factors and the differ-ential effects of glucocorticoids in distinct cell types, a link between glucocorticoid administration and annexin A1 expres-sion has been confirmed in Anxa1–/– mice bearing a LacZ reporter construct under the control of the Anxa1 promoter44. Treatment of these mice with dexametha-sone increased the activity of the AnxA1 promoter in circulating neutrophils and monocytes, leading to a significant increase in LacZ expression as early as 2 hours after dexamethasone injection45. The timing of the increase in promoter activity in these cells following dexamethasone injection correlates with that of annexin A1 expres-sion in cells from wild-type mice. This further supports the idea that annexin A1 expression is regulated by glucocorticoids (as shown in the human studies), although the molecular mechanism underlying this process remains unclear.

Glucocorticoids have also recently been shown to induce the expression of AlXR. Incubation of human monocytes with dexa methasone or other synthetic gluco-corticoids induced the de novo synthesis of AlXR, leading to an increase in mRnA levels after 4–6 hours and protein levels after 12–24 hours46. A microarray study of human monocytes that had been treated with the glucocorticoid fluticasone for 16 hours showed that FPR1 mRnA (which encodes a receptor that is structurally related to AlXR) was upregulated, but ALXR mRnA was not47. However, it is worth noting that fluticasone-mediated induction of ANXA1 mRnA was also not detected under the conditions used in this study. Another study showed that dexamethasone treatment led to the upregulation of AlXR expression by human neutrophils, as well as by skin tissues in a mouse model of dermatitis48. We think these data support the hypothesis that the upregulation of specific anti-inflammatory GPCRs (such as AlXR) is involved in a mechanism that mediates the anti-inflammatory effects of glucocorti-coids. In addition, we propose that the link between glucocorticoids and the expression of annexin A1 and AlXR involves a genomic mechanism, and therefore requires de novo protein synthesis, although this needs to be further substantiated experimentally.

Finally, the finding that glucocorti-coids can also cause a rapid non-genomic mobilization and secretion of annexin A1 by target cells49 has led to the hypothesis that, during an inflammatory response, glucocorticoids first rapidly increase

the availability of annexin A1 at the cell surface (where its receptor is located) through non-genomic mechanisms. They then (within 2–4 hours) upregulate the expression of the genes that encode annexin A1 and AlXR through genomic mechanisms. In the context of resolution of inflammation, recent work indicates that the interaction between annexin A1 and AlXR has a role in controlling leuko-cyte apoptosis and leukocyte clearance by macrophages (BOX 3).

In conclusion, we believe that gluco-corticoids positively regulate the annexin A1–AlXR pathway, thereby assuring an appropriate level of activation of innate immune cells while limiting the duration of the pro-inflammatory response.

Effects in adaptive immunityThe widespread use of glucocorticoids as a treatment for inflammatory diseases is largely due to their ability to simultaneously block both innate and adaptive immune

Nature Reviews | Immunology

Antibacterialactivities

Phagocytosis

Transmigration

Detachment

Glucocorticoids↑ Neutrophil detachment↓ Neutrophil transmigration↓ Endothelial-cell adhesion molecule

expression↑ L-selectin shedding↑ Neutrophil annexin A1 expression

Annexin A1 and its bioactive peptides↑ Neutrophil detachment↓ Neutrophil transmigration↑ L-selectin shedding

Glucocorticoids↓ Neutrophil apoptosis↑ Phagocytosis of apoptotic

neutrophils↑ Macrophage annexin A1 expression↑ Macrophage ALXR expression↑ Annexin A1 secretion

Annexin A1 ↑ Neutrophil apoptosis↑ ANXA1 expression

(neutrophils and macrophages)↑ Phagocytosis of apoptotic

neutrophils

Neutrophil

Apoptotic neutrophil

Blood vessel

Inflammatoryexudate

Endothelial cell

Monocyte

MacrophageAnnexin A1

Figure 2 | leukocyte trafficking and fate in inflammation: annexin A1–AlXr checkpoints. During an inflammatory response, leukocytes, such as neutrophils and monocytes, are recruited to the site of inflammation. Glucocorticoids control the interaction of leukocytes with the vessel wall by affect-ing both the level of leukocyte responsiveness and endothelial-cell reactivity. Glucocorticoid-induced upregulation of the expression of annexin A1 by circulating leukocytes is one possible mechanism by which glucocorticoids might inhibit leukocyte responses. Pharmacological administration of annexin A1 can also mimic the effect of glucocorticoids, resulting in the detachment of adherent cells and the inhibition of neutrophil transmigration. shedding of L-selectin could be one of the molecular mecha-nisms that mediate this glucocorticoid- and annexin A1-mediated effect on leukocytes20. in the inflam-matory exudate, monocytes differentiate into macrophages and the lifespan of neutrophils is prolonged by 3–5 days, which allows sufficient time for them to exert their antibacterial and phagocytic actions. in addition, the intracellular concentration of annexin A1 is restored in the exudate by neutrophils through de novo protein synthesis. At the end of this period, the neutrophils die by apoptosis, which limits the release of annexin A1 and toxins, and prevents further tissue damage. Following glucocorti-coid therapy, the secretion of annexin A1 by macrophages is also increased. in addition, the production of annexin A1 by other cell types, such as mast cells and monocytes, promotes the clearance of apop-totic neutrophils and cell bodies by macrophages. it is unclear whether this effect is mediated by ALXr (also known as FPr2), although other ALXr ligands, such as lipoxin A

4, can also promote the clearance

of apoptotic cells. Phagocytosis of apoptotic neutrophils by macrophages leads to the transduction of anti-inflammatory signals, including those mediated by transforming growth factor-β.

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responses50. With regard to the inhibition of adaptive immune responses, glucocorticoids have marked effects on T-cell activation and differentiation pathways. In naive T cells, glu-cocorticoids negatively modulate early T-cell receptor (TCR)-initiated signalling events51,52 and inhibit the activation of key downstream transcription factors, such as activator pro-tein 1 (AP1), nuclear factor-κB (nF-κB) and nuclear factor of activated T cells (nFAT)53. In vitro studies using isolated CD4+ T cells and in vivo studies in mice have shown that glucocorticoids promote T helper 2 (TH2)-type responses directly and indirectly54–56, an effect that would explain their therapeutic efficacy in TH1-cell-driven autoimmune dis-eases. Glucocorticoids are also widely used for the treatment of TH2-cell-driven diseases, such as asthma, in which they act by suppress-ing the production of cytokines that propagate inflammation in these diseases, such as Il-5 and Il-13, while increasing the synthesis of anti-inflammatory cytokines, such as Il-10 (reFs 56,57). Recent work has identified new effects of annexin A1 on T cells; surprisingly, however, these effects are the opposite to those exerted by glucocorticoids.

Annexin A1 and T‑cell activation. Until recently, little was known about how annexin A1 influences the adaptive immune response, possibly because T cells express lower levels of annexin A1 (reF. 42) than myeloid cells8.

Indeed, naive T cells contain ~100-fold less ANXA1 mRnA and protein than neutrophils and macrophages42; however, annexin A1 expression is upregulated in TH cells following activation and differentiation (F.D’A., unpub-lished observations). The first evidence for a role of annexin A1 in T-cell function came from the studies of its effects, at nanomolar

concentrations, on T cells that had been stimulated with CD3- and CD28-specific antibodies58. Exposure to annexin A1 had no effect on naive T cells, as they express neg-ligible levels of AlXR. However, following stimulation with CD3- and CD28-specific antibodies, annexin A1 led to the upregula-tion of AlXR expression on the surface of the T cells in a time- and concentration-dependent manner. Interestingly, this occurred in parallel with the mobilization and release of endogenous annexin A1 (reF. 58). Upregulation of the annexin A1–AlXR pathway in T cells following treat-ment with annexin A1 led to increased and prolonged phosphorylation of AKT and ERK after the cells were stimulated with CD3- and CD28-specific antibodies. This is in agree-ment with the observation that T cells from Anxa1–/– mice show a lower (suboptimal) signalling response following stimulation than wild-type mice59. The increase in ERK activation by annexin A1 also occurs in neutro phils and other cells in the innate immune system. However, the consequences of this increase differ between cell types — ERK and AKT activation in T cells leads to cell proliferation, whereas ERK activation in neutrophils leads to a loss of cell adhesion and can have an anti-inflammatory effect. The divergent outcomes of the ERK signal-ling pathways suggest that many checkpoints exist to control cell behaviour and responses60, and highlight the importance of cell type and of the microenvironment in determining the biological function of a molecule.

Box 2 | Pharmacophysiology of ALXR

The receptor for lipoxin A4 (ALXR; also known as FPR2), which is also thought to be the receptor

for annexin A1, is a seven-membrane-spanning G-protein-coupled receptor (GPCR) that is structurally related to formyl peptide receptor 1 (FPR1)31. ALXR can recognize various different types of ligand, including several short-lived lipids, such as lipoxin A

4 and its stable analogues, as

well as proteins and peptides, such as serum amyloid protein A (SAA) and those derived from viral glycoproteins74,75. Although it was originally cloned from monomyelocytic cells, ALXR is expressed by a wide range of cells, including epithelial cells, endothelial cells, T cells and cells of the central nervous system (CNS). In the CNS, ALXR is expressed by microglial cells, in which it mediates the activating and pro-inflammatory properties of amyloid-β peptide75.

The long list of ALXR agonists underlies the versatility of this GPCR and its ability to bind a wide range of molecules (such as lipids, proteins and peptides)76. It is probable that different receptor regions, and perhaps conformations, might be required for, or are induced after, binding to each distinct agonist77. Consistent with this hypothesis, a comparison of lipoxin A

4- and SAA-mediated

responses in human neutrophils33 showed that SAA activated the pro-inflammatory transcription factor nuclear factor-κB, whereas lipoxin A

4 inhibited the effect of SAA. In addition, many ALXR

agonists, but not lipoxin A4, can induce a rapid and transient increase in the concentration of

intracellular calcium.Both annexin A1 and lipoxin A

4 induce rapid phosphorylation of extracellular-signal-regulated

kinase31. More recent studies have used genomic and proteomic approaches to determine the downstream targets of annexin A1-induced signalling. For example, CC-chemokine receptor 10 (CCR10) has been shown to be one such target in an epithelial cell line and in cells from the mouse trachea and ileum78. In addition, annexin A1 has been shown to upregulate IL1RA (interleukin-1 receptor antagonist) and downregulate CCR2 among the genes it regulates in human monocytes79.

Small chemical ligands that function as selective agonists for ALXR but not FPR1 have recently been developed80. Importantly, these small molecules showed anti-inflammatory properties in mice80, which supports the hypothesis that ALXR is an important anti-inflammatory GPCR during acute inflammation.

Box 3 | Annexin A1 and ALXR in leukocyte apoptosis

Endogenous glucocorticoids control the rate of clearance of apoptotic neutrophils by macrophages, as shown by the delay in apoptotic-cell clearance in the absence of 11-β-hydroxysteroid dehydrogenase type 1, an enzyme that is required to convert endogenous glucocorticoids into bioactive corticosterone81. Such a mechanism is consistent with the well-known effects of exogenous glucocorticoids in inhibiting neutrophil apoptosis, while inducing eosinophil and lymphocyte apoptosis, and promoting the non-inflammatory removal of apoptotic cells by macrophages82.

Three main effects of annexin A1 in leukocyte apoptosis have been described. First, exogenous administration of annexin A1 promotes human neutrophil apoptosis owing to transient calcium fluxes and dephosphorylation of the pro-apoptotic protein BCL-2-antagonist of cell death (BAD)35. Interestingly, glucocorticoids inhibit neutrophil apoptosis, revealing a surprising converse effect to that of annexin A1 (discussed in reF. 6). Second, endogenous annexin A1 is released from apoptotic neutrophils and acts on macrophages to promote the phagocytosis and removal of the apoptotic cells83. Third, glucocorticoid-treated macrophages secrete annexin A1, which then acts in a autocrine or paracrine manner to increase the engulfment of apoptotic neutrophils84. The molecular mechanisms that are responsible for these effects of annexin A1 have not been fully elucidated and may involve the activation of ALXR (also known as FPR2) and/or binding to phosphatidylserine, a phospholipid that is externalized on the outer leaflet of the plasma membrane of apoptotic cells that can interact with a specific receptor on the phagocyte, thereby favouring the engulfment of apoptotic cells.

Although these observations need to be confirmed in vivo, they suggest that annexin A1 might alter the fate of the extravasated neutrophils and promote their removal by macrophages, thereby contributing to the resolution of inflammation.

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Other downstream signalling responses can also be modulated (both positively and negatively) by exogenous annexin A1 or by its deletion in T cells. For example, stimula-tion of T cells with annexin A1 together with CD3- and CD28-specific antibodies increases the activation of several key transcriptional factors that are downstream of the TCR58: AP1, nF-κB and nFAT. Accordingly, reduced activation of these transcription factors was detected in Anxa1–/– T cells following stimulation59. So, in contrast to glucocorticoids, these studies indicate that annexin A1 transduces a stimulatory signal to promote T-cell activation, probably through AlXR (FIG. 3).

Annexin A1 and effector TH‑cell differentia‑tion. Many factors, including the micro-environment where T cells encounter their antigen and the affinity of the antigen for the TCR, have an important role in the early phase of TCR signalling and therefore in determining the activation of specific gene programmes that lead to the differentia-tion of naive T cells into effector TH cells61. Understanding how subtle changes in the strength of TCR signalling modulate T-cell differentiation is of particular importance for understanding and treating many auto-immune diseases, in which T cells become responsive to signals that would otherwise be ignored. Over the past 20 years, the number

of molecules that have been shown to be involved in the regulation of early events in TCR signalling has increased enormously, and in many cases a link has been found between the dysfunction of these molecules and the development of autoimmune dis-eases62. Recent work suggests that annexin A1 might belong to the group of molecules that fine-tune TCR signalling (FIG. 3).

Activation of naive T cells in non-polarizing conditions (TCR stimulation and Il-2) or TH1-cell-polarizing conditions (TCR stimulation and Il-2, Il-12 and Il-4-specific blocking antibodies) in the presence of exogenous annexin A1 promotes the differentiation of Il-2- and interferon-γ (IFnγ)-producing TH1 cells and suppresses the production of the TH2-type cytokines Il-4 and Il-13 (reF. 58). Consistent with these findings, in vitro differentiation of T cells from Anxa1–/– mice revealed a reduction in the production of TH1-type cytokines and increased skewing to a TH2-cell pheno-type59. In agreement with these in vitro data, systemic treatment of mice with annexin A1 immediately after immunization with collagen exacerbated the severity of collagen-induced arthritis at disease onset58. However, these observations are in contrast to those from studies of Anxa1–/– mice with antigen-induced arthritis26, which showed increased disease severity in the absence of annexin A1. These apparently contradictory results could

Nature Reviews | Immunology

TCR

CD3ALXR

Annexin A1

a Physiological condition b Pathological condition c Pathological conditionAppropriate T-cell activation T-cell hyperactivation

(such as in autoimmune disease)Insufficient T-cell activation (such as immunodeficiency)

Strength of TCR signal: ++++ Strength of TCR signal: ++++++ Strength of TCR signal: ++

MAPK MAPK MAPK MAPK MAPK MAPKMAPK MAPK MAPK

Cytoplasm

Nucleus

Transcription factor

Figure 3 | The annexin A1–AlXr pathway and strength of T-cell receptor signalling. engagement of the T-cell receptor (Tcr) triggers several signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway. Activation of this pathway can lead to the expression of genes such as interleukin-2 (iL-2), which is responsible for T-cell activation and proliferation. Activation of T cells also results in the release of annexin A1 and the expression of its receptor ALXr. This pathway seems to fine-tune the

strength of Tcr signalling and exert homeostatic control on T-cell activation under physiological conditions (a). Higher expression of annexin A1 during pathological conditions could increase the strength of Tcr signalling through the MAPK signalling pathway, thereby causing a state of hyperactivation of T cells (b). By contrast, low levels of annexin A1 expression, which occurs during pathological immunodeficiency, could be associated with a hyporesponsive state of T cells owing to the decreased strength of Tcr signalling (c).

stem from the fact that a single time point (7 days after challenge) was assessed in the Anxa1–/– mice with antigen-induced arthritis and that this disease is monoarticular (it only affects the injected joint). In addition, the comparison of two such different effects — the pharmacological effects of annexin A1 administration in collagen-induced arthritis and the effect of the absence of annexin A1 expression in antigen-induced arthritis — could further confound any conclusions drawn. It is therefore difficult at this stage to determine whether either model might be useful for understanding the role of annexin A1 in human disease. Of interest in this regard, and in agreement with the studies of collagen-induced arthritis, annexin A1 has recently been shown to have an activating effect on synovial fibroblasts from patients with rheumatoid arthritis, in which it com-plements the actions of tumour-necrosis factor and contributes to the release of metalloproteinase 1 (reF. 63). More studies in these and other models are needed to clarify the role of the annexin A1–AlXR pathway in arthritis and in distinct phases of the experimental arthritic response.

Further studies of human cells provide clues that annexin A1-mediated stimulation of T cells might be relevant to human disease. First, ANXA1 mRnA and protein levels were shown to be higher in circulating T cells from patients with rheumatoid arthritis than those

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from healthy individuals58. Second, synovial T cells expressed higher levels of annexin A1 than tonsil T cells from the same patient64. Therefore, it is tempting to speculate that the dysregulated expression of annexin A1 in T cells might contribute to the hyper-sensitivity of these cells to antigen stimulation and to the development of an overexuberant immune response in disease states.

Glucocorticoids and annexin A1 in T cells. The modulation of the annexin A1–AlXR pathway in innate immune cells by glucocor-ticoids (as described above) prompted us to investigate the effects of glucocorticoids on annexin A1 expression in T cells. Incubation of mouse and human T cells with dexam-ethasone induced a time-dependent decrease in both ANXA1 mRnA and annexin A1 expression levels64. These results contrast with those that were obtained from studies of innate immune cells, but may explain how the immunosuppressive effects of glucocorticoids can be achieved given the stimulatory effects of annexin A1 on T cells. It might therefore be possible that glucocorticoids inhibit T-cell activation and proliferation by reducing the expression of annexin A1. Two pieces of evidence support this hypothesis: first, T cells from Anxa1–/– mice proliferate less in response to CD3 and CD28 stimulation59,

and second, incubation of dexamethasone-pretreated human T cells with recombinant annexin A1 for 12 hours almost completely reversed the suppressive effects of dexametha-sone pretreatment and restored their ability to proliferate following stimulation with CD3- and CD28-specific antibodies64.

The clinical relevance of these findings is supported by a recent study in humans. Treatment of five patients with rheumatoid arthritis with the synthetic glucocorticoid methylprednisolone acetate was shown to reduce the expression of annexin A1 in cir-culating T cells by ≥ 50% (reF. 64). Together, these results suggest that inhibition of annexin A1 expression might be one of the mechanisms responsible for the immuno-suppressive effects of glucocorticoids on T cells, and that glucocorticoids exert dif-ferent effects on annexin A1 expression in innate and adaptive immune cells.

The modulation of annexin A1 expres-sion in T cells by glucocorticoids might also explain a well-known but poorly understood aspect of glucocorticoid pharmacology, the cell-specific time latency of their beneficial effects. The inhibitory effects of glucocor-ticoids on macrophages and neutrophils requires short incubation times (less than 2 hours), whereas much longer incubation times (more than 6–12 hours) are needed

for the efficient inhibition of T-cell activa-tion. We propose that this time-dependent difference reflects the differential effects of glucocorticoids on annexin A1 expression in these cells. Future investigation will be needed to determine the molecular mecha-nisms that are responsible for the differential modulation of annexin A1 expression by glucocorticoids in the innate and adaptive immune system. In this regard, a thorough and detailed analysis of the transcriptional regulation of ANXA1 in innate and adap-tive immune cells might provide some clues, such as the involvement of cell-specific transcription factors. Indeed, we have found three consensus GATA-binding protein 3 (GATA3)-binding sites in the Anxa1 pro-moter (F.D’A., unpublished observations). This transcription factor is preferentially expressed by T cells but not monocytes65 and is a well-known regulator of TH2-cell dif-ferentiation66. Interestingly, glucocorticoids have been reported to inhibit GATA3 tran-scriptional activity67. We speculate that inhi-bition of GATA3 activity by glucocorticoids occurs upstream of their suppressive action on annexin A1 expression in T cells.

conclusionsOn the basis of recent studies of the adap-tive immune response in Anxa1–/– mice and previous studies of the innate immune system, we propose a new hypothesis to explain the link between annexin A1 and glucocorticoids. The model suggests that part of the anti-inflammatory effect of gluco-corticoids on the innate immune system is mediated through the release of annexin A1 and the subsequent activation of AlXR, in an autocrine or paracrine manner, in neutro-phils and macrophages. By contrast, the immunosuppressive effect of glucocorticoids on the adaptive immune system is medi-ated through the inhibition of annexin A1 expression by T cells (FIG. 4). We speculate that daily treatment with glucocorticoids or intermittent therapy with high doses of these drugs would induce a constant release of annexin A1 by innate immune cells, and this would have a pro-resolving effect on the inflammatory response. In parallel, gluco-corticoids would reduce the co-stimulatory effect of annexin A1 on T-cell activation, thereby lowering the strength of the TCR signal (FIG. 3) and maintaining these cells in an unresponsive state.

In this model, annexin A1 has important opposing properties during innate and adap-tive immune responses, as it inhibits innate immune cells and promotes T-cell activa-tion, which is a property that is shared with

Adrenalgland

Nature Reviews | Immunology

↑ Annexin A1 ↓ Annexin A1

↓ Innate immune response ↓ Adaptive immune response

↓ Neutrophiltrafficking

↑ Neutrophilapoptosis

↓ Mast-celldegranulation

↓ Mast-cellactivation

No effect reported

Endogenousglucocorticoids

Syntheticglucocorticoids

Adaptive immune cellsInnate immune cells

↑ Phagocytosisof apoptoticcells bymacrophages

↓ T-cell proliferation↓ T-cell activation↓ TH1-cell differentiation↑ TH2-cell differentiation

NeutrophilMonocyte

MacrophageMast cell

T cell B cell

Figure 4 | model of glucocorticoid modulation of the annexin A1 pathway in immune regulation. We propose that endogenous and synthetic glucocorticoids can control innate and adaptive immune responses by modulating the expression and release of annexin A1. exposure of innate immune cells, such as neutrophils, monocytes, macrophages and mast cells, to glucocorticoids induces the release of annexin A1. This, in turn, acts in a paracrine or autocrine manner to maintain the level (and duration) of cell activation. in a dynamic system such as the inflammatory reaction, in which spatial and temporal factors can influence the final outcome, this could promote the resolution of the inflammatory reaction by actively maintaining homeostatic control of innate immune cells. By contrast, exposure of T cells to glucocorticoids leads to a reduction in annexin A1 expression and consequently inhibition of T-cell activation and the differentiation of T cells towards a T helper 2 (T

H2)-cell phenotype.

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other immune regulators, such as galectin 1 (reF. 68) and T-cell immunoglobulin domain and mucin domain 3 (TIM3)69. A complete understanding of the effects of glucocorti-coids and annexin A1 on immune responses, and the identification of AlXR as its main receptor, should aid the development of selective agonists for the resolution of inflam-matory responses and antagonists for the control of adaptive immune responses.

Mauro Perretti and Fulvio D’Acquisto are at the William Harvey Research Institute, Barts and

The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, UK.

Correspondence to M.P. e-mail: m.perretti@qmul.ac.uk

doi:10.1038/nri2470

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AcknowledgementsWork in our laboratory is funded by the Arthritis Research Campaign UK, the Wellcome Trust, the British Heart Foundation and the Medical Research Council UK. We apolo‑gize to the many colleagues whose work could not be cited here owing to space limitations.

Competing financial interestsThe authors declare competing financial interests: see web version for details.

DAtABAsEsentrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneAKT | ANXA1 | DusP1 | erK1 | erK2 | FPr1 | FPr2 | FPr3 | GATA3 | iL-1 | iL-6

FURtHER inFoRMAtionMauro Perretti’s homepage: http://www.whri.qmul.ac.uk/staff/perretti.htmlFulvio D’Acquisto’s homepage: http://www.whri.qmul.ac.uk/staff/D’Acquisto.html

All lInks ArE AcTIvE In ThE onlInE pdf

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