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The annexin 1 receptor(s): Is the plot unravelling?
Abstract
Recent studies have proposed a functional link between annexin 1 (ANXA1), an endogenous anti-inflammatory mediator, and receptors of the formyl-peptide family. In particular, exogenous and endogenous ANXA1 and its peptidomimetics interact with one member of this family, the formyl-peptide-receptor-like 1. Further analyses of the interactions between ANXA1 and this and other members of this receptor family, and a better characterization of the ANXA1 receptor systems in models of inflammation, might clarify their mechanism of anti-inflammatory effects. This line of research will facilitate the development of ANXA1 mimetics and take advantage of >20 years of biological research into the functions of this glucocorticoid-modulated protein.
... Expressed in a variety of cell types, ANXA1 is particularly abundant in neutrophils. The protein is primarily cytosolic, but it may also be secreted through a nonclassic secretory process and found on the outer cell surface, causing leukocyte detachment and thereby inhibiting their transendothelial migration (for review, see Perretti, 2003). At low concentrations, both ANXA1 holoprotein and its Nterminal peptides (Ac2-26, Ac-AMVSEFLKQAWFIEN-EEQEYVQTVK, and Ac9 -25) elicit Ca 2ϩ transients through FPR1 without fully activating the MAPK pathway (Ernst et al., 2004a), causing neutrophil desensitization and inhibition of transendothelial migration induced by other chemoattractants such as the chemokine IL-8 (CXCL8). ...
... In contrast, at high concentrations, the ANXA1 peptides fully activate neutrophils in vitro and become potent proinflammatory stimulants. The antimigratory activity of exogenous and endogenous ANXA1 has been shown in both acute and chronic models of inflammation (Perretti, 2003). Fpr1 knockout mice exhibit normal neutrophil accumulation during thioglycolate-elicited peritonitis (Perretti et al., 2001). ...
... In contrast, FPR2/ALX ligands such as LXA4 and ANXA1 exhibit anti-inflammatory activities (Perretti et al., 2002;Chiang et al., 2006). ANXA1 has been shown to cause detachment of leukocytes and prevent transendothelial migration (diapedesis) (Perretti, 2003). In comparison, the anti-inflammatory effect of LXA4 is shown to involve suppression of proinflammatory gene expression. ...
Formyl peptide receptors (FPRs) are a small group of seven-transmembrane domain, G protein-coupled receptors that are expressed mainly by mammalian phagocytic leukocytes and are known to be important in host defense and inflammation. The three human FPRs (FPR1, FPR2/ALX, and FPR3) share significant sequence homology and are encoded by clustered genes. Collectively, these receptors bind an extraordinarily numerous and structurally diverse group of agonistic ligands, including N-formyl and nonformyl peptides of different composition, that chemoattract and activate phagocytes. N-formyl peptides, which are encoded in nature only by bacterial and mitochondrial genes and result from obligatory initiation of bacterial and mitochondrial protein synthesis with N-formylmethionine, is the only ligand class common to all three human receptors. Surprisingly, the endogenous anti-inflammatory peptide annexin 1 and its N-terminal fragments also bind human FPR1 and FPR2/ALX, and the anti-inflammatory eicosanoid lipoxin A4 is an agonist at FPR2/ALX. In comparison, fewer agonists have been identified for FPR3, the third member in this receptor family. Structural and functional studies of the FPRs have produced important information for understanding the general pharmacological principles governing all leukocyte chemoattractant receptors. This article aims to provide an overview of the discovery and pharmacological characterization of FPRs, to introduce an International Union of Basic and Clinical Pharmacology (IUPHAR)-recommended nomenclature, and to discuss unmet challenges, including the mechanisms used by these receptors to bind diverse ligands and mediate different biological functions.
... To date, the only investigations of annexin-1 peptides in myocardial ischaemia/reperfusion injury have focussed on reduced infiltration of neutrophils into the heart (D' Amico et al., 2000;La et al., 2001). In contrast, we have previously shown that in the absence of neutrophils, the N-terminal annexin-1 peptide Anx-1 2-26 is capable of preventing the loss of contractile function response of the myocardium induced by the inflammatory mediators endotoxin (lipopolysaccharide, LPS) and interferong (IFNg) in vitro (Ritchie et al., 1999;2003). ...
... Thus, annexin-1 peptides are clearly effective in limiting the neutrophil-mediated component of ischaemia/reperfusion in-jury, which certainly contributes to cardioprotection. From our studies both in cardiac myocytes subjected to metabolic inhibition in the present investigation, and in myocardial preparations suppressed by endotoxin and IFN-g (Ritchie et al., 1999;2003), it is clear, however, that annexin-1 peptides also exert direct, neutrophil-independent, cardioprotective actions. Development of new treatment strategies that protect against both neutrophil-dependent and neutrophil-independent mechanisms of ischaemia/reperfusion injury could have major clinical impact, and targeting annexin-1-related mechanisms represents such a strategy. ...
... Evidence is now emerging from both receptor antagonists (Boc 1, Boc 2) and transgenic knockout approaches that the annexin-1 peptides bind and activate the formyl peptide receptor family of receptors to exert their antiinflammatory, antineutrophil and other actions . This family comprises formyl peptide receptors (FPR) and the FPR-like receptors, aspirin-triggered lipoxin A 4 receptors (known as FPR-like receptor subtype 1, FPRL-1 or ALXR) and FPR-like receptor subtype 2 (FPRL-2), all of which have seven transmembrane-spanning domains functionally coupled to guanine nucleotide-binding protein G i (Perretti, 2003). Homology between these receptors is relatively high at both the nucleotide and amino-acid levels . ...
The anti‐inflammatory properties of annexin‐1 peptides have been largely ascribed to their powerful antineutrophil actions in vivo . We have recently reported that the N‐terminal fragment of annexin‐1, Anx‐1 2–26 , preserves contractile function of cardiac muscle in vitro . The aim of the present study was to determine if Anx‐1 2–26 elicits protective actions specifically on the cardiac myocyte (in the absence of neutrophils), using a model of metabolic inhibition to simulate ischaemia.
Metabolic inhibition of cardiac myocytes (4 h incubation at 37°C in HEPES‐containing buffer supplemented with 2‐deoxy‐ D ‐glucose, D , L ‐lactic acid and pH adjusted to 6.5) followed by 2.5 h recovery in normal medium markedly increased creatine kinase (CK) and lactate dehydrogenase (LDH) levels by 179±39 and 26±7 IU L ⁻¹ (both n =40, P <0.001), respectively. However, cellular injury was significantly decreased when Anx‐1 2–26 (0.3 μ M ) was present during metabolic inhibition, CK by 74±10% and LDH by 71±6% (both n =31, P <0.001), respectively.
Boc 2 (10 μ M ), a nonselective formyl peptide receptor antagonist, present during metabolic inhibition, abolished the cardioprotective effect of Anx‐1 2–26 .
Addition of chelerythrine (10 μ M ), 5‐hydroxydecanoate (500 μ M ) or SB202190 (1 μ M ) during metabolic inhibition also abolished Anx‐1 2–26 ‐induced cardioprotection.
Cellular injury induced by metabolic inhibition was also largely prevented when myocytes were incubated with Anx‐1 2–26 for 5 min with 10 min recovery prior to the insult, or when Anx‐1 2–26 was present only during the recovery period following drug‐free metabolic inhibition.
In conclusion, the annexin‐1 peptide Anx‐1 2–26 potently prevents cardiac myocyte injury induced by metabolic inhibition, an action that was dependent at least in part on the activation of the formyl peptide receptor family of G‐protein‐coupled receptors, protein kinase C, p38 mitogen‐activated protein kinase and ATP‐sensitive potassium channels.
British Journal of Pharmacology (2005) 145 , 495–502. doi: 10.1038/sj.bjp.0706211
... The biological effects of ANXA1 and its cleavage product Ac 2-26 peptide are mediated by formyl peptide receptors (FPRs). In humans, three FPRs (FPR1, FPR2, and FPR3) regulate innate inflammatory responses [9][10][11][12]. ...
... 4.6. Infection with T. gondii and Ac 2-26 treatment can reduce COX-2 and PGE2 levels in placental explants of the third trimester of pregnancy ANXA1 is a protein that displays potent anti-inflammatory properties by inhibiting the prostaglandin synthesis and limiting COX-2 [10]. The relevance of these molecules in placental explants infected with T. gondii and treated with Ac 2-26 mimetic peptide of ANXA1 was investigated through the immunolocalization of COX-2 and measuring PGE2 levels by ELISA. ...
... ANXA1 exerts its biological responses by activation Formyl peptide receptors (FPRs) (Perretti, 2003;Perretti and D'Acquisto, 2009). The three human FPRs (FPR1, FPR2/ALX, and FPR3) are G protein-coupled receptors that share significant sequence homology (Ye et al., 2009). ...
... Several pro-and anti-inflammatory ligands bind FPR1 and FPR2/ALX (Le et al., 2002). The biological response of the ANXA1 protein and its cleavage product Ac2-26 peptide are mediated by FPR1 and FPR2/ALX (Leoni et al., 2015a;Perretti, 2003;Perretti et al., 2002). Intestinal epithelial cells express ANXA1 and its receptors (FPR1 and FPR2/ ALX). ...
Epithelial barriers play an important role in regulating mucosal homeostasis. Upon injury, the epithelium and immune cells orchestrate repair mechanisms that re-establish homeostasis. This process is highly regulated by protein and lipid mediators such as Annexin A1. In this review, we focus on the pro-repair properties of Annexin A1.
... Moreover, short peptide fragments from the ANXA1 N-terminus mimicked the glucocorticoid effect. It would be worth investigating whether these effects are mediated by interaction of exogenous ANXA1 with the membrane receptors reported to bind the protein [17] (see below). ...
... It has been shown that ANXA1 and lipoxin A 4 directly interact with human ALXR/FPRL1 to synergize in inhibiting neutrophil activities in inflammation [42]. The interaction of ANXA1 with different receptors has been recently reviewed by Mauro Perretti [17]. More experimental work is warranted to precisely define the intracellular signalling pathways mediated by these receptors. ...
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 alpha4beta1 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.
... So, the discovery that annexin-1 and its antiinflammatory peptido-mimetics bind to ALX can open the way to the development of novel small-molecule entities based on annexin-1 (REF. 128), thereby, at least in part, retaining some of the anti-inflammatory properties of glucocorticoids that signal the resolution of ongoing inflammatory lesions. ...
... This has consequences for understanding the nature of the endogenous control of resolving inflammation, showing how anti-inflammatory mediators of differing structure and metabolic origin can exert their antiinflammatory effects through the same receptor. On this theme, our ongoing challenge is to identify novel receptors, the activation of which is integral to resolving inflammation, thereby presenting alternative targets for drug development 128 . Other potential pro-resolving lipid mediators for drug targeting are PGD 2 metabolites. ...
Treatment of inflammatory diseases today is largely based on interrupting the synthesis or action
of mediators that drive the host’s response to injury. Non-steroidal anti-inflammatories, steroids
and antihistamines, for instance, were developed on this basis. Although such small-molecule
inhibitors have provided the main treatment for inflammatory arthropathies and asthma, they are
not without their shortcomings. This review offers an alternative approach to the development of
novel therapeutics based on the endogenous mediators and mechanisms that switch off acute
inflammation and bring about its resolution. It is thought that this strategy will open up new
avenues for the future management of inflammation-based diseases.
... When recombinant preparations of the protein or peptido-mimetic drawn from the N-terminal region were tested, acute models of neutrophil extravasation insensitive to inhibitors of lipid metabolism, displayed a marked inhibitory effect [7,8]. These pharmacological studies opened the way to several others, such that today we refer to the 'ANXA1 system' as an endogenous biochemical process, which operates in the context of the adherent extravasating neutrophil [9,10], briefly summarized below. ...
... In resting neutrophils, ANXA1 is predominantly localized in the cytosol, and a good proportion of this is localized in cytoplasmic granules [11], and possibly other organelles, such that it can be rapidly mobilized to the cell surface when the neutrophil adheres on to endothelium [12]. On the plasma membrane, ANXA1 interacts in an autocrine/paracrine fashion with receptors that belong to a specific family of 'chemotactic' G-protein-coupled receptors, most probably the lipoxin A 4 receptor [10,13]. From other post-receptor mechanisms, the end-point is controlled neutrophil activation and detachment from the post-capillary venule endothelium [14,15]. ...
ANXA1 (annexin 1), a member of the 'annexin' family of calcium- and phospholipid-binding proteins, was originally identified as an endogenous mediator of the anti-inflammatory actions of glucocorticoids. However, this protein exerts multiple inhibitory effects on the host inflammatory response, including a preferential regulation of the adhesion step of blood-borne neutrophil within the microenvironment of an inflamed vasculature. It is now emerging that ANXA1 is endowed with other roles, since the protein is abundant in inflammatory exudates as it is produced and released by the extravasated neutrophil. In the present paper, we review the novel proapoptotic effect of ANXA1 and discuss its potential with respect to the pathophysiology of inflammation and leucocyte recruitment.
... Furthermore, passive immunisation strategies (Perretti et al. 1996a) and the development of ANXA1 null mice (Hannon et al. 2003) have allowed a better definition of the roles played by the en-dogenous protein in several cellular functions, including phagocytosis, extravasation, and mediator generation. Over the past decade, our own studies have detailed the mobilization and the function that this protein plays with respect to the process of neutrophil recruitment, so much that we now refer to the "annexin 1 system" as an endogenous biochemical process that operates in the context of the adherent, extravasating neutrophil (Perretti 2003). However, it is well accepted that ANXA1 can affect several other cell types and systems as well, as recently reviewed (John et al. 2004, Parente et al. 2004, Perretti & Flower 2004). ...
... There was no indication of a receptor mediated-mechanism however it is of interest that most if not all of these effects could be reproduced with peptides drawn from the ANXA1 N-terminal region (Croxtall et al. 1993(Croxtall et al. , 1998. Recent indication suggest that the effects of peptide Ac2-26 on A549 cells are also mediated by FPR (Rescher et al. 2002), though the question of the specific receptor mechanism responsible for ANXA1 effects remains open also on this cell type (Perretti 2003). ...
The concept of anti-inflammation is currently evolving with the definition of several endogenous inhibitory circuits that are important in the control of the host inflammatory response. Here we focus on one of these pathways, the annexin 1 (ANXA1) system. Originally identified as a 37 kDa glucocorticoid-inducible protein, ANXA1 has emerged over the last decade as an important endogenous modulator of inflammation. We review the pharmacological effects of ANXA1 on cell types involved in inflammation, from blood-borne leukocytes to resident cells. This review reveals that there is scope for more research, since most of the studies have so far focused on the effects of the protein and its peptido-mimetics on neutrophil recruitment and activation. However, many other cells central to inflammation, e.g. endothelial cells or mast cells, also express ANXA1: it is foreseen that a better definition of the role(s) of the endogenous protein in these cells will open the way to further pharmacological studies. We propose that a more systematic analysis of ANXA1 physio-pharmacology in cells involved in the host inflammatory reaction could aid in the design of novel anti-inflammatory therapeutics based on this endogenous mediator.
... leukocytes. 10,11 The downstream molecular mechanisms by which ANXA1 modulates these cellular responses are unclear although this protein modulates the activity of extracellular signal regulated kinase (ERK), a mitogenactivated protein kinase (MAPK). 12,13 Lipopolysaccharide (LPS) activates Toll-like Receptor-4 (TLR-4), in a complex with CD14, LPS-binding protein and MD-2 14 to activate a number of intracellular signalling proteins, including the MAPK p38, ERK1, ERK2 and c-jun N-terminal kinase (JNK). ...
... In leukocytes, exogenous administration of ANXA1 peptidomimetics activates G-protein-coupled receptors, such as the lipoxin A4 and FPR receptors, to inhibit inflammatory effects. 10,11 ANXA1 N-terminal peptides desensitize FPR receptors. Therefore, we speculate that transient administration of exogenous ANXA1 activates a G-protein- coupled receptor to inhibit LPS-induced iNOS production in macrophages. ...
Annexin-1 (ANXA1) is a glucocorticoid-regulated protein that modulates the effects of bacterial lipopolysaccharide (LPS) on macrophages. Exogenous administration of peptides derived from the N-terminus of ANXA1 reduces LPS-stimulated inducible nitric oxide synthase (iNOS) expression, but the effects of altering the endogenous expression of this protein are unclear. We transfected RAW264.7 murine macrophage-like cell lines to over-express constitutively ANXA1 and investigated whether this protein modulates the induction of iNOS, cyclooxygenase-2 (COX-2) and tumour necrosis factor-alpha (TNF-alpha) in response to LPS. In contrast to exogenous administration of N-terminal peptides, endogenous over-expression of ANXA1 results in up-regulation of LPS-induced iNOS protein expression and activity. However, levels of iNOS mRNA are unchanged. ANXA1 has no effect on COX-2 or TNF-alpha production in response to LPS. In experiments to investigate the mechanisms underlying these phenomena we observed that activation of signalling proteins classically associated with iNOS transcription was unaffected. Over-expression of ANXA1 constitutively activates extracellular signal regulated kinase (ERK)-1 and ERK-2, components of a signalling pathway not previously recognized as regulating LPS-induced iNOS expression. Inhibition of ERK activity, by the inhibitor U0126, reduced LPS-induced iNOS expression in our cell lines. Over-expression of ANXA1 also modified LPS-induced phosphorylation of the ERK-regulated translational regulation factor eukaryotic initiation factor 4E. Our data suggest that ANXA1 may modify iNOS levels by post-transcriptional mechanisms. Thus differential effects on iNOS expression in macrophages are seen when comparing acute administration of ANXA1 peptides versus the chronic endogenous over-expression of ANXA1.
... ANXA1 is one of the molecules released following mucosal injury and plays a role in maintaining intestinal hemostasis [3]. ANXA1 is a 37 kDa protein that is regulated via glucocorticoids, it binds to the formyl peptide receptors (FPRs), especially FPR1 and FPR2, and mediates the healing effect on the epithelial layers [3,4]. The expression of FPR1 and FPR2 in the intestine controls the inflammatory responses and disease progression [5]. ...
Objective and Design
Annexin A1 (ANXA1) plays a role in maintaining intestinal hemostasis, especially following mucosal inflammation. The published data about ANXA1 was derived from experimental animal models where there is an overlapping between epithelial and immune cells. There is no in vitro gut epithelial model that can assess the direct effect of ANXA1 on the gut epithelium.
Methods
We developed high-throughput stem-cell-based murine epithelial cells and bacterial lipopolysaccharides (LPS) were used to induce inflammation. The impact of ANXA1 and its functional part (Ac2-26) was evaluated in the inflamed model. Intestinal integrity was assessed by the transepithelial electrical resistance (TEER), and FITC-Dextran permeability. Epithelial junction proteins were assessed using confocal microscopy and RT-qPCR. Inflammatory cytokines were evaluated by RT-qPCR and ELISA.
Results
LPS challenge mediated a damage in the epithelial cells as shown by a drop in the TEER and an increase in FITC-dextran permeability; reduced the expression of epithelial junctional proteins (Occludin, ZO-1, and Cadherin) and increased the expression of the gut leaky protein, Claudin − 2. ANXA1 and Ac2-26 treatment reduced the previous damaging effects. In addition, ANXA1 and Ac2-26 inhibited the inflammatory responses mediated by the LPS and increased the transcription of the anti-inflammatory cytokine, IL-10.
Conclusion
ANXA1 and Ac2-26 directly protect the epithelial integrity by affecting the expression of epithelial junction and inflammatory markers. The inflamed gut model is a reliable tool to study intestinal inflammatory diseases, and to evaluate the efficacy of potential anti-inflammatory drugs and the screening of new drugs that could be candidates for inflammatory bowel disease.
... 16,17 Moreover, FPR1 interacts with other endogenous ligands including annexin A1 (ANXA1), a ubiquitous protein contained in the cytosol of all nucleated cells that leak into the extracellular space when cells die. [18][19][20][21][22][23] Thus, FPR1 plays a major role in the response to pathogens as well as in the regulation of immune and inflammatory responses. 24 The roles of FPR1 in the response to infectious pathogens can be either positive or negative. ...
Formyl peptide receptor 1 (FPR1) is a pattern-recognition receptor that detects bacterial as well as endogenous danger-associated molecular patterns to trigger innate immune responses by myeloid cells. A single nucleotide polymorphism, rs867228 (allelic frequency 19–20%), in the gene coding for FPR1 accelerates the manifestation of multiple carcinomas, likely due to reduced anticancer immunosurveillance secondary to a defect in antigen presentation by dendritic cells. Another polymorphism in FPR1, rs5030880 (allelic frequency 12–13%), has been involved in the resistance to plague, correlating with the fact that FPR1 is the receptor for Yersinia pestis. Driven by the reported preclinical effects of FPR1 on lung inflammation and fibrosis, we investigated whether rs867228 or rs5030880 would affect the severity of coronavirus disease-19 (COVID-19). Data obtained on patients from two different hospitals in Paris refute the hypothesis that rs867228 or rs5030880 would affect the severity of COVID-19.
... Annexin A1 is an effective inhibitor in both acute and chronic inflammation [57,58], which has a significant effect on the leukocyte/ endothelial interaction promoted by ischemia-reperfusion (I/R) procedures [59]. There is evidence that the fragment of annexin A1 (188 amino acids) has a protective effect on the rat model of cerebral ischemia [60]. ...
Annexin A is a kind of calcium-dependent phospholipid-binding proteins, which contributes to the formation of the cell membranes and cytoskeleton and played a part as a membrane skeleton to stabilize lipid bilayer. Autophagy is one of the most important programmed cell death mechanisms. And recently some reports suggest that annexin A family protein is associated with autophagy for annexin A can regulate the formation of vesicular lipid membranes and promote cell exocytosis. In this review, we summarized the roles of annexin A protein family in autophagy regulation and targeted medical treatment for better diagnoses and therapies.
... The most recent studies suggested a significant role for FPR ligands in the mechanisms that maintain the balance between proinflammatory and anti-inflammatory pathways to retain homeostasis following ischemic insult. Ligands such as AnxA1 and its mimetic peptides, e.g., N-terminalderived Ac2-26, are of particular interest [176,177]. The prevailing view is that AnxA1 regulates cell apoptosis, proliferation, and differentiation [178] but also diminishes leu-kocyte adhesion and migration, thus inhibiting proinflammatory cytokine release under ischemic conditions [175,179]. ...
Chronic inflammatory processes within the central nervous system (CNS) are in part responsible for the development of neurodegenerative and psychiatric diseases. These processes are associated with, among other things, the increased and disturbed activation of microglia and the elevated production of proinflammatory factors. Recent studies indicated that the disruption of the process of resolution of inflammation (RoI) may be the cause of CNS disorders. It is shown that the RoI is regulated by endogenous molecules called specialized pro-resolving mediators (SPMs), which interact with specific membrane receptors. Some SPMs activate formyl peptide receptors (FPRs), which belong to the family of seven-transmembrane G protein-coupled receptors. These receptors take part not only in the proinflammatory response but also in the resolution of the inflammation process. Therefore, the activation of FPRs might have complex consequences.
This review discusses the potential role of FPRs, and in particular the role of FPR2 subtype, in the brain under physiological and pathological conditions and their involvement in processes underlying neurodegenerative and psychiatric disorders as well as ischemia, the pathogenesis of which involves the dysfunction of inflammatory processes.
... We suggest a role for ANXA1 in mesoglycan-induced keratinocyte motility as both migration and invasion could be strongly inhibited by ANXA1 knockdown in mesoglycan treated cells. This is consistent with the notion that ANXA1 may act in an autocrine or paracrine/juxtacrine manner by possible interaction with formyl peptide receptors (Perretti, 2003). Moreover, mesoglycan could be able, as it does with other factors, to mediate ANXA1 extracellular interactions. ...
Wound healing is a dynamic process comprising multiple events, such as inflammation, re‐epithelialization, and tissue remodeling. Re‐epithelialization phase is characterized by the engagement of several cell populations, mainly of keratinocytes that sequentially go through cycles of migration, proliferation, and differentiation to restore skin functions. Troubles can arise during the re‐epithelialization phase of skin wound healing particularly in keratinocyte migration, resulting in chronic non‐healing lesions, which represent a serious clinical problem. Over the last decades, the efforts aimed to find new pharmacological approaches for wound care were made, yet almost all current therapeutic strategies used remain inadequate or even ineffective. As such, it is crucial to identify new drugs that can enable a proper regeneration of the epithelium in wounded skin. Here, we have investigated the effects of the fibrinolytic drug mesoglycan, a glycosaminoglycans mixture derived from porcine intestinal mucosa on HaCaT human keratinocytes that were used as in vitro experimental model of skin re‐epithelialization. We found that mesoglycan induces keratinocyte migration and early differentiation by triggering the syndecan‐4/PKCα pathway and that these effects were at least in part, because of the formation of the annexin A1/S100A11 complex. Our data suggest that mesoglycan may be useful as a new pro‐healing drug for skin wound care.
... 3,4 Annexin A1 -also known as lipocortin 1, lipomodulin, maerocortin or renocortin -is a 37-kD protein that is encoded by the ANNA1 gene in humans. 5,6 It is a member of the superfamily of the Ca + -dependent phospholipid-binding structuralrelated protein. [7][8][9][10][11] Annexin A1 is involved in mediating several host physiological 474 lai et al and pathological processes such as endocytosis and exocytosis, signal transduction, proliferation, differentiation, apoptosis, invasion, migration, inflammation, tumor, and immune function. ...
Purpose
Fibrosis in peripheral airways is responsible for airflow limitation in chronic obstructive pulmonary disease (COPD). Annexin A1 modulates several key biological events during inflammation. However, little is known about its role in airway fibrosis in COPD. We investigated whether levels of Annexin A1 were upregulated in patients with COPD, and whether it promoted airway fibrosis.
Methods
We quantified serum Annexin A1 levels in never-smokers (n=12), smokers without COPD (n=11), and smokers with COPD (n=22). Correlations between Annexin A1 expression and clinical indicators (eg, lung function) were assessed. In vitro, human bronchial epithelial (HBE) cells were exposed to cigarette smoke extract (CSE) and Annexin A1 expression was assessed. Primary human lung fibroblasts were isolated from patients with COPD and effects of Annexin A1 on fibrotic deposition of lung fibroblasts were evaluated.
Results
Serum Annexin A1 was significantly higher in patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines stage III or IV than in those with GOLD stages I or II (12.8±0.8 ng/mL versus 9.8±0.7 ng/mL; p=0.016). Annexin A1 expression was negatively associated with airflow obstruction (forced expiratory volume in one second % predicted; r=−0.72, p<0.001). In vitro, Annexin A1 was significantly increased in CSE-exposed HBE cells in a time- and concentration-dependent manner. Annexin A1 promoted lung fibroblasts proliferation, migration, differentiation, and collagen deposition via the ERK1/2 and p38 mitogen-activated protein kinase pathways.
Conclusion
Annexin A1 expression is upregulated in patients with COPD and affects lung fibroblast function. However, more studies are needed to clarify the role of Annexin A1 in airway fibrosis of COPD.
... The polypeptide chain of anxA1 comprises a C-terminal core domain, which is conserved amongst all members of the family and harbors the Ca 2+ /phospholipid binding sites, and an N-terminal tail that varies between annexin members. The N-terminal tail of anxA1 can interact with the receptor for formylated peptides (FPR2/ALXR) [11,[13][14][15][16]. In absence of calcium and a phospholipid surface, the tail is concealed within the core domain. ...
Objective:
To investigate therapeutic effects of annexin A1 (anxA1) on atherogenesis in LDLR-/- mice.
Methods:
Human recombinant annexin A1 (hr-anxA1) was produced by a prokaryotic expression system, purified and analysed on phosphatidylserine (PS) binding and formyl peptide receptor (FPR) activation. Biodistribution of 99mTechnetium-hr-anxA1 was determined in C57Bl/6J mice. 12 Weeks old LDLR-/- mice were fed a Western Type Diet (WTD) during 6 weeks (Group I) or 12 weeks (Group P). Mice received hr-anxA1 (1 mg/kg) or vehicle by intraperitoneal injection 3 times per week for a period of 6 weeks starting at start of WTD (Group I) or 6 weeks after start of WTD (Group P). Total aortic plaque burden and phenotype were analyzed using immunohistochemistry.
Results:
Hr-anxA1 bound PS in Ca2+-dependent manner and activated FPR2/ALX. It inhibited rolling and adherence of neutrophils but not monocytes on activated endothelial cells. Half lives of circulating 99mTc-hr-anxA1 were <10 minutes and approximately 6 hours for intravenously (IV) and intraperitoneally (IP) administered hr-anxA1, respectively. Pharmacological treatment with hr-anxA1 had no significant effect on initiation of plaque formation (-33%; P = 0.21)(Group I) but significantly attenuated progression of existing plaques of aortic arch and subclavian artery (plaque size -50%, P = 0.005; necrotic core size -76% P = 0.015, hr-anxA1 vs vehicle) (Group P).
Conclusion:
Hr-anxA1 may offer pharmacological means to treat chronic atherogenesis by reducing FPR-2 dependent neutrophil rolling and adhesion to activated endothelial cells and by reducing total plaque inflammation.
... The upregulation of IL-4 by CH might be another key event for CH to exert its protective role against stroke because IL-4 can promote tissue repair by exerting its neuroprotective role, and IL-4 deficiency exacerbates ischemic brain injuries and neurological impairments in mice [33]. ANXA1 is a potent inhibitor in both acute and chronic inflammation [34,35], with a significant efficacy on leukocyte/endothelium interactions promoted by ischemia-reperfusion (I/R) procedures [36]. Evidence showed an ANXA1 fragment (188amino acid long) was protective in a rat model of cerebral ischemia [37]. ...
Preconditioning is promising for treating cerebral ischemic stroke. Annexin A1 (ANXA1) is a homeostatic antiinflammatory mediator that participates in countering against ischemic injuries. We investigated whether chloral hydrate preconditioning (CH) exerts neuroprotection via regulation of ANXA1 in stroke.
Adult male C57BL/6J mice or ANXA1 knockout (ANXA1(-/-) ) mice were randomly allocated to control (NCH) and CH groups [2%, 6%, and 10% chloral hydrate (i.p.) 1 h before the middle cerebral artery occlusion (MCAO)]. Neurological performances were evaluated by modified 7-point neurological scales and rotarod test. Cerebral infarction was analyzed by triphenyltetrazolium chloride (TTC) staining and MRI. The expression of ANXA1, pro-inflammatory (TNF-α, IL-1β, IL-6), and antiinflammatory (IL-4, IL-10, TGF-β) cytokines was investigated by RT-PCR, western blot, and immunofluorescence.
Chloral hydrate preconditioning significantly improved the neurological outcomes and reduced the infarction and brain edema after ischemia. In addition, CH increased the expression of ANXA1 in the microglia, decreased the levels of TNF-α, IL-1β, and IL-6, while elevated the levels of IL-4, IL-10, and TGF-β in the MCAO mice. Furthermore, both ANXA1 blocker Boc1 (5 mg/kg, i.c.v.) or ANXA1 gene deficiency restrained the protective effects of CH against stroke.
Chloral hydrate preconditioning protects against ischemic injuries through upregulating the expression of ANXA1, and the followed antiinflammatory mechanisms.
© 2015 John Wiley & Sons Ltd.
... Administration of ANX-A1 protein or Ac2-26 induces apoptosis of neutrophils (as discussed later in this review), suppresses leukocyte adhesion and migration, and inhibit the activity of PLA 2 , prostaglandin E 2 and myeloperoxidase (as reviewed in detail by Perretti and D'Acquisto (2009), Perretti and Dalli (2009). Considerable evidence suggests that ANX-A1 and Ac2-26 bind to the formyl peptide receptor (FPR) family of G-protein-coupled receptor (GPCR)s to exert their biological effects, with the FPR2 subtype in neutrophils receiving the majority of research attention in this regard (Walther et al., 2000;Perretti et al., 2002;Perretti, 2003;Gastardelo et al., 2009). As discussed in Section 3 of this review, it is now widely accepted that formyl peptide receptors (FPR) mediate the anti-inflammatory actions of ANX-A1. ...
... Several peptides deriving from its Nterminal domain potently inhibit neutrophil recruitment (Perretti et al., 2002). Upon exposure to the cell surface, annexin 1 interacts with the formyl peptide receptor (FPR), the high affinity receptor of fMLP, as well as the lipoxin A4 receptor (ALX or FPR-like receptor) (El Kebir et al., 2008;Perretti and Flower, 2004;Perretti et al., 2002;Perretti, 2003). Glucocorticoids promote annexin 1 synthesis, and annexin 1 is thereby thought to mediate many of the anti-inflammatory effects of these steroids (Perretti and Flower, 2004). ...
Infection or sterile inflammation triggers site-specific attraction of leukocytes. Leukocyte recruitment is a process comprising several steps orchestrated by adhesion molecules, chemokines, cytokines and endogenous regulatory molecules. Distinct adhesive interactions between endothelial cells and leukocytes and signaling mechanisms contribute to the temporal and spatial fine-tuning of the leukocyte adhesion cascade. Central players in the leukocyte adhesion cascade include the leukocyte adhesion receptors of the β2-integrin family, such as the αLβ2 and αMβ2 integrins, or of the β1-integrin family, such as the α4β1-integrin. Given the central involvement of leukocyte recruitment in different inflammatory and autoimmune diseases, the leukocyte adhesion cascade in general, and leukocyte integrins in particular, represent key therapeutic targets. In this context, the present review focuses on the role of leukocyte integrins in the leukocyte adhesion cascade. Experimental evidence that has implicated leukocyte integrins as targets in animal models of inflammatory disorders, such as experimental autoimmune encephalomyelitis, psoriasis, inflammatory bone loss and inflammatory bowel disease as well as preclinical and clinical therapeutic applications of antibodies that target leukocyte integrins in various inflammatory disorders are presented. Finally, we review recent findings on endogenous inhibitors that modify leukocyte integrin function, which could emerge as promising therapeutic targets.
... In human embryonic kidney-293 (HEK-293) cells, AnxA1 interacts with FPR2/ALXR specifically. 118,119 AnxA1 is present in the nervous system and is involved in nociceptive sensation. 120,121 It has also analgesic effects in inflammatory pain through an interaction with FPR2/ ALXR. ...
The resolution of inflammation (RoI), once believed to be a passive process, has lately been shown to be an active and delicately orchestrated process. During the resolution phase of acute inflammation, novel mediators, including lipoxins and resolvins, which are members of the specialized pro-resolving mediators of inflammation, are produced. FPR2/ALXR, a receptor modulated by some of these lipids as well as by peptides (e.g., annexin A1), has been shown to be one of the receptors involved in the RoI. The aim of this perspective is to present the concept of the RoI from a medicinal chemistry point of view and to highlight the effort of the research community to discover and develop anti-inflammatory/pro-resolution small molecules to orchestrate inflammation by activation of FPR2/ALXR.
... The biological effects of ANXA1 and its cleavage product Ac2-26 peptide are mediated by formyl peptide receptors (FPRs) (2,17,18). In humans 3 FPRs (FPR1, FPR2, and FPR3) regulate innate inflammatory responses. ...
N-formyl peptide receptors (FPRs) are critical regulators of host defense in phagocytes and are also expressed in epithelia. FPR signaling and function have been extensively studied in phagocytes, yet their functional biology in epithelia is poorly understood. We describe a novel intestinal epithelial FPR signaling pathway that is activated by an endogenous FPR ligand, annexin A1 (ANXA1), and its cleavage product Ac2-26, which mediate activation of ROS by an epithelial NADPH oxidase, NOX1. We show that epithelial cell migration was regulated by this signaling cascade through oxidative inactivation of the regulatory phosphatases PTEN and PTP-PEST, with consequent activation of focal adhesion kinase (FAK) and paxillin. In vivo studies using intestinal epithelial specific Nox1-/-IEC and AnxA1-/- mice demonstrated defects in intestinal mucosal wound repair, while systemic administration of ANXA1 promoted wound recovery in a NOX1-dependent fashion. Additionally, increased ANXA1 expression was observed in the intestinal epithelium and infiltrating leukocytes in the mucosa of ulcerative colitis patients compared with normal intestinal mucosa. Our findings delineate a novel epithelial FPR1/NOX1-dependent redox signaling pathway that promotes mucosal wound repair.
... Yet, it remains to be seen whether such a lack of function would also be seen in knockouts for the AnxA1 receptors.5 43 As an example, in preliminary observations, mice nullified for FPR2 displayed higher arthritic score upon injection of the arthritogenic serum.44 ...
To establish the role and effect of glucocorticoids and the endogenous annexin A1 (AnxA1) pathway in inflammatory arthritis.
Ankle joint mRNA and protein expression of AnxA1 and its receptors were analysed in naive and arthritic mice by real-time PCR and immunohistochemistry. Inflammatory arthritis was induced with the K/BxN arthritogenic serum in AnxA1(+/+) and AnxA1(-/-) mice; in some experiments, animals were treated with dexamethasone (Dex) or with human recombinant AnxA1 or a protease-resistant mutant (termed SuperAnxA1). Readouts were arthritic score, disease incidence, paw oedema and histopathology, together with pro-inflammatory gene expression.
All elements of the AnxA1 pathway could be detected in naive joints, with augmentation during ongoing disease, due to the infiltration of immune cells. No difference in arthritis intensity of profile could be observed between AnxA1(+/+) and AnxA1(-/-) mice. Treatment of mice with Dex (10 µg intraperitoneally daily from day 2) afforded potent antiarthritic effects highly attenuated in the knockouts: macroscopic changes were mirrored by histopathological findings and pro-inflammatory gene (eg, Nos2) expression. Presence of proteinase 3 mRNA in the arthritic joints led the authors to test AnxA1 and the mutant SuperAnxA1 (1 µg intraperitoneally daily in both cases from day 2), with the latter one being able to accelerate the resolving phase of the disease.
AnxA1 is an endogenous determinant for the therapeutic efficacy of Dex in inflammatory arthritis. Such an effect can be partially mimicked by application of SuperAnxA1 which may represent the starting point for novel antiarthritic therapeutic strategies.
... This system has been better dissected for the polymorphonuclear leukocyte and a model for an autocrine/ paracrine circuit by which ANXA1 and its receptor control leukocyte activation and adhesion has been proposed (Perretti, 2003). However, the model is likely to be applicable to other cell systems, including macrophages (Maderna et al., 2005) and specialized epithelial cells Babbin et al., 2006). ...
Annexin A1 (ANXA1, lipocortin-1) is the first characterized member of the annexin superfamily of proteins, so called since their main property is to bind (i.e., to annex) to cellular membranes in a Ca(2+) -dependent manner. ANXA1 has been involved in a broad range of molecular and cellular processes, including anti-inflammatory signalling, kinase activities in signal transduction, maintenance of cytoskeleton and extracellular matrix integrity, tissue growth, apoptosis, and differentiation. New insights show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. This suggests that ANXA1 may contribute to the regeneration of skeletal muscle tissue and may have therapeutic implications with respect to the development of ANXA1 mimetics.
... We investigated the internalization of transiently expressed FPR2/ALX receptor in response to LXA 4 (1 nM) and Ac2-26 peptide (30 M), well-described agonists of the receptor with anti-inflammatory and proresolution activity (7,24,25,29,31,44). In unstimulated cells, FPR2/ALX was visualized primarily at the plasma membrane (Fig. 1A). ...
Lipoxins (LXs) are endogenously produced eicosanoids with well-described anti-inflammatory and proresolution activities, stimulating nonphlogistic phagocytosis of apoptotic cells by macrophages. LXA(4) and the glucocorticoid-derived annexin A1 peptide (Ac2-26) bind to a common G-protein-coupled receptor, termed FPR2/ALX. However, direct evidence of the involvement of FPR2/ALX in the anti-inflammatory and proresolution activity of LXA(4) is still to be investigated. Here we describe FPR2/ALX trafficking in response to LXA(4) and Ac2-26 stimulation. We have transfected cells with HA-tagged FPR2/ALX and studied receptor trafficking in unstimulated, LXA(4) (1-10 nM)- and Ac2-26 (30 μM)-treated cells using multiple approaches that include immunofluorescent confocal microscopy, immunogold labeling of cryosections, and ELISA and investigated receptor trafficking in agonist-stimulated phagocytosis. We conclude that PKC-dependent internalization of FPR2/ALX is required for phagocytosis. Using bone marrow-derived macrophages (BMDMs) from mice in which the FPR2/ALX ortholog Fpr2 had been deleted, we observed the nonredundant function for this receptor in LXA(4) and Ac2-26 stimulated phagocytosis of apoptotic neutrophils. LXA(4) stimulated phagocytosis 1.7-fold above basal (P<0.001) by BMDMs from wild-type mice, whereas no effect was found on BMDMs from Fpr2(-/-) mice. Similarly, Ac2-26 stimulates phagocytosis by BMDMs from wild-type mice 1.5-fold above basal (P<0.05). However, Ac2-26 failed to stimulate phagocytosis by BMDMs isolated from Fpr2(-/-) mice relative to vehicle. These data reveal novel and complex mechanisms of the FPR2/ALX receptor trafficking and functionality in the resolution of inflammation.
... 10 -12 GCs induce the Anx-A1 gene in many cells and also increase secretion of the protein from existing intracellular pools by stimulating protein kinase C (PKC) activity. 13 Once secreted, the protein acts in a paracrine/autocrine fashion, using formyl peptide receptors (FPRs) 14,15 to produce its biological effects. The N-terminal region of Anx-A1 bears the biological activity of the protein, and phosphorylation on the serine 27 residue is crucial for protein export and secretion. ...
To determine whether the inhibitory action of the antiallergic cromone "mast cell stabilizing" drugs on polymorphonuclear leukocyte (PMN) trafficking is mediated through an annexin-A1 (Anx-A1) dependent mechanism.
Intravital microscopy was used to monitor the actions of cromones in the inflamed microcirculation. Reperfusion injury provoked a dramatic increase in adherent and emigrated leukocytes in the mesenteric vascular bed, associated with augmented tissue levels of myeloperoxidase. Nedocromil, 2 to 20 mg/kg, significantly (P<0.05) inhibited cell adhesion and emigration, as well as myeloperoxidase release, in wild-type but not Anx-A1(-/-) mice. Short pretreatment of human PMNs with nedocromil, 10 nmol/L, inhibited cell adhesion (P<0.05) in the flow chamber assay, and this effect was reversed by specific anti-AnxA1 or a combination of antiformyl peptide receptors 1 and 2, but not irrelevant control, antibodies. Western blotting experiments revealed that cromones stimulate protein kinase C-dependent phosphorylation and release Anx-A1 in human PMNs.
We propose a novel mechanism to explain the antiinflammatory actions of cromones on PMN trafficking, an effect that has long puzzled investigators.
... The C-terminal domain of the annexins is highly conserved and mediates the calcium-regulated binding of the protein to membranes, whereas the unique N-terminal part defines the precise function of the protein. In annexin I, the N terminus is sensitive to cleavage by endogenous proteases (12,13), and peptides derived from this domain possess immunoregulatory effects (14). An N-terminally acetylated annexin I peptide corresponding to the sequence Gln 9 -Lys 25 has been shown to mediate both activating and inhibitory effects on neutrophil function (7,10). ...
The N-terminal part of the calcium-regulated and phospholipid-binding protein annexin AI contains peptide sequences with pro-
and anti-inflammatory activities. We have earlier shown that a proinflammatory signal triggered by one of these peptides,
Gln9–Lys25, is mediated by FPR1, a member of the formyl peptide receptor family expressed in human neutrophils. To determine the core
structure in Gln9–Lys25, smaller peptides were generated, and their capacity to activate neutrophils was determined. A peptide spanning from amino
acid Glu14 to Lys25 was inactive, whereas the activity was retained in the Gln9–Tyr20 peptide. Removal of amino acids from the C and N terminus of Gln9–Tyr20 revealed that the first amino acid (Gln9) was of the utmost importance for activity. The core structure that activated the neutrophil NADPH oxidase to release superoxide
anions was Gln9-Ala10-Trp11-Phe12. This peptide also inhibited the activity induced by N-formyl-Met-Leu-Phe and WKYMVM. A structural model of the peptide agonist-FPR1 complex suggests that the transmembrane part
of the binding pocket of the receptor binds optimally to a tetrapeptide. According to the model and the results presented,
the N-terminal amino acid glutamine in Gln9–Phe12 is located close to the bottom of the binding cleft, leaving for steric reasons insufficient space to extend the peptide
at the N terminus. The addition of amino acids at the C terminus will not affect binding. The model presented may be helpful
in developing specific FPR1 ligands.
... One potential marker of tumor progression is annexin A1 (ANXA1), a protein with diverse roles in apoptosis, proliferation, and epidermal growth factor receptor signaling. [4][5][6][7][8][9][10] Alterations in ANXA1 expression have been observed in a variety of tumors and the role of ANXA1 in carcinogenesis seems to be tissue specific. For instance, studies have shown increased ANXA1 expression in pancreatic, 11 hepatic, 12 glial, 13 and stomach cancers. ...
Annexin A1 (ANXA1) is a potential marker of development of breast cancer. However, previous studies of ANXA1 expression in primary breast carcinoma and lymph node metastasis have yielded conflicting results. Therefore, to accurately characterize the ANXA1 expression pattern, we used microarray analysis and matched patient samples to evaluate progressive alterations in ANXA1 protein expression during malignant transformation and metastasis.
We constructed a tissue microarray using 82 pairs of primary breast cancers and lymph node metastases from archival materials. We also identified 21 cases of breast carcinoma for which a single slide contained the entire progression from benign breast tissue, carcinoma in situ, to invasive carcinoma. Immunohistochemical staining for ANXA1 and various prognostic markers was performed.
Microarray analysis revealed that ANXA1 expression was lost in 79% of breast carcinomas, and there was no difference in ANXA1 expression between primary breast carcinoma and lymph node metastasis. Most ANXA1-negative tumors were positive for estrogen and progesterone receptors but negative for HER2/neu and epidermal growth factor receptor. In contrast, most ANXA1-positive tumors were negative for estrogen, progesterone, and HER2/neu. In the whole tissue sections, ANXA1 is heterogeneously expressed in benign epithelium and is lost in both in situ carcinoma and invasive carcinoma.
The lack of ANXA1 expression in the majority of breast carcinomas and the early loss of ANXA1 expression in in situ carcinoma, which is maintained in both invasive and metastatic tumors, suggests a possible role for ANXA1 in the early events of malignant transformation.
... This interaction is mediated via the N-terminal domain of annexin 1 (36). In fact, N-terminal peptides of annexin 1 can mimic anti-inflammatory activity of annexin 1 (reviewed in Ref. 37). Therefore, it is possible that phosphorylation of annexin 1 at Ser 5 can also modulate its interaction with formyl peptide receptor. ...
TRPM7 is an unusual bifunctional molecule consisting of a TRP ion channel fused to a protein kinase domain. It has been shown
that TRPM7 plays a key role in the regulation of intracellular magnesium homeostasis as well as in anoxic neuronal death.
TRPM7 channel has been characterized using electrophysiological techniques; however, the function of the kinase domain is
not known and endogenous substrates for the kinase have not been reported previously. Here we have identified annexin 1 as
a substrate for TRPM7 kinase. Phosphorylation of annexin 1 by TRPM7 kinase is stimulated by Ca2+ and is dramatically increased in extracts from cells overexpressing TRPM7. Phosphorylation of annexin 1 by TRPM7 kinase occurs
at a conserved serine residue (Ser5) located within the N-terminal amphipathic α-helix of annexin 1. The N-terminal region plays a crucial role in interaction
of annexin 1 with other proteins and membranes, and therefore, phosphorylation of annexin 1 at Ser5 by TRPM7 kinase may modulate function of annexin 1.
... Recently, the peptide Ac2-26 and annexin 1 have been shown to activate the lipoxin A 4 receptor, suggesting that annexin 1 peptide derivatives and lipoxin A 4 may act in concert at this G protein-coupled receptor to inhibit formation of mediators of inflammation and leukocyte recruitment (Perretti, 2003). Interestingly, the annexin 1/lipoxin A 4 receptor is also expressed in rats (Chiang et al., 2003), and several parameters of rat allergic pleurisy, including edema, eotaxin formation, and eosinophil accumulation, were inhibited by lipoxin A 4 stable analogs (Bandeira-Melo et al., 2000a,b). ...
Previous investigations have provided evidence that the N-terminal peptide of annexin 1 (peptide Ac2-26) has the capacity of reproducing the anti-inflammatory actions of the full-length protein in many systems. In the current study, we report the effectiveness of the peptide Ac2-26 as an antiallergic tool in a model of rat pleurisy and provide indication for some of the mechanisms involved. In rats inflamed by injection of ovalbumin into the pleural cavity 14 days postsensitization, peptide Ac2-26 (50-200 microg/cavity) inhibited mast cell degranulation, plasma protein leakage, and the accumulation of both neutrophils and eosinophils. Treatment with either peptide Ac2-26 (200 microg/cavity) or dexamethasone (1 mg/kg i.p.) inhibited ovalbumin-induced eotaxin release in the pleural effluents. In vitro, peptide Ac2-26 inhibited ovalbumin-evoked histamine release from subcutaneous tissue fragments obtained from sensitized rats (33-66 microM) and interleukin-13-evoked eotaxin generation from cultured rat mesothelial cells (16-33 microM) but not eosinophil chemotaxis. This work demonstrates that the annexin 1 mimetic peptide Ac2-26 prevents allergen-evoked eosinophilic inflammatory response in rats. Combined analysis of the in vivo and in vitro experiments presented herein suggests that the blockade of secretion of pivotal mediators for the allergic response, such as histamine and eotaxin, could be responsible for the inhibitory actions displayed by peptide Ac2-26.
... Glucocorticoids cause rapid serine phosphorylation and translocation of the protein to the cell surface where it appears to suppress the secretion of ACTH by acting on membranebound receptors. The nature of these receptors awaits definition, but data from studies on both the host defence and neuroendocrine systems suggest that members of the formyl peptide receptor family may be important in this regard (Perretti, 2003;John et al., 2004). ANXA1 may thus target S264 J.C. Buckingham ...
Well over 80 years ago Philip Smith described the beneficial clinical effects of adrenocortical extracts in animal models of adrenal insufficiency. In the ensuing years, scientists across the globe have sought to understand the mechanisms by which adrenal hormones and their synthetic analogues produce their complex and varied actions. Particular attention has focused on the glucocorticoids, partly because they have a vital place in the treatment of inflammatory and autoimmune disorders but also because dysregulation of the secretion and/or activity of endogenous glucocorticoids is increasingly implicated in a number of common disorders that pose a growing clinical burden, such as obesity, type II diabetes, the metabolic syndrome, hypertension and depression. This review considers some of the key advances that have been made in our understanding of the physiology, pathology and pharmacology of the glucocorticoids. Emphasis is placed on the molecular mechanisms of glucocorticoid signalling and the complex mechanisms that regulate the access of steroids in the systemic circulation to their receptors in their various target cells and tissues. In addition, consideration is given to the irreversible ‘organisational’ actions of glucocorticoids in perinatal life and to the potential role of the steroids in the aetiology of disease.
British Journal of Pharmacology (2006) 147, S258–S268. doi:10.1038/sj.bjp.0706456
For the longest time, the contribution of the immune system in chemotherapy has been disregarded as cytotoxic drugs are generally believed to be immunosuppressive [1, 2, 3, 4, 5]. Consequently, evaluation of new chemotherapeutic agents involved screening of drug candidates upon xenografted tumors in immunodeficient mice which neglects any possible immune contribution.
Inflammation is essential to protect the host from exogenous and endogenous dangers that ultimately lead to tissue injury. The consequent tissue repair is intimately associated with the fate of the inflammatory response. Restoration of tissue homeostasis is achieved through a balance between pro-inflammatory and anti-inflammatory/pro-resolving mediators. In chronic inflammatory diseases such balance is compromised resulting in persistent inflammation and impaired healing. During the last two decades the glucocorticoid-regulated protein Annexin A1 (AnxA1) has emerged as a potent pro-resolving mediator acting on several facets of the innate immune system. Here, we review the therapeutic effects of AnxA1 on tissue healing and repairing together with the molecular targets responsible for these complex biological properties.
Bacterial/mitochondrial fMLF analogs bind FPR1, driving accumulation/activation of PMN at sites of infection/injury, while promoting wound healing in epithelia. We quantified levels of UFPR1 and TFPR1 in isolated PMN by use of phosphosensitive NFPRb and phosphorylation-independent NFPRa antibodies. UFPR1 and total TFPR were assessed inflamed mucosa, observed in human IBD. In isolated PMN after fMLF stimulation, UFPR1 declined 70% ((fMLF)EC50 = 11 ± 1 nM; t1/2 = 15 s) and was stable for up to 4 h, whereas TFPR1 changed only slightly. Antagonists (tBoc-FLFLF, CsH) and metabolic inhibitor NaF prevented the fMLF-dependent UFPR1 decrease. Annexin A1 fragment Ac2-26 also induced decreases in UFPR1 ((Ac2-26)EC50 ∼ 3 µM). Proinflammatory agents (TNF-α, LPS), phosphatase inhibitor (okadaic acid), and G-protein activator (MST) modestly increased (fMLF)EC50, 2- to 4-fold, whereas PTX, Ca(2+) chelators (EGTA/BAPTA), H2O2, GM-CSF, ENA-78, IL-1RA, and LXA4 had no effect. Aggregation-inducing PAF, however, strongly inhibited fMLF-stimulated UFPR1 decreases. fMLF-driven PMN also demonstrated decreased UFPR1 after traversing monolayers of cultured intestinal epithelial cells, as did PMN in intestinal mucosal samples, demonstrating active inflammation from UC patients. Total TFPR remained high in PMN within inflamed crypts, migrating through crypt epithelium, and in the lamina propria-adjoining crypts, but UFPR1 was only observed at some peripheral sites on crypt aggregates. Loss of UFPR1 in PMN results from C-terminal S/T phosphorylation. Our results suggest G protein-insensitive, fMLF-dependent FPR1 phosphorylation in isolated suspension PMN, which may manifest in fMLF-driven transmigration and potentially, in actively inflamed tissues, except at minor discrete surface locations of PMN-containing crypt aggregates.
There is growing consensus that the clinical therapeutic efficacy of some chemotherapeutic agents depends on their off-target immune-modulating effects. Pt anticancer drugs have previously been identified to be potent immunomodulators of both the innate and the adaptive immune system. Nevertheless, there has been little development in the rational design of Pt-based chemotherapeutic agents to exploit their immune-activating capabilities. The FPR1/2 formyl peptide receptors are highly expressed in immune cells, as well as in many metastatic cancers. Herein, we report a rationally designed multimodal Pt(IV) prodrug containing a FPR1/2-targeting peptide that combines chemotherapy with immunotherapy to achieve therapeutic synergy and demonstrate the feasibility of this approach.
There is growing consensus that the clinical therapeutic efficacy of some chemotherapeutic agents depends on their off-target immune-modulating effects. Pt anticancer drugs have previously been identified to be potent immunomodulators of both the innate and the adaptive immune system. Nevertheless, there has been little development in the rational design of Pt-based chemotherapeutic agents to exploit their immune-activating capabilities. The FPR1/2 formyl peptide receptors are highly expressed in immune cells, as well as in many metastatic cancers. Herein, we report a rationally designed multimodal PtIV prodrug containing a FPR1/2-targeting peptide that combines chemotherapy with immunotherapy to achieve therapeutic synergy and demonstrate the feasibility of this approach.
Objective:
Wheezing is a common and challenging health issue in infancy and early childhood. Asthma diagnosis is frequent in patients with a history of recurrent wheezing. A relationship has been reported between asthma and anti-inflammatory mediators such as lipoxin A4 and annexin A1. However, this remains uncertain in wheezy infants. The aim of the present study was to determine lipoxin A4 and annexin A1 levels in wheezy infants.
Materials and methods:
Eighty-seven patients aged 6-36 months were included in this study. Demographic characteristics, clinical features, laboratory data, clinical diagnoses, and treatments, if present, were recorded. Patients were divided into 2 groups: patients with wheezing (n = 59) and healthy controls (n = 28). Blood samples were taken and lipoxin A4 and annexin A1 levels were evaluated by ELISA.
Results:
Lipoxin A4 and annexin A1 levels were significantly lower in the wheezing group than in the control group (p < 0.05). A significant correlation was found between the serum total immunoglobulin E (IgE) level and the percentage and absolute number of eosinophils (p < 0.05). No significant correlation was found in terms of lipoxin A4 and annexin A1 levels, the serum total IgE level, and the percentage and absolute number of eosinophils among groups (p > 0.05).
Conclusion:
This is the first study to assess lipoxin A4 and annexin A1 levels in wheezy infants. The levels of lipoxin A4 and annexin A1 were found to be low in wheezy infants. We hope that these results will lead to novel therapeutic options for asthma in cases where an optimal treatment modality is lacking.
Leukocytes accumulate at sites of inflammation and immunological reaction in response to locally existing chemotactic mediators. The first chemotactic factors structurally defined were N-formyl peptides. Subsequently, numerous ligands were identified to activate formyl peptide receptors (FPRs) that belong to the seven-transmembrane G protein-coupled receptor superfamily. FPRs interact with this menagerie of structurally diverse pro- and anti-inflammatory ligands to possess important regulatory effects in multiple diseases, including inflammation, amyloidosis, Alzheimer's disease, prion disease, acquired immunodeficiency syndrome, obesity, diabetes, and cancer. How these receptors recognize diverse ligands and how they contribute to disease pathogenesis and host defense are basic questions currently under investigation that would open up new avenues for the future management of inflammation-related diseases.
It is increasingly apparent that effective host defence involves biphasic production of mediators. An initial acute response
involves leukocyte activation and recruitment, a second phase is characterised by the production of mediators regulating phagocytic
clearance of apoptotic cells and the active suppression of the initial inflammatory response [1]–[10]. Eicosanoid production in inflammation tightly regulates these processes. During the initial phase, proinflammatory mediators
including leukotriene (LT) B4, the cysteinyl LTs and prostaglandins (PG) evoke potent chemotactic responses of leukocytes whose activation is coupled to
the production of proinflammatory (Th1-derived cytokines) at sites of inflammation [11]. To facilitate resolution, a second phase of lipid mediators may be produced favouring agents with “pro-resolution activities”,
including lipoxins (LXs) and the more recently described resolvins and protectins [5, 12–20].
It is now evident that several endogenous anti-inflammatory pathways are activated in parallel with the host inflammatory
response to maintain a homeostatic control. From this idea has arisen the concept of anti-inflammation, a term used to describe
the balance that exists between pro-inflammatory and anti-inflammatory mediators/pathways that operate in concert to initiate,
maintain and finally resolve the inflammatory reaction.
Annexin 1 (ANXA1) has analgesic effects in inflammatory pain. We aimed to investigate the anti-nociceptive role of ANXA1, at the dorsal root ganglion (DRG) level, through an interaction with formyl-peptide-receptor-like 1 (FPR2/ALX).
Inflammatory pain was evoked by injecting complete Freund's adjuvant (CFA, 50 μl) into the hindpaw of male Sprague-Dawley rats. The distribution of ANXA1 and FPR2/ALX in L4/5 DRGs was evaluated by immunofluorescence. The expression of ANXA1 was measured by western blot. The involvement of FPR2/ALX in the anti-nociception of ANXA1 was investigated by thermal (irradiant heat) and mechanical (von Frey filament) pain tests with intrathecal (i.t.) ANXA1-derived peptide (Anxa1(2-26)), FPR2/ALX agonist 5(S)-6(R)-7-trihydroxyheptanoic-acid-methyl-ester (BML-111), and antagonist N-t-Boc-Phe-Leu-Phe-Leu-Phe (Boc1).
ANXA1 and FPR2/ALX localized in the satellite glial cells and neurones in L4/5 DRGs. CFA treatment (n=20) increased ANXA1 expression in L4/5 DRGs within 7 days (P<0.01). I.T. Anxa1(2-26) (20 and 100 µg µl(-1)) and BML-111 (10 and 100 nmol) reduced CFA-induced thermal and mechanical nociception within 48 h (n=40) (P<0.05). However, i.t. Boc1 10 µg intensified inflammatory pain (P<0.05) and reversed the anti-nociceptive effect of Anxa1(2-26) (n=25) (P<0.05). Moreover, ANXA1 expression increased in L4/5 DRGs after i.t. Anxa1(2-26) (20 µg µl(-1)) (P<0.05) and BML-111 (10 nmol) (P<0.01) but decreased after i.t. Boc1 (10 and 100 µg) alone (P<0.01) or Boc1 (10 µg) co-injection with Anxa1(2-26) (20 µg µl(-1)) (P<0.05).
Endogenous ANXA1 expression at the DRG level is involved in CFA-induced inflammatory pain, and i.t. ANXA1 20 µg µl(-1) produces its anti-nociceptive effect through FPR2/ALX.
Annexin 1 is a 37-kDa protein that has complex intra- and extracellular effects. To discover whether the absence of this protein alters bone development, we monitored this event in the annexin-A1 null mice in comparison with littermate wild-type controls.
Radiographic and densitometry methods were used for the assessment of bone in annexin-A1 null mice at a gross level. We used whole-skeleton staining, histological analysis, and Western blotting techniques to monitor changes at the tissue and cellular levels.
There were no gross differences in the appendicular skeleton between the genotypes, but an anomalous development of the skull was observed in the annexin-A1 null mice. This was characterized in the newborn annexin-A1 null animals by a delayed intramembranous ossification of the skull, incomplete fusion of the interfrontal suture and palatine bone, and the presence of an abnormal suture structure. The annexin-A1 gene was shown to be active in osteocytes during this phase and COX-2 was abundantly expressed in cartilage and bone taken from annexin-A1 null mice.
Expression of the annexin-A1 gene is important for the normal development of the skull in mice, possibly through the regulation of osteoblast differentiation and a secondary effect on the expression of components of the cPLA2-COX-2 system.
To investigate the effects of exogenous annexin-1 (ANXA1) on lipopolysaccharide(LPS)-induced proliferation, reactive oxygen species (ROS) production, and calcium signal transduction in RAW264.7 macrophages.
RAW264.7 macrophages were treated with or without LPS in the absence or presence of ANXA1. The proliferation effects were detected by Cell Counting Kit-8 assay. ROS were quantified by flow cytometry and fluorescence microscopy. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was analyzed by laser confocal scanning microscopy. IkappaBalpha degradation and NF-kappaB translocation were tested by Western blot.
Exogenous ANXA1 inhibited LPS-induced proliferation and ROS production in a dose-dependent manner. LPS evoked [Ca(2+)](i) increase through CRAC channels, and ANXA1 suppressed LPS-induced [Ca(2+)](i) increase in a dose-dependent manner. The CRAC channels were associated with LPS-induced proliferation and ROS production. Exogenous ANXA1 had no effect on LPS-induced IkappaB degradation and NF-kappaB translocation.
ANXA1 inhibited LPS-induced proliferation and ROS production in RAW264.7 macrophages partially through modulation of CRAC channels but independent of the NF-kappaB pathway.
The purpose of this study was twofold: to reveal cellular events associated with the protective role of endogenous annexin A1 (AnxA1) in inflammation and to highlight the potential involvement of members of the formyl peptide receptor (Fpr) family in this process. We found that wild-type, AnxA1-null, and Fpr1-null mice all displayed an intense neutrophil recruitment into the peritoneal cavity as assessed 4 hours after carrageenin injection, and that this recruitment was most pronounced in AnxA1-null mice. In addition, this cell influx could be inhibited by the AnxA1 pharmacophore peptide, Ac2-26, in wild-type, AnxA1-null, and Fpr1-null mice, but was restored when co-treated with the pan-receptor antagonist Boc2. Using the LacZ gene reporter assay, an enhancement of AnxA1 gene promoter activity in extravasated neutrophils was evident in AnxA1-null mice; again this response was reduced after peptide treatment. The lack of functional involvement of Fpr1 prompted us to monitor the structurally related receptor Fpr2. We report, for the first time, the ultrastructural immunocytochemical co-localization of Fpr2 with AnxA1 in neutrophils that migrate into the mesenteric microcirculation and extravasate into the peritoneal fluid. Collectively, these data provide in vivo support to the hypothesis that endogenous AnxA1 is an essential effector of endogenous anti-inflammation and provide an ultrastructural indication that this mediator interacts with Fpr2 in murine neutrophils. We believe that these findings could significantly affect the development of novel therapeutics, which are modeled after the anti-migratory actions of AnxA1.
Inflammation is the response of vascularized tissues to injury, irritation and infection. Nearly always, the inflammatory response is successfully resolved and, when necessary, a process of wound healing is initiated. Nowhere in the body is this homeostatic process more challenging than in the gastrointestinal (GI) tract, where the microbial flora sits in very close proximity to the mucosal immune system, separated only by an epithelial cell barrier. Delicate regulatory systems of the mucosal immune system determine mucosal permeability and response to bacterial flora, and aberrations in this system result in acute or chronic inflammatory conditions. Examples of such are two commonly occurring inflammatory GI disorders: inflammatory bowel disease and postoperative ileus. Inflammatory bowel disease is the result of a chronic and excessive mucosal immune response, whereas postoperative ileus represents a transient condition of GI tract paralysis that is the result of an inflammatory response to abdominal surgery. The clinical management of both conditions is very challenging and depends heavily on the possibility of modulating the host immune response. In this brief report, we highlight the role of neuropeptides in GI physiology and immune regulation, discuss a recently discovered endogenous anti-inflammatory pathway mediated by the ChemR23 receptor and speculate on the therapeutic potential of peptides that bind G-protein-coupled receptors in the management of inflammation in the GI tract.
Dendritic cells play a key role in the adaptive immune system by influencing T-cell differentiation. Annexin-1 (Anx-A1) has recently been shown to modulate the adaptive immune response by regulating T-cell activation and differentiation. Here we investigated the role of endogenous Anx-A1 in dendritic cells as major cellular counterpart of T-cell-driven immune response. We found that Anx-A1(-/-) bone marrow-derived dendritic cells show an increased number of CD11c(+) cells expressing high levels of some maturation markers, such as CD40, CD54, and CD80, coupled to a decreased capacity to take up antigen compared to control Anx-A1(+/+) cells. However, analysis of LPS-treated dendritic cells from Anx-A1(-/-) mice demonstrated a diminished up-regulation of maturation markers, a decreased migratory activity in vivo, and an attenuated production of the inflammatory cytokines interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and IL-12. This defect was also accompanied by impaired nuclear factor (NF)-kappaB/DNA-binding activity and lack of Anx-A1 signaling, as demonstrated by the reduced activation of extracellular-signal regulated kinase (ERK)1/2 and Akt compared to cells from control littermates. As a consequence of this phenotype, Anx-A1(-/-) dendritic cells showed an impaired capacity to stimulate T-cell proliferation and differentiation in mixed leukocyte reaction. Together, these findings suggest that inhibition of Anx-A1 expression or function in dendritic cells might represent a useful way to modulate the adaptive immune response and pathogen-induced T-cell-driven immune diseases.
The essential fatty acid eicosapentaenoic acid (EPA) present in fish oils displays beneficial effects in a range of human disorders associated with inflammation including cardiovascular disease. Resolvin E1 (RvE1), a new bioactive oxygenated product of EPA, was identified in human plasma and prepared by total organic synthesis. Results of bioaction and physical matching studies indicate that the complete structure of RvE1 is 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-EPA. At nanomolar levels, RvE1 dramatically reduced dermal inflammation, peritonitis, dendritic cell (DC) migration, and interleukin (IL) 12 production. We screened receptors and identified one, denoted earlier as ChemR23, that mediates RvE1 signal to attenuate nuclear factor-kappaB. Specific binding of RvE1 to this receptor was confirmed using synthetic [(3)H]-labeled RvE1. Treatment of DCs with small interference RNA specific for ChemR23 sharply reduced RvE1 regulation of IL-12. These results demonstrate novel counterregulatory responses in inflammation initiated via RvE1 receptor activation that provide the first evidence for EPA-derived potent endogenous agonists of antiinflammation.
The inflammatory response is a life-saving protective process mounted by the body to overcome pathogen infection and injury; however, in chronic inflammatory pathologies, this response can become deregulated. Aspirin and glucocorticoids are two examples of drugs developed over the years to rectify deregulated inflammatory reactions. Interestingly, both these prototypes of anti-inflammatory therapeutics have been 'borrowed' from Mother Nature, identified from the plant and animal world, respectively. In the past century, systematic organic chemistry has been the major approach for producing new drugs, and vast quantities of aspirin and prednisolone have been synthesized, packaged and sold. However, the fascination provoked by these often life-saving drugs has not subsided, and recent work into the endogenous control of the host inflammatory response has revitalized these compounds. Thus, epi-lipoxins, produced after aspirin acetylation of inducible cyclooxygenase-2, and glucocorticoid-regulated annexin 1 appear to be important endogenous mediators of their respective anti-inflammatory effects. In addition, aspirin-triggered epi-lipoxins and glucocorticoid-regulated annexin 1 might act on the same G-protein-coupled receptor, thus rendering this shared receptor a more likely and worthwhile target for fruitful drug discovery.
We have tested the effects of annexin 1 (ANXA1) and its N-terminal peptide Ac2-26 on polymorphonuclear leukocyte (PMN) recruitment under flow. Differential effects of the full-length protein and its peptide were observed; ANXA1 inhibited firm adhesion of human PMNs, while Ac2-26 significantly attenuated capture and rolling without effect on firm adhesion. Analysis of the effects of ANXA1 and Ac2-26 on PMN adhesion molecule expression supported the flow chamber results, with Ac2-26 but not ANXA1 causing l-selectin and PSGL-1 shedding. ANXA1 and its peptide act via the FPR family of receptors. This was corroborated using HEK-293 cells transfected with FPR or FPRL-1/ALX (the 2 members of this family expressed by human PMNs). While Ac2-26 bound both FPR and FPRL-1/ALX, ANXA1 bound FPRL-1/ALX only. ANXA1 and Ac2-26 acted as genuine agonists; Ac2-26 binding led to ERK activation in both FPR- and FPRL-1/ALX-transfected cells, while ANXA1 caused ERK activation only in cells transfected with FPRL-1/ALX. Finally, blockade of FPRL-1/ALX with a neutralizing monoclonal antibody was found to abrogate the effects of ANXA1 in the flow chamber but was without effect on Ac2-26-mediated inhibition of rolling. These findings demonstrate for the first time distinct mechanisms of action for ANXA1 and its N-terminal peptide Ac2-26.
Lipocortin 1 (annexin 1) is a calcium- and phospholipid-binding protein that modulates anti-inflammatory responses including those induced by lipopolysaccharide. To investigate the precise role of lipocortin 1 in regulating the lipopolysaccharide-induced signal transduction pathways, we generated stable RAW 264.7 macrophage cell lines expressing decreased and increased lipocortin 1 protein. Several RAW 264.7 clones with increased lipocortin 1 protein levels showed constitutive activation of the mitogen-activated protein kinase extracellular signal-regulated kinase, which was down-regulated following lipopolysaccharide treatment. Conversely, clones with decreased lipocortin 1 protein expression showed prolonged extracellular signal-regulated kinase activity, following lipopolysaccharide activation. Lipocortin 1 specifically regulates the components of the extracellular signal-regulated kinase pathway, since changes in lipocortin 1 protein expression had no affect on the related mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Lipocortin 1 modulated upstream components of the extracellular signal-regulated kinase pathway and associated with the adaptor protein growth factor binding protein. The downstream consequences of altered extracellular signal-regulated kinase activity were independent of the proinflammatory transcription factor nuclear factor kappa B. These data indicate that lipocortin 1 specifically regulates proximal signaling components of the extracellular signal-regulated kinase signal transduction pathway, resulting in the modulation of biochemical functions in RAW macrophages.
The velocity of rolling leukocytes is thought to be determined by the expression of adhesion molecules and the prevailing wall shear stress. Here, we investigate whether rapid cleavage of L-selectin may be an additional physiologic regulatory parameter of leukocyte rolling. A unique protease in the membrane of leukocytes cleaves L-selectin after activation, resulting in L-selectin shedding. The hydroxamic acid–based metalloprotease inhibitor KD-IX-73-4 completely prevented L-selectin shedding in vitro and significantly decreased the rolling velocity of leukocytes in untreated wild-type C57BL/6 mice from 55 to 35 μm/s in vivo. When E-selectin was expressed on the endothelium (tumor necrosis factor [TNF]-α treatment 2.5–3 h before the experiment), rolling velocity was 4 μm/s and did not change after the application of KD-IX-73-4. However, KD-IX-73-4 decreased mean rolling velocity by 29% from 23 to 16 μm/s in E-selectin–deficient mice treated with TNF-α. The reduction of velocity caused by KD-IX-73-4 was immediate (<5 s) after injection of KD-IX-73-4 as shown by a novel method using a local catheter. These results establish a role for L-selectin shedding in regulating leukocyte rolling velocity in vivo.
Lipoxin A4 stimulates rapid lipid remodeling and a pertussis toxin-sensitive release of arachidonic acid in polymorphonuclear leukocytes (PMN) (Nigam, S., Fiore, S., Luscinskas, F.W., and Serhan, C.N. (1990) J. Cell. Physiol. 143, 512-523) and has been shown to inhibit leukocyte responses in several systems. To examine the basis underlying these actions, we have prepared [11,12-3H]lipoxin A4 (LXA4) and characterized its interactions with human PMN. Time course studies (0-90 min) with intact PMN demonstrated cell association of 3H label which was specific and reversible. PMN bound [3H]LXA4 with a Kd of 0.5 +/- 0.3 nM, representing approximately 1,830 sites/PMN, and the Hill plot value of 1.9 suggests cooperative binding. [3H]LXA4 binding was stereoselective since neither leukotriene B4 (LTB4), lipoxin B4 (LXB4), (6S)-LXA4, 11-trans-LXA4, nor SKF 104353 competed for [3H]LXA4-specific binding while LTD4 and LTC4 partially competed. Subcellular fractionation revealed that specific binding with [3H]LXA4 was associated with membrane (42.1%)-, granule (34.5%)-, and nuclear (23.3%)-enriched fractions, a distribution distinct from that of [14,15-3H] LTB4 binding. [11,12-3H]LXA4-specific binding was modulated by guanosine analogs, suggesting the involvement of G proteins. A fluorescent LXA4 derivative (methyl-7-methoxycoumarin-LXA4) competed with [3H]LXA4 binding to intact PMN and showed specific and reversible binding as monitored by flow cytometric analysis. These results indicate that PMN possess specific recognition sites for LXA4 which may mediate its actions.
Transfection of either the C5a receptor or the formyl peptide receptor into undifferentiated U937 cells generated continuously growing cell lines that stably expressed these receptors. The transfected cells displayed significant numbers of cell surface receptors that had ligand binding properties similar to fully differentiated U937 cells. Undifferentiated transfected U937 cells were capable of a ligand-specific calcium flux and showed migratory responses that were qualitatively and quantitatively similar to differentiated cells and were specific for each chemoattractant. Moreover, the chemotactic response could be desensitized by preincubating the cells in a high concentration of ligand and could be blocked by pertussis toxin. These results demonstrate that undifferentiated U937 cells possess the subcellular signaling apparatus and machinery necessary to generate a motile response and that the only missing component for chemotaxis is expression of a chemoattractant receptor. In addition, the results demonstrate that undifferentiated U937 cells transfected with chemoattractant receptors provide a defined model system to study receptor structure/function relationships and may be used to investigate receptor-mediated chemotactic responses in a relevant human myeloid cell.
Lipoxins are bioactive eicosanoids that are immunomodulators. In human myeloid cells, lipoxin (LX) A4 actions are mediated by interaction with a G protein-coupled receptor. To explore functions of LXA4 and aspirin-triggered 5(S),6(R),15(R)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (15-epi-LXA4) in vivo, we cloned and characterized a mouse LXA4 receptor (LXA4R). When expressed in Chinese hamster ovary cells, the mouse LXA4R showed specific binding to [3H]LXA4 (K(d) approximately 1.5 nM), and with LXA4 activated GTP hydrolysis. Mouse LXA4R mRNA was most abundant in neutrophils. In addition to LXA4 and 15-epi-LXA4, bioactive LX stable analogues competed with both [3H]LXA4 and [3H]leukotriene D4 (LTD4)-specific binding in vitro to neutrophils and endothelial cells, respectively. Topical application of LXA4 analogues and novel aspirin-triggered 15-epi-LXA4 stable analogues to mouse ears markedly inhibited neutrophil infiltration in vivo as assessed by both light microscopy and reduced myeloperoxidase activity in skin biopsies. The 15(R)-16-phenoxy-17,18, 19,20-tetranor-LXA4 methyl ester (15-epi-16-phenoxy-LXA4), an analogue of aspirin triggered 15-epi-LXA4, and 15(S)-16-phenoxy-17,18,19,20-tetranor-LXA4 methyl ester (16-phenoxy-LXA4) were each as potent as equimolar applications of the anti-inflammatory, dexamethasone. Thus, we identified murine LXA4R, which is highly expressed on murine neutrophils, and showed that both LXA4 and 15-epi-LXA4 stable analogues inhibit neutrophil infiltration in the mouse ear model of inflammation. These findings provide direct in vivo evidence for an anti-inflammatory action for both aspirin-triggered LXA4 and LXA4 stable analogues and their site of action in vivo.
In this study we investigated, using intravital microscopy, how neutrophil extravasation across mouse mesenteric postcapillary venules is inhibited by the glucocorticoid-regulated protein lipocortin (LC; also termed annexin) 1. Intraperitoneal injection of 1 mg of zymosan into mice induced neutrophil rolling on the activated mesenteric endothelium followed by adhesion (maximal at 2 hr: 5-6 cells per 100-micrometers of vessel length) and emigration (maximal at 4 hr: 8-10 cells per high-powered field). Treatment of mice with human recombinant LC1 (2 mg/kg s.c.) or its mimetic peptide Ac2-26 (13 mg/kg s.c.) did not modify cell rolling but markedly reduced (>/=50%) the degree of neutrophil adhesion and emigration (P < 0.05). Intravenous treatment with peptide Ac2-26 (13 mg/kg) or recombinant human LC1 (0.7-2 mg/kg) promoted detachment of neutrophils adherent to the endothelium 2 hr after zymosan administration, with adherent cells detaching within 4.12 +/- 0.75 min and 2.36 +/- 0.31 min, respectively (n = 20-25 cells). Recruitment of newly adherent cells to the endothelium was unaffected. The structurally related protein LC5 was inactive in this assay, whereas a chimeric molecule constructed from the N terminus of LC1 (49 aa) attached to the core region of LC5 produced cell detachment with kinetics similar to LC1. Removal of adherent neutrophils from activated postcapillary endothelium is a novel pharmacological action, and it is at this site where LC1 and its mimetics operate to down-regulate this aspect of the host inflammatory response.
We have previously reported (Badolato, R., J.M. Wang, W.J. Murphy, A. R. Lloyd, D.F. Michiel, L.L. Bausserman, D.J. Kelvin, and J.J. Oppenheim. 1994. J. Exp. Med. 180:203; Xu, L., R. Badolato, W.J. Murphy, D.L. Longo, M. Anver, S. Hale, J.J. Oppenheim, and J.M. Wang. 1995. J. Immunol. 155:1184.) that the acute phase protein serum amyloid A (SAA) is a potent chemoattractant for human leukocytes in vitro and mouse phagocytes in vivo. To identify the signaling mechanisms, we evaluated patterns of cross-desensitization between SAA and other leukocyte chemoattractants. We found that the chemotactic bacterial peptide, N-formyl- methionyl-leucyl-phenylalanine (fMLP), was able to specifically attenuate Ca2+ mobilization in human phagocytes induced by SAA, but only at very high concentrations, suggesting that SAA uses a low affinity fMLP receptor. Here we demonstrate that SAA selectively induced Ca2+ mobilization and migration of HEK cells expressing FPRL1, a human seven-transmembrane domain phagocyte receptor with low affinity for fMLP, and high affinity for lipoxin A4. Furthermore, radiolabeled SAA specifically bound to human phagocytes and FPRL1-transfected 293 cells. In contrast, SAA was not a ligand or agonist for FPR, the high affinity fMLP receptor. Thus, SAA is the first chemotactic ligand identified for FPRL1. Our results suggest that FPRL1 mediates phagocyte migration in response to SAA.
N-formylpeptides derive from bacterial and mitochondrial proteins, and bind to specific receptors on mammalian phagocytes. Since binding induces chemotaxis and activation of phagocytes in vitro, it has been postulated that N-formylpeptide receptor signaling in vivo may be important in antimicrobial host defense, although direct proof has been lacking. Here we test this hypothesis in mice lacking the high affinity N-formylpeptide receptor (FPR), created by targeted gene disruption. FPR-/- mice developed normally, but had increased susceptibility to challenge with Listeria monocytogenes, as measured by increased mortality compared with wild-type littermates. FPR-/- mice also had increased bacterial load in spleen and liver 2 d after infection, which is before development of a specific cellular immune response, suggesting a defect in innate immunity. Consistent with this, neutrophil chemotaxis in vitro and neutrophil mobilization into peripheral blood in vivo in response to the prototype N-formylpeptide fMLF (formyl-methionyl-leucyl-phenylalanine) were both absent in FPR-/- mice. These results indicate that FPR functions in antibacterial host defense in vivo.
It is of wide interest to understand how opposing extracellular signals (positive or negative) are translated into intracellular signaling events. Receptor-ligand interactions initiate the generation of bioactive lipids by human neutrophils (PMN), which serve as signals to orchestrate cellular responses important in host defense and inflammation. We recently identified a novel polyisoprenyl phosphate (PIPP) signaling pathway and found that one of its components, presqualene diphosphate (PSDP), is a potent negative intracellular signal in PMN that regulates superoxide anion generation by several stimuli, including phosphatidic acid. We determined intracellular PIPP signaling by autocoids with opposing actions on PMN: leukotriene B4 (LTB4), a potent chemoattractant, and lipoxin A4 (LXA4), a 'stop signal' for recruitment. LTB4 receptor activation initiated a rapid decrease in PSDP levels concurrent with activation of PLD and cellular responses. In sharp contrast, activation of the LXA4 receptor reversed LTB4-initiated PSDP remodeling, leading to an accumulation of PSDP and potent inhibition of both PLD and superoxide anion generation. Thus, an inverse relationship was established for PSDP levels and PLD activity with two PMN ligands that evoke opposing responses. In addition, PSDP directly inhibited both isolated human recombinant (Ki = 6 nM) and plant (Ki = 20 nM) PLD. Together, these findings link PIPP remodeling to intracellular regulation of PMN function and suggest a role for PIPPs as lipid repressors in signal transduction, a novel mechanism that may also explain aspirin's suppressive actions in vivo in cell signaling.
A D-methionine-containing peptide, Trp-Lys-Tyr-Met-Val-D-Met-NH(2) (WKYMVm), featuring a unique receptor specificity was investigated with respect to its ability to activate neutrophil effector functions. The peptide was found to be more potent than the N-formylated peptide N-formyl-Met-Leu-Phe (fMLF) at inducing neutrophil chemotaxis, mobilization of neutrophil complement receptor 3 (CR3), and activation of the neutrophil NADPH-oxidase. The fact that binding of fML[(3)H]F was inhibited by both fMLF and WKYMVm suggests that N-formyl peptide receptor (FPR) is shared by these peptides. However, the neutrophil response induced by the WKYMVm peptide was insensitive to the fMLF antagonists, cyclosporin H, and Boc-FLFLF that specifically block the function of the FPR. These results suggest that even though WKYMVm may bind FPR the cells are activated preferentially through a receptor distinct from the FPR. Using transfected HL-60 cells expressing either the FPR or its neutrophil homologue FPRL1, also referred to as LXA(4)R because it has been shown to bind lipoxin A(4), we show that WKYMVm is about 300-fold more active at mobilizing intracellular calcium through FPRL1 than through FPR. The WKYMVm activates FPRL1-expressing cells in a cyclosporin H-independent manner with an EC(50 )of around 75 pmol/L, whereas it activates FPR-expressing cells with an EC(50 )of around 25 nmol/L. The observation that exudated cells are primed in their response to WKYMVm suggests that FPRL1/LXA(4)R like FPR is stored in mobilizable organelles. (Blood. 2000;95:1810-1818)
Lipoxin (LX) A(4) and aspirin-triggered LX (ATL) are endogenous lipids that regulate leukocyte trafficking via specific LXA(4) receptors (ALXRs) and mediate antiinflammation and resolution. ATL analogues dramatically inhibited human neutrophil (polymorphonuclear leukocyte [PMN]) responses evoked by a potent necrotactic peptide derived from mitochondria as well as a rogue synthetic chemotactic peptide. These bioactive lipid analogues and small peptides each selectively competed for specific (3)H-LXA(4) binding with recombinant human ALXR, and its N-glycosylation proved essential for peptide but not LXA(4) recognition. Chimeric receptors constructed from receptors with opposing functions, namely ALXR and leukotriene B(4) receptors (BLTs), revealed that the seventh transmembrane segment and adjacent regions of ALXR are essential for LXA(4) recognition, and additional regions of ALXR are required for high affinity binding of the peptide ligands. Together, these findings are the first to indicate that a single seven-transmembrane receptor can switch recognition as well as function with certain chemotactic peptides to inhibitory with ATL and LX (lipid ligands). Moreover, they suggest that ALXR activation by LX or ATL can protect the host from potentially deleterious PMN responses associated with innate immunity as well as direct effector responses in tissue injury by recognition of peptide fragments.
Annexin 1 (ANX1), a calcium-binding protein, participates in the regulation of early inflammatory responses. Whereas some of its effects depend on intracellular interactions, a growing number of observations indicate that ANX1 may also act via autocrine/paracrine functions following externalization to the outer side of the plasma membrane. We studied the effects of ANX1 on leukocyte adhesion to endothelial cells using as a model system the monocytic cell line U937 and human bone marrow microvascular endothelial cells. Exogenous rANX1, as well as endogenous ANX1 externalized by U937 differentiated in vitro, inhibited monocyte firm adhesion to vascular endothelium. Both binding of ANX1 to U937 cells and ANX1-mediated inhibition of cell adhesion involved the short N-terminal domain of the ANX1 molecule. Under experimental conditions in which ANX1 inhibited U937 adhesion to human bone marrow microvascular endothelial cells, this protein specifically colocalized with the alpha 4 integrin, and a direct interaction between ANX1 and the alpha 4 integrin could be documented by immunoprecipitation experiments. Moreover, ANX1 competed with the endothelial integrin counterreceptor, VCAM-1, for binding to alpha 4 integrin. These results indicate that ANX1 plays an important physiological role in modulating monocyte firm adhesion to the endothelium.
Formyl peptides are potent neutrophil chemoattractants. In humans and rabbits, the formyl peptide receptor (FPR) binds N-formyl-Met-Leu-Phe (fMLF) with high affinity (K(d) approximately 1 nM). The mouse FPR (mFPR) is a low-affinity receptor for fMLF (K(d) approximately 100 nM); therefore, other agonists for this receptor may exist. Using mFPR-transfected rat basophilic leukemia cells, we found that a recently identified synthetic peptide Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) is a potent agonist for mFPR. WKYMVm induced calcium mobilization with an EC(50) of 1.2-1.5 nM. Optimal chemotaxis was achieved with 1 nM of WKYMVm, but it required 100 nM of fMLF. WKYMVm stimulated rapid and potent phosphorylation of the mitogen-activated protein kinases extracellular signal-related kinases 1 and 2 when used at 50 nM. Pertussis toxin only partially blocked calcium mobilization and production of inositol 1,4,5-trisphosphate in the stimulated mFPR cells, suggesting the possibility that this receptor couples to Galpha proteins other than Gi and Go. Competitive binding and desensitization data suggest that both peptides interact with the same receptor but may use nonoverlapping binding sites because WKYMVm was unable to effectively displace [(3)H]fMLF bound to mFPR. These results provide evidence for the presence of an alternative potent agonist for mFPR, and suggest a potential usage of WKYMVm for probing the ligand-receptor interactions with the murine formyl peptide receptor homologs.
We recently showed that a class of novel carboxylated N:-glycans was constitutively expressed on endothelial cells. Activated, but not resting, neutrophils expressed binding sites for the novel glycans. We also showed that a mAb against these novel glycans (mAbGB3.1) inhibited leukocyte extravasation in a murine model of peritoneal inflammation. To identify molecules that mediated these interactions, we isolated binding proteins from bovine lung by their differential affinity for carboxylated or neutralized glycans. Two leukocyte calcium-binding proteins that bound in a carboxylate-dependent manner were identified as S100A8 and annexin I. An intact N terminus of annexin I and heteromeric assembly of S100A8 with S100A9 (another member of the S100 family) appeared necessary for this interaction. A mAb to S100A9 blocked neutrophil binding to immobilized carboxylated glycans. Purified human S100A8/A9 complex and recombinant human annexin I showed carboxylate-dependent binding to immobilized bovine lung carboxylated glycans and recognized a subset of mannose-labeled endothelial glycoproteins immunoprecipitated by mAbGB3.1. Saturable binding of S100A8/A9 complex to endothelial cells was also blocked by mAbGB3.1. These results suggest that the carboxylated glycans play important roles in leukocyte trafficking by interacting with proteins known to modulate extravasation.
Neutrophils express the G protein-coupled N-formyl peptide receptor (FPR) and its homologue FPRL1, whereas monocytes express FPR, FPRL1, and FPRL2, an orphan receptor
sharing 83% amino acid identity with FPRL1. FPRL1 is a promiscuous receptor activated by serum amyloid A and by different
synthetic peptides, including the hexapeptide Trp-Lys-Tyr-Met-Val-d-Met-NH2 (WKYMVm). By measuring calcium flux in HL-60 cells transfected with FPR, FPRL1, or FPRL2, we show that WKYMVm activated all
three receptors, whereas thel-conformer WKYMVM activated exclusively FPRL1 and FPRL2. The functionality of FPRL2 was further assessed by the ability of
HL-60-FPRL2 cells to migrate toward nanomolar concentrations of hexapeptides. The half-maximal effective concentrations of
WKYMVM for calcium mobilization in HL-60-FPRL1 and HL-60-FPRL2 cells were 2 and 80 nm, respectively. Those of WKYMVm were 75 pmand 3 nm. The tritiated peptide WK[3,5-3H2]YMVM bound to FPRL1 (K
D ∼ 160 nm), but not to FPR. The two conformers similarly inhibited binding of 125I-labeled WKYMVm to FPRL2-expressing cells (IC50 ∼ 2.5–3 μm). Metabolic labeling with orthophosphoric acid revealed that FPRL1 was differentially phosphorylated upon addition of the
l- or d-conformer, indicating that it induced different conformational changes. In contrast to FPRL1, FPRL2 was already phosphorylated
in the absence of agonist and not evenly distributed in the plasma membrane of unstimulated cells. However, both receptors
were internalized upon addition of either of the two conformers. Taken together, the results indicate that neutrophils are
activated by WKYMVM through FPRL1 and that FPRL2 is a chemotactic receptor transducing signals in myeloid cells.
Aspirin-triggered 15-epi-lipoxin A4 (ATL) is an endogenous lipid mediator that mimics the actions of native lipoxin A4, a putative "stop signal" involved in regulating resolution of inflammation. A metabolically more stable analog of ATL, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A4 analog (ATLa), inhibits neutrophil recruitment in vitro and in vivo and displays potent anti-inflammatory actions. ATLa binds with high affinity to the lipoxin A4 receptor, a G protein-coupled receptor on the surface of leukocytes. In this study, we used freshly isolated human neutrophils to examine ATLa's potential for initiating rapid nuclear responses. Using differential display reverse transcription polymerase chain reaction, we identified a subset of genes that was selectively up-regulated upon short exposure of polymorphonuclear leukocytes to ATLa but not to the chemoattractant leukotriene B4 or vehicle alone. We further investigated ATLa regulation of one of the genes, NAB1, a transcriptional corepressor identified previously as a glucocorticoid-responsive gene in hamster smooth muscle cells. Treatment of human neutrophils with pertussis toxin blocked ATLa up-regulation of NAB1. In addition, ATLa stimulated NAB1 gene expression in murine lung vascular smooth muscle in vivo. These findings provide evidence for rapid transcriptional induction of a cassette of genes via an ATLa-stimulated G protein-coupled receptor pathway that is potentially protective and overlaps with the anti-inflammatory glucocorticoid regulatory circuit.
Efficacy has been defined in receptor pharmacology as a proportionality factor denoting the amount of physiological response a given ligand imparts to a biological system for a given amount of receptor occupancy. While first defined in terms of response, the concept can be expanded to a wide variety of G protein-coupled receptor (GPCR) behaviors, which includes pleiotropic interaction with multiple G proteins, internalization, oligomerization, desensitization, and interaction with membrane auxilliary proteins. Thus, there can be numerous types of efficacy, and different ligands can have a range of efficacies for different receptor behaviors. This review discusses the use of the efficacy concept in GPCR models based on the thermodynamic linkage theory and also in terms of the protein ensemble theory, in which macroaffinity of ligands for an ensemble of receptor microstates produces a new ligand-bound ensemble. The pharmacological characteristics of the ligand emerge from the intersection of the ligand-bound ensemble with the various ensembles defining pharmacological receptor behaviors. Receptor behaviors discussed are activation of G proteins; ability to be phosphorylated, desensitized, and internalized; formation of dimers and oligomers; and the interaction with auxiliary membrane and cytosolic proteins. The concepts of ligand-specific receptor conformation and conditional efficacy are also discussed in the context of ligand control of physiological response.
Mutations at the Nramp1 gene cause susceptibility to infections with intracellular pathogens. In human blood, polymorphonuclear (PMN) leukocytes are the most abundant site of NRAMP1 messenger RNA (mRNA) expression, suggesting that NRAMP1 plays an important role in the activity of these cells. By Northern blot analysis, NRAMP1 mRNA was only detected in most mature neutrophils from bone marrow (band and segmented cells). A high-affinity polyclonal rabbit antihuman NRAMP1 antibody directed against the amino terminus of the protein was produced and used to study cellular and subcellular localization of the protein in primary human neutrophils. Subcellular fractionation of granule populations together with immunoblotting studies with granule-specific markers indicate that NRAMP1 expression is primarily in tertiary granules. These granules are positive for the matrix enzyme gelatinase and the membrane subunit of the vacuolar H(+)/ATPase and can be recruited for exocytosis by treatment of neutrophils with phorbol myristate acetate. Immunogold studies by cryoelectron microscopy with primary neutrophils confirm that a majority (75%) of NRAMP1-positive granules are also positive for gelatinase, but they also suggest further heterogeneity in this granule population. Presence of NRAMP1 in tertiary granules is in agreement with the late-stage appearance of NRAMP1 mRNA during neutrophil maturation in bone marrow. Finally, immunofluorescence studies of Candida albicans-containing phagosomes formed in neutrophils indicate that NRAMP1 is recruited from tertiary granules to the phagosomal membrane on phagocytosis, supporting a role for NRAMP1 in the antimicrobial defenses of human neutrophils.
We have examined in whole blood the actions of 2 lipoxin A4 (LXA4) stable analogs, 15-R/S-methyl-LXA4 and 16-phenoxy-LXA4, for their impact on the expression of adhesion molecules on human leukocytes and coronary artery endothelial cells (HCAEC) and on neutrophil adhesion to HCAEC in vitro. Both LXA4 analogs in nanomolar to micromolar concentrations prevented shedding of L-selectin and downregulated CD11/CD18 expression on resting neutrophils, monocytes, and lymphocytes. Changes in CD11/CD18 expression were blocked by the mitogen-activated protein kinase kinase inhibitor PD98059. The LXA4 analogs also attenuated changes in L-selectin and CD11/CD18 expression evoked by platelet-activating factor (PAF), interleukin-8, or C-reactive protein-derived peptide 201-206 with IC50 values of 0.2 to 1.9 μmol/L, whereas they did not affect lipopolysaccharide (LPS)– or tumor necrosis factor-–stimulated expression of E-selectin and intercellular adhesion molecule-1 on HCAEC. These LXA4analogs markedly diminished adhesion of neutrophils to LPS-activated HCAEC. Inhibition of adhesion was additive with function blocking anti–E-selectin and anti–L-selectin antibodies, but was not additive with anti-CD18 antibody. Combining LXA4 analogs with dexamethasone (100 nmol/L) almost completely inhibited PAF-induced changes in adhesion molecule expression on leukocytes and gave additive inhibition of neutrophil adhesion to HCAEC. Culture of HCAEC with dexamethasone, but not with LXA4 analogs, also decreased neutrophil attachment. Together, these results indicate that LXA4 stable analogs modulate expression of both L-selectin and CD11/CD18 on resting and immunostimulated leukocytes and inhibit neutrophil adhesion to HCAEC by attenuating CD11/CD18 expression. These actions are additive with those of glucocorticoids and may represent a novel and potent regulatory mechanism by which LXA4 and aspirin-triggered 15-epi-LXA4 modulate leukocyte trafficking.
The glucocorticoid-regulated protein annexin I (lipocortin I) has been shown to mediate antiinflammatory activities of glucocorticoids, but the molecular basis of its action has remained elusive. Here we show that annexin I acts through the formyl peptide receptor (FPR) on human neutrophils. Peptides derived from the unique N-terminal domain of annexin I serve as FPR ligands and trigger different signaling pathways in a dose-dependent manner. Lower peptide concentrations possibly found in inflammatory situations elicit Ca2+ transients without fully activating the MAP kinase pathway. This causes a specific inhibition of the transendothelial migration of neutrophils and a desensitization of neutrophils toward a chemoattractant challenge. These findings identify annexin I peptides as novel, endogenous FPR ligands and establish a mechanistic basis of annexin I–mediated antiinflammatory effects.
British Journal of Pharmacology (1999) 126, 537–550; doi:10.1038/sj.bjp.0702328
Annexin I, a member of the calcium- and phospholipid-binding annexin superfamily of proteins, is largely present in human neutrophils. To determine its exact intracellular distribution a combination of flow cytometry, confocal microscopy and electron microscopy analyses were performed on resting human neutrophils as well as on cells which had been activated. In resting neutrophils, annexin I was found to be present in small amounts in the nucleus, in the cytoplasm and partially also associated with the plasma membrane. The cytoplasmic pool of annexin I was predominant, and the protein was co-localized with gelatinase (marker of gelatinase granules), but not with human serum albumin or CD35 (markers of secretory vesicles), or with lysosomes. Electron microscopy showed the presence of annexin I inside the gelatinase granules. Neutrophil adhesion to monolayers of endothelial cells, but not phagocytosis of particles of opsonized zymosan, provoked an intense mobilization of annexin I, with a marked externalization on the outer leaflet of the plasma membrane. Remaining intracellular annexin I was also found in proximity of the plasma membrane. These results provide a novel mechanism for annexin I secretion from human neutrophils, which is via a degranulation event involving gelatinase granules.
A multi-faceted approach was used to investigate the effect of an anti-inflammatory peptide derived from human lipocortin 1 N-terminus region (amino acid 2–26; termed human Ac2–26) on human neutrophil activation in vitro. When incubated with purified human neutrophils. human Ac2–26 produced a concentration-dependent inhibition of elastase release stimulated by formyl-Met-Leu-Phe (fMLP), platelet-activating factor, or leukotriene B4, with an approximate EC50 of 33 μM (100 μg/ml). At this concentration, human Ac2–26 also inhibited (77%) the release of [3H]-arachidonic acid from neutrophils stimulated with fMLP. The peptide, however, did not inhibit the up-regulation of the β2-integrin CD11b and the concomitant shedding of L-selectin from neutrophil plasma membrane induced by fMLP. In adhesion experiments, human Ac2–26 inhibited neutrophil adhesion to endothelial monolayers when this was stimulated with fMLP, but not when this followed endothelial cell activation with histamine or platelet-activating factor. Again, the effect of the peptide was concentration-dependent, and an approximate EC50 of 33 μM was calculated. When a preparation of 125I-labeled human Ac2–26 was incubated with the neutrophils, the peptide was internalised in an energy-dependent fashion. All together, these observations lead us to propose a model in which this peptide derived from the N-terminus of human lipocortin 1 alters a common cellular mechanism producing a selective inhibition of neutrophil activation.
N-formyl peptides, such as fMet-Leu-Phe, are one of the most potent chemoattractants for phagocytic leukocytes. The interaction of N-formyl peptides with their specific cell surface receptors has been studied extensively and used as a model system for the characterization of G-protein-coupled signal transduction in phagocytes. The cloning of the N-formyl peptide receptor cDNA from several species and the identification of homologous genes have allowed detailed studies of structural and functional aspects of the receptor. Recent findings that the receptor is expressed in nonhematopoietic cells and that nonformylated peptides can activate the receptor suggest potentially novel functions and the existence of additional ligands for this receptor.
The formylpeptide receptor (FPR), previously found only on polymorphonuclear leukocytes and monocytes/macrophages, responds to both synthetic N-formyl oligopeptides and those produced by bacteria. The cDNA for human FPR has been cloned and a rabbit polyclonal antiserum directed against a synthetic 11-amino-acid peptide corresponding to the deduced carboxy-terminus has been produced. We have now extensively characterized and used the antibody to detect FPR on normal human tissues and cell types. The receptor antigen is present on some epithelial cells, especially those with a secretory function, and on some endocrine cells, e.g., follicular cells of the thyroid and cortical cells of the adrenal. Liver hepatocytes and Kupffer cells are positive. Smooth muscle and endothelial cells are also generally positive. In the brain and spinal cord, the neurons of the motor, sensory, and cerebellar systems, and those of the parasympathetic and sympathetic systems stain positively. These data suggest that the putative endogenous agonist for FPR or an antigenically similar receptor reacts with cellular targets in the neuromuscular, vascular, endocrine, and immune systems.
Significant future developments in the effective treatment of inflammatory diseases may arise from non-toxic dual inhibitors of both cyclooxygenase and lipoxygenase pathways in the arachidonate cascade. Inhibition of phospholipase A2(PLA2)(EC3.1.1.4), may provide such a dual action and recent research has concentrated on the role of PLA2-inhibitory proteins as possible anti-inflammatory agents. Blastokinin or uteroglobin is a steroid-induced rabbit secretory protein with PLA2-inhibitory activity. Its biochemical and biological properties have been extensively studied and its crystallographic structure has been resolved at 1.34 A (refs 15, 16). Lipocortins are a family of related proteins, which, it has been suggested, mediate the anti-inflammatory effects of glucocorticoids (for a review, see ref. 23). Some proteins of this group have been purified and the complementary DNA sequences of two human lipocortins are known. Lipocortins inhibit PLA2 in vitro, although their mechanism of action is still unclear. Recombinant lipocortin I inhibits eicosanoid synthesis in isolated perfused lungs from the guinea pig. Here, we report that synthetic oligopeptides corresponding to a region of high amino-acid sequence similarity between uteroglobin and lipocortin I have potent PLA2 inhibitory activity in vitro and striking anti-inflammatory effects in vivo.
The glucocorticoids inhibit our 'defence reactions' at many levels. One way in which they achieve this is by inhibiting the synthesis of chemicals involved in the promotion of the inflammatory response. The production of many mediators involved in the response to infection, injury, haemorrhage or metabolic disturbances are under glucocorticoid control such that elevated levels of hormone in the blood suppresses their formation. In many cases the action of these mediators is blocked as well. It might be thought that the glucocorticoids act simply by decreasing the synthesis rate of these protein regulators of inflammation such as the lymphokines, or of the enzymes which make prostaglandins. Whilst this undoubtedly does occur, another mechanism is also employed: that is, the glucocorticoid-induced synthesis of inhitibory proteins. Lipocortin (and possibly other related proteins) then is a sort of 'second messenger' of the glucocorticoids. It is only one of many such regulatory proteins but it is an important one, controlling as it does the mediators which promote development of the symptoms of the inflammatory response. It is undoubtedly the significant component of the inbuilt mechanism for terminating the inflammatory response which the physician exploits, for when he gives his patients relatively large doses of steroids to control an inflammatory response, he is in reality increasing the synthesis of these 'second mesenger' proteins such as lipocortin to a near maximum. All the early studies on lipocortin were performed in vitro, that is under conditions in which steroids were not normally present. Under these circumstances the generation and appearance of lipocortin seemed absolutely dependent upon the presence of glucocorticoids in the perfusing medium. These findings have led some to the erroneous notion that lipocortin was only present following treatment with exogenous steroids. Of course, all healthy mammals have circulating glucocorticoids and thus it is more rational to expect that lipocortin is a normal constituent of plasma and tissues (as indeed it appears to be), although the amount present in the cells can be increased by raising the concentration of endogenous or exogenous steroids. There has been a corresponding change in our appreciation of the function of lipocortin. Originally, it was regarded mainly as an 'anti-inflammatory protein' but today it seems more likely that this protein is present in most cells, and that its function is to control phospholipase A2 activity and to allow lipid hydrolysis only under strictly defined circumstances. This reversible inhibitory function of lipocortin could well be controlled by the phosphorylation and dephosphorylation cycle described above. Naturally, during inflammation, phospholipase is substantially activated and thus there is a requirement for a greater than normal supply of the inhibitory protein, hence the relationship between the rate of synthesis and the release of steroid hormones. The discovery, characterisation, islation, sequencing and cloning of lipocortin has opened up an entirely new and exciting chapter in cell biology and also holds out a strong promise for the future of anti-inflammatory therapy. In addition to their beneficial clinical effects, steroids produce a wide spectrum of side effects which preclude the use of these drugs for long periods of time except in the very seriously ill. These side effects are caused by changes in the transcription of specific genes in the same way as the anti-inflammatory effects. It has long been an article of a faith of scientists working in this are that if we could identify and isolate the 'second messengers' of steroid action that are responsible for the anti-inflammatory effects, it should be possible to produce drugs which possess many of the beneficial action of steroids without incurring the heavy penalty of side effects. The real value of this work is that it enables us to take our first stpes in that direction.
This study investigates the effect of dexamethasone on leukocyte extravasation in the post-capillary venules of the hamster cheek pouch, using an intravital microscopy technique, and seeks to clarify the potential involvement of the steroid-inducible protein lipocortin 1. Topical application of FMLP (10 nmol), or substance P (10 nmol), to the superfused cheek pouch induced at the level of the post-capillary venules the three characteristic phenomena of leukocyte rolling, adhesion, and transmigration. Pretreatment of hamsters with an anti-inflammatory dose of dexamethasone (1 mg/kg) increased lipocortin 1 levels in circulating leukocytes as assessed by flow cytometry, but did not modify either leukocyte rolling or the number of adherent cells; however approximately 65% of the adherent leukocytes subsequently detached and returned to the blood stream, whereas those that entered into the diapedesis process exhibited a long latency (approximately three- to fourfold longer than in control animals) before transmigration. In hamsters passively immunized with a polyclonal anti-lipocortin 1 serum, leukocyte diapedesis started at similar times in both control and dexamethasone-treated animals, whereas a significant prolongation was observed in those animals treated with a non-immune sheep serum. These observations indicate that 1) lipocortin 1 is elevated in circulating leukocytes following dexamethasone treatment; 2) the step of leukocyte extravasation affected by dexamethasone in the actual transmigration process, and 3) this specific effect upon leukocyte diapedesis is mediated by endogenous lipocortin 1.
Polymorphonuclear leukocyte (PMN) migration into sites of inflammation is fundamental to the host defense response. Activation of endothelial cells and PMNs increases the expression or activation of adhesion molecules, culminating in rolling and subsequent adherence of these cells to the vascular wall. Further activation of adherent PMNs, possibly by endothelial cell ligands, leads, within a few minutes, to extravasation itself. This process is not clearly understood, but adhesion molecules or related proteins, as well as endogenous chemokines, may play an important role. The anti-inflammatory glucocorticoids delay extravasation, which implies that an inhibitory regulatory system exists. Resting PMNs contain abundant cytoplasmic lipocortin 1 (LC1, also called annexin I)', and the activity profile of this protein suggests that it could reduce PMN responsiveness. To investigate this we have assessed neutrophil transmigration both in vivo and in vitro and examined the content and subcellular distribution of LC1 in PMNs by fluorescence-activated cell-sorting (FACS) analysis, western blotting and confocal microscopy. We report that LC1 is mobilized and externalized following PMN adhesion to endothelial monolayers in vitro or to venular endothelium in vivo and that the end point of this process is a negative regulation of PMN transendothelial passage.
Leukocyte extravasation occurs in many pathophysiological conditions, including inflammation, neoplasia and asthma. In recent years many studies have elucidated the steps that promote the initial interaction between extravasating cells and endothelium of the post-capillary venule; the sequential role of several classes of adhesion molecules (cell-specific chemokines) and activators (multipotent cytokines) is well established. In this review, Mauro Perretti focuses on a less well investigated mechanism by which the host downregulates extravasation at the leukocyte-endothelium interface. The neutrophilic polymorphonuclear leukocyte is used as the prime example of a leukocyte that interacts with the endothelium, and particular emphasis is given to the possibility that novel anti-inflammatory therapies might be developed from a better understanding of the inhibitory mechanisms activated by endogenous mediators such as adenosine, lipocortin 1, NO, prostacyclin and cathepsin G.
The human formylpeptide receptor (FPR) gene cluster has three members: FPR1 and FPRL1, which are expressed in neutrophils and monocytes and encode seven-transmembrane-domain chemotactic receptors specific for N-formylpeptides, and FPRL2, whose function is unknown. The FPRL1 receptor is also a lipoxin A4 receptor. Using probes for the three human genes we have cloned six distinct mouse genes, designated Fpr1 and Fpr-rs1 through Fpr-rs5, which form a cluster on chromosome 17 in a region of conserved synteny with human chromosome 19. Fpr1 encodes a functional receptor and is clearly the orthologue of FPR1. Both Fpr-rs1 and Fpr-rs2 have higher sequence homology to FPRL1 than to FPRL2; Fpr-rs1 is 97% identical in amino acid sequence to a previously reported cDNA that encodes a lipoxin A4 receptor, whereas the putative ligand for Fpr-rs2 is unknown. Fpr-rs3, Fpr-rs4, and Fpr-rs5 appear to lack human counterparts and are most similar in sequence to FPRL1. RNA for Fpr1, Fpr-rs1, and Fpr-rs2 is present in leukocytes, spleen, and lung, whereas RNA for Fpr-rs3 was detected only in skeletal muscle. We did not detect Fpr-rs4 or Fpr-rs5 RNA in any tissue tested. Moreover, Fpr-rs5 has a stop codon in the protein-coding region corresponding to transmembrane domain VI and may not encode a functional receptor. These results suggest that the FPR gene cluster has undergone differential expansion in mammals with FPRL2, Fpr-rs2, Fpr-rs3, Fpr-rs4, and Fpr-rs5 arising after divergence of human and mouse.
Migration of blood-derived leukocytes to tissue sites of inflammation is a hallmark of the response that the host organizes to counteract an insult or a trauma or an infection. A cascade of events is then activated to allow interaction between the leukocyte and the endothelium of postcapillary venule, and this cascade is finely regulated such that mechanisms of negative control are operating side by side with pathways that promote and sustain the extravasation process. Examples of both these positive and negative regulatory systems are discussed here.
Annexin I is an endogenous antiinflammatory mediator, expressed in rheumatoid arthritis (RA) synovium, the contribution of which to autoregulation of the synovial inflammatory response has not been examined in models of RA. We investigated the antiinflammatory role of annexin I in rat adjuvant arthritis.
Rats with adjuvant-induced arthritis (AIA) were treated with a specific anti-annexin I monoclonal antibody (mAb), isotype control IgG, and/or dexamethasone. Clinical outcomes and synovial synthesis of tumor necrosis factor alpha (TNFalpha), prostaglandin E2 (PGE2), and nitric oxide were examined, and annexin I expression was assessed by flow cytometry and reverse transcription-polymerase chain reaction.
Anti-annexin I mAb reversed the effects of dexamethasone on the clinical features of AIA and exacerbated AIA in the absence of exogenous glucocorticoid. Clinical exacerbation of AIA by anti-annexin I mAb was accompanied by significantly increased synovial TNFalpha and PGE2, suggesting that annexin I tonically inhibits the production of these mediators. Anti-annexin I mAb treatment was associated with significantly reduced leukocyte intracellular annexin I, despite increased annexin I messenger RNA expression, consistent with a depletion effect of extracellular mAb via the cell surface.
Annexin I is a key endogenous inhibitory mediator of arthritis via mechanisms that include inhibition of cytokine and effector molecule production. Moreover, a synthesis-independent depletion of intracellular annexin I by extracellular antibody supports the hypothesis that externalization of annexin I is involved in its mode of action.
We have examined in whole blood the actions of 2 lipoxin A(4) (LXA(4)) stable analogs, 15-R/S-methyl-LXA(4) and 16-phenoxy-LXA(4), for their impact on the expression of adhesion molecules on human leukocytes and coronary artery endothelial cells (HCAEC) and on neutrophil adhesion to HCAEC in vitro. Both LXA(4) analogs in nanomolar to micromolar concentrations prevented shedding of L-selectin and downregulated CD11/CD18 expression on resting neutrophils, monocytes, and lymphocytes. Changes in CD11/CD18 expression were blocked by the mitogen-activated protein kinase kinase inhibitor PD98059. The LXA(4) analogs also attenuated changes in L-selectin and CD11/CD18 expression evoked by platelet-activating factor (PAF), interleukin-8, or C-reactive protein-derived peptide 201-206 with IC(50) values of 0.2 to 1.9 micromol/L, whereas they did not affect lipopolysaccharide (LPS)- or tumor necrosis factor-alpha-stimulated expression of E-selectin and intercellular adhesion molecule-1 on HCAEC. These LXA(4) analogs markedly diminished adhesion of neutrophils to LPS-activated HCAEC. Inhibition of adhesion was additive with function blocking anti-E-selectin and anti-L-selectin antibodies, but was not additive with anti-CD18 antibody. Combining LXA(4) analogs with dexamethasone (100 nmol/L) almost completely inhibited PAF-induced changes in adhesion molecule expression on leukocytes and gave additive inhibition of neutrophil adhesion to HCAEC. Culture of HCAEC with dexamethasone, but not with LXA(4) analogs, also decreased neutrophil attachment. Together, these results indicate that LXA(4) stable analogs modulate expression of both L-selectin and CD11/CD18 on resting and immunostimulated leukocytes and inhibit neutrophil adhesion to HCAEC by attenuating CD11/CD18 expression. These actions are additive with those of glucocorticoids and may represent a novel and potent regulatory mechanism by which LXA(4) and aspirin-triggered 15-epi-LXA(4) modulate leukocyte trafficking.
Antiflammin-1 and antiflammin-2 are nonapeptides corresponding to the region of highest similarity between glucocorticoid-inducible proteins lipocortin-1 and uteroglobin. We have studied whether antiflammins could affect expression of adhesion molecules on human leukocytes and coronary artery endothelial cells (HCAEC) and binding of neutrophils (PMNs) to HCAEC. Although neither antiflammin-1 nor antiflammin-2 affected expression of adhesion molecules on resting PMNs, monocytes, and lymphocytes in whole blood, they attenuated changes in L-selectin and CD11/CD18 expression evoked by platelet-activating factor or interleukin-8 with IC(50) values of 4-20 micromol/l. The maximum inhibition was similar to those seen with human recombinant lipocortin-1 (100 microgram/ml). Unlike dexamethasone (100 nmol/l), the antiflammins had little effect on LPS-stimulated expression of E-selectin and ICAM-1 on HCAEC. Consistently, culture of HCAEC with dexamethasone, but not with antiflammins, decreased PMN binding to endothelial cells. Preincubation of PMNs with antiflammins markedly decreased their adhesion to LPS-activated HCAEC. Inhibition of adhesion was additive with function blocking anti-E-selectin and anti-L-selectin antibodies, but was not additive with anti-CD18 antibody. These results show that antiflammins inhibit PMN adhesion to HCAEC by attenuating activation-induced up-regulation of CD11/CD18 expression on leukocytes, and suggest that antiflammins may represent a novel therapeutic approach in blocking leukocyte trafficking in host defense and inflammation.
The glucocorticoid-regulated protein annexin I (lipocortin I) has been shown to mediate antiinflammatory activities of glucocorticoids, but the molecular basis of its action has remained elusive. Here we show that annexin I acts through the formyl peptide receptor (FPR) on human neutrophils. Peptides derived from the unique N-terminal domain of annexin I serve as FPR ligands and trigger different signaling pathways in a dose-dependent manner. Lower peptide concentrations possibly found in inflammatory situations elicit Ca2+ transients without fully activating the MAP kinase pathway. This causes a specific inhibition of the transendothelial migration of neutrophils and a desensitization of neutrophils toward a chemoattractant challenge. These findings identify annexin I peptides as novel, endogenous FPR ligands and establish a mechanistic basis of annexin I-mediated antiinflammatory effects.
L-selectin has been shown to be important in mediating leukocyte recruitment during inflammatory responses. Although there are numerous in vitro studies demonstrating that engagement of L-selectin leads to the activation of several signaling pathways potentially contributing to subsequent adhesion, emigration, or even migration through the interstitium, whether this actually induces cellular events in vivo is completely unknown. Therefore, we used intravital microscopy to visualize the role of L-selectin in downstream leukocyte adhesion, emigration, and interstitial migration events in wild-type and L-selectin-deficient (L-selectin(-/-)) mice. The cremaster muscle was superfused with the chemotactic inflammatory mediators platelet-activating factor or KC. Leukocyte rolling, adhesion, and emigration in postcapillary venules were examined, and the migration of emigrated leukocytes was recorded continuously using time-lapse videomicroscopy. Platelet-activating factor increased leukocyte adhesion to a similar level in both wild-type and L-selectin(-/-) mice. In contrast, both the number of emigrated leukocytes and the distance of extravascular migration were significantly reduced in L-selectin(-/-) mice. A similar pattern was observed in response to the superfusion of KC. Because superfusion of these mediators induced chemokinesis, we developed a new in vivo chemotaxis assay using slow release of KC from an agarose gel positioned 350 microm from a postcapillary venule. These experiments showed that L-selectin(-/-) leukocytes were also severely impaired in their ability to respond to a directional cue. These findings indicate that L-selectin is important in enabling leukocytes to respond effectively to chemotactic stimuli in inflamed tissues.
Aspirin-triggered lipoxin A(4) (ATL, 15-epi-LXA(4)) and leukotriene D(4) (LTD(4)) possess opposing vascular actions mediated via receptors distinct from the LXA(4) receptor (ALX) that is involved in leukocyte trafficking. Here, we identified these receptors by nucleotide sequencing and demonstrate that LTD(4) receptor (CysLT(1)) is induced in human vascular endothelia by interleukin-1beta. Recombinant CysLT(1) receptor gave stereospecific binding with both [(3)H]-LTD(4) and a novel labeled mimetic of ATL ([(3)H]-ATLa) that was displaced with LTD(4) and ATLa ( approximately IC(50) 0.2 to 0.9 nmol/L), but not with a bioinactive ATL isomer. The clinically used CysLT(1) receptor antagonist, Singulair, showed a lower rank order for competition with [(3)H]-ATLa (IC(50) approximately 8.3 nmol/L). In contrast, LTD(4) was an ineffective competitive ligand for recombinant ALX receptor with [(3)H]-ATLa, and ATLa did not compete for [(3)H]-LTB(4) binding with recombinant LTB(4) receptor. Endogenous murine CysLT(1) receptors also gave specific [(3)H]-ATLa binding that was displaced with essentially equal affinity by LTD(4) or ATLa. Systemic ATLa proved to be a potent inhibitor (>50%) of CysLT(1)-mediated vascular leakage in murine skin (200 microg/kg) in addition to its ability to block polymorphonuclear leukocyte recruitment to dorsal air pouch (4 microg/kg). These results indicate that ATL and LTD(4) bind and compete with equal affinity at CysLT(1), providing a molecular basis for aspirin-triggered LXs serving as a local damper of both vascular CysLT(1) signals as well as ALX receptor-regulated polymorphonuclear leukocyte traffic.
Annexin 1 (ANX-A1) exerts antimigratory actions in several models of acute and chronic inflammation. This is related to its ability to mimic the effect of endogenous ANX-A1 that is externalized on neutrophil adhesion to the postcapillary endothelium. In the present study we monitored ANX-A1 expression and localization in intravascular and emigrated neutrophils, using a classical model of rat peritonitis. For this purpose, a pair of antibodies raised against the ANX-A1 N-terminus (ie, able to recognize intact ANX-A1) or the whole protein (ie, able to interact with all ANX-A1 isoforms) was used by immunofluorescence and immunocytochemistry analyses. The majority ( approximately 50%) of ANX-A1 on the plasma membrane of intravascular neutrophils was intact. Extravasation into the subendothelial matrix caused loss of this pool of intact protein (to approximately 6%), concomitant with an increase in total amount of the protein; only approximately 25% of the total protein was now recognized by the antibody raised against the N-terminus (ie, it was intact). In the cytoplasm of these cells, ANX-A1 was predominantly associated with large vacuoles, possibly endosomes. In situ hybridization confirmed de novo synthesis of ANX-A1 in the extravasated cells. In conclusion, biochemical pathways leading to the externalization, proteolysis, and synthesis of ANX-A1 are activated during the process of neutrophil extravasation.
Glucocorticoids can dampen inflammatory responses by inhibiting neutrophil recruitment to tissue sites. The detailed mechanism by which glucocorticoids exert this affect on neutrophils is unknown. L-selectin is a leukocyte cell surface receptor that is implicated in several steps of neutrophil recruitment. Recently, several studies have shown that systemic treatment of animals and humans with glucocorticoids induces decreased L-selectin expression on neutrophils, suggesting one mechanism by which inflammation may be negatively regulated. However, when neutrophils are treated in vitro with glucocorticoids, no effect on L-selectin expression is observed. Thus, the existence of an additional mediator is plausible. In this study, we investigate whether annexin 1 (ANX1), a recognized second messenger of glucocorticoids, could be such a mediator. We show that ANX1 induces a dose- and time-dependent decrease in L-selectin expression on both peripheral blood neutrophils and monocytes but has no effect on lymphocytes. The loss of L-selectin from neutrophils is due to shedding that is mediated by a cell surface metalloprotease ("sheddase"). Using cell shape and a beta(2) integrin activation epitope, we show that the ANX1-induced shedding of L-selectin appears to occur without overt cell activation. These data may provide the basis for further understanding of mechanisms involved in the down-regulation of inflammatory responses.
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.
Lipoxins (LXs) are endogenously produced eicosanoids typically generated during cell-cell interactions. In this article, the compelling evidence from in vitro and in vivo model systems that LXs actively promote the resolution of inflammation is reviewed. Of particular interest are indications that stable synthetic analogues of LXs and aspirin-triggered 15-epi-LXs can mimic many of the desirable anti-inflammatory, "pro-resolution" actions of native LXs. Given the enhanced stability and efficacy of these compounds a role as novel anti-inflammatory therapeutics is proposed.
This study exploited established immunoneutralization protocols and an N-terminal annexin 1 peptide (annexin 1Ac2 – 26) to advance our knowledge of the role of annexin 1 as a mediator of acute glucocorticoid action in the rat neuroendocrine system in vivo.
Rats were treated with corticosterone (500 μg kg−1, i.p.) or annexin 1Ac2 – 26 (0.1 – 10 ng rat−1, i.c.v.) and 75 min later with interleukin 1β (IL-1β, 10 ng rat−1, i.c.v. or 500 μg kg−1, i.p). Blood was collected 1 h later for hormone immunoassay. Where appropriate, anti-annexin 1 polyclonal antiserum (pAb) was administered subcutaneously or centrally prior to the steroid challenge.
Corticosterone did not affect the resting plasma corticotrophin (ACTH) concentration but suppressed the hypersecretion of ACTH induced by IL-1β (i.p. or i.c.v.). Its actions were quenched by anti-annexin 1 pAb (s.c. or i.c.v) and mimicked by annexin 1Ac2 – 26.
By contrast, corticosterone provoked an increase in serum growth hormone (GH) which was ablated by central but not peripheral administration of anti-annexin 1 pAb. IL-1β (i.c.v. or i.p.) did not affect basal GH but, when given centrally but not peripherally, it abolished the corticosterone-induced hypersecretion of GH. Annexin 1Ac2 – 26 (i.c.v.) also produced an increase in serum GH which was prevented by central injection of IL-1β.
The results support the hypothesis that the acute regulatory actions of glucocorticoids on hypothalamo-pituitary-adrenocortical function require annexin 1. They also provide novel evidence that the positive influence of the steroids on GH secretion evident within this timeframe is effected centrally via an annexin 1-dependent mechanism which is antagonized by IL-1β.
British Journal of Pharmacology (2001) 134, 887–895; doi:10.1038/sj.bjp.0704324
Myocardial reperfusion injury is associated with the infiltration of blood-borne polymorphonuclear leukocytes. We have previous described the protection afforded by annexin 1 (ANXA1) in an experimental model of rat myocardial ischemia-reperfusion (IR) injury. We examined the 1) amino acid region of ANXA1 that retained the protective effect in a model of rat heart IR; 2) changes in endogenous ANXA1 in relation to the IR induced damage and after pharmacological modulation; and 3) potential involvement of the formyl peptide receptor (FPR) in the protective action displayed by ANXA1 peptides. Administration of peptide Ac2-26 at 0, 30, and 60 min postreperfusion produced a significant protection against IR injury, and this was associated with reduced myeloperoxidase activity and IL-1beta levels in the infarcted heart. Western blotting and electron microscopy analyses showed that IR heart had increased ANXA1 expression in the injured tissue, associated mainly with the infiltrated leukocytes. Finally, an antagonist to the FPR receptor selectively inhibited the protective action of peptide ANXA1 and its derived peptides against IR injury. Altogether, these data provide further insight into the protective effect of ANXA1 and its mimetics and a rationale for a clinical use for drugs developed from this line of research.
Inappropriate or prolonged inflammation is the main cause of many diseases; for this reason it is important to understand the physiological mechanisms that terminate inflammation in vivo. Agonists for several Gs-protein-coupled receptors, including cell-surface adenosine purinergic receptors, can increase levels of immunosuppressive cyclic AMP in immune cells; however, it was unknown whether any of these receptors regulates inflammation in vivo. Here we show that A2a adenosine receptors have a non-redundant role in the attenuation of inflammation and tissue damage in vivo. Sub-threshold doses of an inflammatory stimulus that caused minimal tissue damage in wild-type mice were sufficient to induce extensive tissue damage, more prolonged and higher levels of pro-inflammatory cytokines, and death of male animals deficient in the A2a adenosine receptor. Similar observations were made in studies of three different models of inflammation and liver damage as well as during bacterial endotoxin-induced septic shock. We suggest that A2a adenosine receptors are a critical part of the physiological negative feedback mechanism for limitation and termination of both tissue-specific and systemic inflammatory responses.
Annexins are Ca2+ and phospholipid binding proteins forming an evolutionary conserved multigene family with members of the family being expressed throughout animal and plant kingdoms. Structurally, annexins are characterized by a highly alpha-helical and tightly packed protein core domain considered to represent a Ca2+-regulated membrane binding module. Many of the annexin cores have been crystallized, and their molecular structures reveal interesting features that include the architecture of the annexin-type Ca2+ binding sites and a central hydrophilic pore proposed to function as a Ca2+ channel. In addition to the conserved core, all annexins contain a second principal domain. This domain, which NH2-terminally precedes the core, is unique for a given member of the family and most likely specifies individual annexin properties in vivo. Cellular and animal knock-out models as well as dominant-negative mutants have recently been established for a number of annexins, and the effects of such manipulations are strikingly different for different members of the family. At least for some annexins, it appears that they participate in the regulation of membrane organization and membrane traffic and the regulation of ion (Ca2+) currents across membranes or Ca2+ concentrations within cells. Although annexins lack signal sequences for secretion, some members of the family have also been identified extracellularly where they can act as receptors for serum proteases on the endothelium as well as inhibitors of neutrophil migration and blood coagulation. Finally, deregulations in annexin expression and activity have been correlated with human diseases, e.g., in acute promyelocytic leukemia and the antiphospholipid antibody syndrome, and the term annexinopathies has been coined.