No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Inhibition of neutrophil and monocyte recruitment by endogenous and exogenous lipocortin 1
British Journal of Pharmacology
Abstract
The role played by endogenous lipocortin 1 in the anti-migratory action exerted by dexamethasone (Dex) on monocyte recruitment in an in vivo model of acute inflammation was investigated by use of several neutralizing polyclonal antibodies raised against lipocortin 1 or a lipocortin 1-derived N-terminus peptide (peptide Ac2-26). The efficacy of peptide Ac2-26 in inhibiting monocyte and polymorphonuclear leucocyte (PMN) recruitment was also tested.
Intraperitoneal (i.p.) injection of zymosan A (1 mg) produced a time-dependent cell accumulation into mouse peritoneal cavities which followed a typical profile of acute inflammation: PMN influx was maximal at 4 h post-zymosan (between 15 and 20×106 cells per mouse), and this was followed by an accumulation of monocytes which peaked at the 24 h time-point (between 10 and 15×106 cells per mouse).
Dex administration to mice reduced zymosan-induced 4 h PMN infiltration and 24 h monocyte accumulation with similar efficacy: approximately 50% of inhibition of recruitment of both cell types was achieved at the dose of 30 μg per mouse (∼1 mg kg−1, subcutaneously (s.c.)). Maximal inhibitions of 64% and 67% on PMN and monocyte recruitment, respectively, were measured after a dose of 100 μg per mouse (∼3 mg kg−1, s.c.).
Dex (30 μg s.c.) inhibited monocyte (53%) and PMN (69%) accumulation in response to zymosan application in mice which had been treated with a non-immune sheep serum (50 μl s.c.). In contrast, the steroid was no longer active in reducing cell accumulation in mice which had been passively immunized against full length human recombinant lipocortin 1 (serum LCS3), or against lipocortin 1 N-terminus peptide.
Treatment of mice with vinblastine (1 mg kg−1, intravenously (i.v.)) produced a remarkable leucopenia as assessed 24 h after administration. This was accompanied by a 60% reduction in 4 h-PMN influx, and by a 27% reduction in 24 h-monocyte accumulation, measured after zymosan administration. The inhibitory effect of Dex on monocyte recruitment was not significantly modified in vinblastine-treated mice, with 36% and 57% of inhibition calculated at the dose of 30 μg Dex, and 70% and 60% of inhibition at 100 μg Dex, in vehicle- and vinblastine-treated mice, respectively.
Treatment of mice with peptide Ac2-26 dose-dependently attenuated PMN influx at 4 h post-zymosan with a significant effect at 100 μg per mouse (45% of inhibition, n=9, P<0.05) and a maximal effect of 61% inhibition at the highest dose tested of 200 μg s.c. (n=14, P<0.05). No effect of peptide Ac2-26 (200 μg s.c.) was seen on zymosan-induced 24 h monocyte recruitment. In contrast, administration of 200 μg peptide Ac2-26 every 6 h was effective in reducing the number of monocytes harvested from the inflamed peritoneal cavities at 24 h post-zymosan: 9.40±0.58×106 monocytes per mouse (n=13) and 5.74±0.34 monocytes per mouse (n=14) in vehicle- and peptide Ac2-26-treated mice, respectively (P<0.05).
Finally, peptide Ac2-26 produced a concentration-dependent inhibition of the rate of phagocytosis of mouse resident peritoneal macrophages as measured by flow cytometry, with a maximal reduction of 34% at the highest concentration tested of 100 μg ml−1 (n=8 experiments performed in duplicate; P<0.05).
In conclusion, this study suggests that in vivo monocyte recruitment during acute inflammation is, at least in part, under the negative modulatory control of endogenous lipocortin 1 (as seen after administration of Dex by using the specific antisera) and exogenous lipocortin 1 mimetics (as observed with peptide Ac2-26). In addition to the neutrophil, we can now propose that the monocyte also can be a target for the in vivo anti-inflammatory action of lipocortin 1.
British Journal of Pharmacology (1997) 120, 1075–1082; doi:10.1038/sj.bjp.0701029
... Lack of FPR2 signaling has been linked to exacerbated injury in animal models of ischemia-reperfusion injury, associated with neutrophil pro-inflammatory activation 24 . In particular, annexin A1, a protein also abundantly expressed in neutrophils 25 , has been linked to the regulation of inflammation and the resolution of inflammatory processes via FPR2 activation [26][27][28] . The antiinflammatory and pro-resolving abilities induced by FPR2 agonists have positioned this receptor as a promising target for drug development This, in particular, is being enhanced by recent structural analysis of ligand-receptor interactions on the atomic scale [29][30][31] . ...
... FPR1 on the other hand has not been assigned anti-inflammatory roles so far; notably, both receptors share an amino acid sequence identity of approximately 69% and hence display a high degree structural similarity ( 6,32 ). Given their homology, structural relation and shared agonist repertoire, including annexin A1 and its mimetic N-terminal peptide Ac2-26 6,26,33,34 , we explored whether and how these closely related receptors with overlapping expression patterns 6,34 convey distinct signaling profiles. Potentially, each receptor might have a distinct signaling profile or recognize FPR agonist subsets with specific activation patterns 35 . ...
... FPR1 mediates a wide array of pro-inflammatory responses 56 . In contrast, FPR2, coupled with its endogenous agonists, has been associated with agonist-induced anti-inflammatory and pro-resolving properties 26 However, several nuclear copies of humanin were identified. Therefore whether the corresponding RNA template is transcribed within mitochondria and translated by the mitochondrial ribosome or instead within the cytoplasm is yet to be conclusively established 64 . ...
Pattern Recognition Receptors are key in identifying pathogenic or damaged cell-related patterns or molecules. Among these, the closely linked formyl peptide receptors FPR1 and FPR2 are believed to hold pivotal yet differing functions in immune regulation. To address the intriguing question of how these highly related receptors with a shared agonist spectrum play differing roles in modulating inflammation, we analyzed the signaling profile for a panel of FPR agonists in vivo and ex vivo settings. Our analysis uncovered a shared core signature for both FPRs across signaling pathways. Whereas formylated peptides generally acted as potent agonists at FPR1, FPR2 agonists, irrespective of N-terminal formylation, displayed consistently low activity ratios, suggesting an underutilized signaling potential of this receptor. Signaling outcomes were defined by specific agonist receptor pairings and no receptor-specific signaling texture was identified. Activation of the FPR signaling axis by fMLF in human neutrophils did impact neutrophil survival. Overall, the distinct characteristics underlying inflammatory, anti-inflammatory, or pro-resolving profiles could not be attributed to a specific receptor isoform, signaling pattern, or a particular class of agonists, challenging assumptions about distinct inflammatory profiles linked to specific receptors, signaling patterns, or agonist classes.
... Several studies using animal models of inflammation have demonstrated that the administration of ANXA1 or Ac2-26 moderates neutrophil recruitment. [54][55][56][57][58] Early studies showed that both ANXA1 and Ac2-26 inhibited adhesion of human neutrophils to activated endothelial cells under static 54,59 and flow conditions 60,61 in vitro and thus impairing neutrophil recruitment. Use of an ANXA1 blocking antibody increased the ability of neutrophils to transmigrate through an endothelial monolayer indicating that ANXA1 is able to impair neutrophil transmigration. ...
... Since most studies focused on the effects of ANXA1 on neutrophils, it is important to remark that also monocyte recruitment is inhibited by ANXA1 or its derived peptides. 56,65,66 Possibly, these findings can be extrapolated to other leukocyte subsets. ...
... In line with these findings ANXA1-deficient mice subjected to various inflammatory stimuli exhibited a pronounced inflammation compare with control mice indicating a protective role of ANXA1. 67,70 Additionally, dexamethasone was shown to be less effective in ANXA1 deficient mice 67 or when mice where immunized against ANXA1 56,69,71 showing the importance of ANXA1 in the anti-inflammatory and immunosuppressive effects of glucocorticoids. Again, evaluation of the cremasteric microcirculation by intravital microscopy revealed ANXA1 as a modulator of the leukocyte adhesion cascade since ANXA1 null mice demonstrated increased transendothelial migration. ...
The inflammatory response protects the human body against infection and injury. However, uncontrolled and unresolved inflammation can lead to tissue damage and chronic inflammatory diseases. Therefore, active resolution of inflammation is essential to restore tissue homeostasis. This review focuses on the pro-resolving molecule annexin A1 (ANXA1) and its derived peptides. Mechanisms instructed by ANXA1 are multidisciplinary and affect leukocytes as well as endothelial cells and tissue resident cells like macrophages and mast cells. ANXA1 has an outstanding role in limiting leukocyte recruitment and different aspects of ANXA1 as modulator of the leukocyte adhesion cascade are discussed here. Additionally, this review details the therapeutic relevance of ANXA1 and its derived peptides in cardiovascular diseases since atherosclerosis stands out as a chronic inflammatory disease with impaired resolution and continuous leukocyte recruitment.
... Besides affecting PMN recruitment in the peritoneal compartment after stimulation with classical inflammogens (20,21), LC1 also modulates neutrophil accumulation into the rat small intestine in a model of reperfusion injury (22). This effect was linked to a reduction in PMN recruitment to the damaged tissue. ...
... This effect was linked to a reduction in PMN recruitment to the damaged tissue. Finally, in most, if not all, of these experimental conditions, LC1 appeared to act as a mediator of the protective effect displayed by glucocorticoids (e.g., dexamethasone [DEX]), inasmuch as anti-LC1 antibodies prevented the inhibition afforded by the steroid (20,22). Therefore, the present study investigated whether myocardial injury could be modulated by systemic treatment with LC1. ...
... Recently, we have demonstrated that administration of LC1 to experimental animals reduced the extravasation of blood-borne neutrophils in simple models of acute inflammation (16,40), apparently by interfering with the process of neutrophil interaction with the activated endothelium (19). Antibodies against LC1 prevented the antimigratory action displayed by DEX (18,20,40). Other groups have confirmed these observations (17,21). ...
... Additionally, protectin D1n-3 docosapentaenoic acid (DPA) and resolving D5n-3 DPA exert similar actions during peritonitis and intestinal inflammation, and this effect is due to the regulation of neutrophil adhesion to the endothelium [20]. In line with this, one of the major protein components of resolution cascade, ANXA1, has been extensively studied by its actions on neutrophil infiltration blockade in acute models of inflammation, such as acute murine MSU-induced gout [21], DSS-induced colitis [22], zymosan-induced peritonitis [23], among others [24]. These studies reveal a prominent role for ANXA1 on neutrophil infiltration, by the inhibition of neutrophil adhesion to the endothelium [25] and the impairment of adequate rolling [26] and extravasation to the sites of inflammation [27]. ...
... Annexin A1 (ANXA1) is a 37-kDa phospholipid-binding protein that acts as a downstream effector of glucocorticoids (GCs). This protein is a well-characterized anti-inflammatory and pro-resolving factor [73,74], which is able to blockade leukocyte recruitment to inflamed tissues [23], inhibit pro-inflammatory mediators release [75], and promote tissue repair [76]. ...
Inflammation is an essential physiological process that is directed to the protection of the organism against invading pathogens or tissue trauma. Most of the existing knowledge related to inflammation is focused on the factors and mechanisms that drive the induction phase of this process. However, since the recognition that the resolution of the inflammation is an active and tightly regulated process, increasing evidence has shown the relevance of this process for the development of chronic inflammatory diseases, such as inflammatory bowel disease. For that reason, with this review, we aimed to summarize the most recent and interesting information related to the resolution process in the context of intestinal inflammation. We discussed the advances in the understanding of the pro-resolution at intestine level, as well as the new mediators with pro-resolutive actions that could be interesting from a therapeutic point of view.
... Thus, vinblastine targets dividing cells and leads to mitotic arrest (25). Treatment of mice with vinblastine induces strong neutropenia (26,27). Nevertheless, this drug is rarely used to study neutrophils in vivo. ...
... Overall, vinblastine exhibits the same disadvantages as cyclophosphamide. It displays a poor selectivity in blood (as illustrated by a respective 35 and 39% reduction in blood monocytes and lymphocytes) (26), and induces cytotoxicity in other cell types such as pancreatic cells (28) and during spermatogenesis (29). Furthermore, vinblastine might affect any cellular process involving microtubule assembly, limiting its use for in vivo experiments. ...
Neutrophils are the most abundant leukocytes in human blood and critical actors of the immune system. Many neutrophil functions and facets of their activity in vivo were revealed by studying genetically modified mice or by tracking fluorescent neutrophils in animals using imaging approaches. Assessing the roles of neutrophils can be challenging, especially when exact molecular pathways are questioned or disease states are interrogated that alter normal neutrophil homeostasis. This review discusses the main in vivo models for the study of neutrophils, their advantages and limitations. The side-by-side comparison underlines the necessity to carefully choose the right model(s) to answer a given scientific question, and exhibit caveats that need to be taken into account when designing experimental procedures. Collectively, this review suggests that at least two models should be employed to legitimately conclude on neutrophil functions.
... It is increased upon glucocorticoid (GC) activation, mediating in part anti-inflammatory activities through inhibition of phospholipase A2 and prevention of subsequent proinflammatory mediators, including leukotrienes and PGs (6)(7)(8). In addition, ANXA1 has been shown to suppress inflammation through the inhibition of leukocyte activation and migration and also is an antiproliferative and proapoptotic protein (9)(10)(11). Deficiency of ANXA1 results in increased neutrophil and monocyte transmigration, and in keeping with this, administration of an ANXA1 peptide prevents neutrophil influx following an inflammatory stimulus (11)(12)(13). ...
... In addition, ANXA1 has been shown to suppress inflammation through the inhibition of leukocyte activation and migration and also is an antiproliferative and proapoptotic protein (9)(10)(11). Deficiency of ANXA1 results in increased neutrophil and monocyte transmigration, and in keeping with this, administration of an ANXA1 peptide prevents neutrophil influx following an inflammatory stimulus (11)(12)(13). The exact role of ANXA1 in the immune response is unclear. ...
TLRs play a pivotal role in the recognition of bacteria and viruses. Members of the family recognize specific pathogen sequences to trigger both MyD88 and TRIF-dependent pathways to stimulate a plethora of cells. Aberrant activation of these pathways is known to play a critical role in the development of autoimmunity and cancer. However, how these pathways are entirely regulated is not fully understood. In these studies, we have identified Annexin-A1 (ANXA1) as a novel regulator of TLR-induced IFN-β and CXCL10 production. We demonstrate that in the absence of ANXA1, mice produce significantly less IFN-β and CXCL10, and macrophages and plasmacytoid dendritic cells have a deficiency in activation following polyinosinic:polycytidylic acid administration in vivo. Furthermore, a deficiency in activation is observed in macrophages after LPS and polyinosinic:polycytidylic acid in vitro. In keeping with these findings, overexpression of ANXA1 resulted in enhanced IFN-β and IFN-stimulated responsive element promoter activity, whereas silencing of ANXA1 impaired TLR3- and TLR4-induced IFN-β and IFN-stimulated responsive element activation. In addition, we show that the C terminus of ANXA1 directly associates with TANK-binding kinase 1 to regulate IFN regulatory factor 3 translocation and phosphorylation. Our findings demonstrate that ANXA1 plays an important role in TLR activation, leading to an augmentation in the type 1 IFN antiviral cytokine response.
... Annexin-A1 acts as an anti-inflammatory mechanism by inhibiting the release of pro-inflammatory mediators and blocking the migration of inflammatory cells. 32,33 Annexin-A1 also plays a significant role in cardiovascular diseases, including its protective role in atherosclerosis and myocardial infarction. 11,[34][35][36] Subsequently, ANXA1sp was synthesized and had strong protective effects on the heart and brain. ...
Sepsis‐induced myocardial injury is one of the most difficult complications of sepsis in intensive care units. Annexin A1 (ANXA1) short peptide (ANXA1sp) protects organs during the perioperative period. However, the protective effect of ANXA1sp against sepsis‐induced myocardial injury remains unclear. We aimed to explore the protective effects and mechanisms of ANXA1sp against sepsis‐induced myocardial injury both in vitro and in vivo. Cellular and animal models of myocardial injury in sepsis were established with lipopolysaccharide. The cardiac function of mice was assessed by high‐frequency echocardiography. Elisa assay detected changes in inflammatory mediators and markers of myocardial injury. Western blotting detected autophagy and mitochondrial biosynthesis‐related proteins. Autophagic flux changes were observed by confocal microscopy, and autophagosomes were evaluated by TEM. ATP, SOD, ROS, and MDA levels were also detected.ANXA1sp pretreatment enhanced the 7‐day survival rate, improved cardiac function, and reduced TNF‐α, IL‐6, IL‐1β, CK‐MB, cTnI, and LDH levels. ANXA1sp significantly increased the expression of sirtuin‐3 (SIRT3), mitochondrial biosynthesis‐related proteins peroxisome proliferator‐activated receptor γ co‐activator 1α (PGC‐1α), and mitochondrial transcription factor A (TFAM). ANXA1sp increased mitochondrial membrane potential (△Ψm), ATP, and SOD, and decreased ROS, autophagy flux, the production of autophagosomes per unit area, and MDA levels. The protective effect of ANXA1sp decreased significantly after SIRT3 silencing in vitro and in vivo, indicating that the key factor in ANXA1sp's protective role is the upregulation of SIRT3. In summary, ANXA1sp attenuated sepsis‐induced myocardial injury by upregulating SIRT3 to promote mitochondrial biosynthesis and inhibit oxidative stress and autophagy.
... The adhesion of neutrophils to the endothelium contributes to monocyte recruitment by depositing chemotactic proteins on the endothelium (Silvestre-Roig et al., 2020). The recruitment of monocytes is also inhibited by AnxA1 or Ac2-26 during acute inflammation (Getting et al., 1997). Leukocyte adhesion to the injured endothelium is a critical step in atherosclerosis development. ...
Cardiovascular diseases (CVD) remain the leading cause of mortality worldwide. The main cause underlying CVD is associated with the pathological remodeling of the vascular wall, involving several cell types, including endothelial cells, vascular smooth muscle cells, and leukocytes. Vascular remodeling is often related with the development of atherosclerotic plaques leading to narrowing of the arteries and reduced blood flow. Atherosclerosis is known to be triggered by high blood cholesterol levels, which in the presence of a dysfunctional endothelium, results in the retention of lipoproteins in the artery wall, leading to an immune-inflammatory response. Continued hypercholesterolemia and inflammation aggravate the progression of atherosclerotic plaque over time, which is often complicated by thrombus development, leading to the possibility of CV events such as myocardial infarction or stroke. Annexins are a family of proteins with high structural homology that bind phospholipids in a calcium-dependent manner. These proteins are involved in several biological functions, from cell structural organization to growth regulation and vesicle trafficking. In vitro gain- or loss-of-function experiments have demonstrated the implication of annexins with a wide variety of cellular processes independent of calcium signaling such as immune-inflammatory response, cell proliferation, migration, differentiation, apoptosis, and membrane repair. In the last years, the use of mice deficient for different annexins has provided insight into additional functions of these proteins in vivo , and their involvement in different pathologies. This review will focus in the role of annexins in CVD, highlighting the mechanisms involved and the potential therapeutic effects of these proteins.
... Thus, ANXA1 serves as a negative regulator of transmigration (Getting, Flower and Perretti, 1997;Chatterjee et al., 2005;de Jong et al., 2017;Sheikh and Solito, 2018). Another major involvement of ANXA1 in anti-inflammatory processes is through initiation of apoptosis and regulation of apoptotic cell removal. ...
Oriented cell divisions (OCDs) represent a fundamental mechanism for tissue morphogenesis, repair and differentiation where the mitotic spindle is oriented along a specific polarity axis. Early research identified the evolutionarily conserved Gαi/LGN/NuMA ternary complex that mediates orientation of the mitotic spindle by being restricted to specific cortical regions. The mechanisms that control the recruitment of these proteins to the cortex remain unfolding, particularly in epithelial systems such as the mammary gland. The mammary gland represents a unique organ that develops predominantly after birth where postnatal morphogenesis of the mammary gland drives dramatic tissue turnover and remodelling. Thus, differentiation and proliferation are constantly balanced to allow normal mammary gland development and homeostasis. How mammary epithelial cells regulate mitotic spindle orientation, hence OCDs, to accompany the rapid and constant tissue turnover is not well understood. This study aimed to identify novel factors that regulate the LGN-mediated spindle orientation machinery and determine how their dysregulation affects OCDs in mammary epithelial cells. By combining co-immunoprecipitation with mass spectrometry, the LGN interactome at the cell cortex of mitotic mammary epithelial cells was characterised and the membrane-associated protein Annexin A1 (ANXA1) was identified as a novel partner of LGN. Confocal and time-lapse microscopy demonstrated a critical role of ANXA1 in regulating the position and planar orientation of the mitotic spindle by instructing the accumulation and restriction of the LGN complex at the lateral cortex. Moreover, loss of ANXA1 leads to mitotic spindle misassembly and chromosome segregation defects, affecting the dynamics and progression of mitosis. Collectively the present study identified ANXA1 as a novel intrinsic cue of OCDs in mammary epithelial cells. Given increasing evidence of a link between OCD and tumorigenesis, this work is not only important for advancing our understanding of normal epithelial biology but also elucidating how imbalance of OCDs can contribute to the abnormal cell behaviour observed in cancer.
... We and others have previously described that AnxA1 is an effective mediator of inflammation resolution involved in various preclinical models of inflammatory diseases such as gout, pleurisy, peritonitis, and arthritis [33, [42][43][44][45]. However, AnxA1 has been less investigated in the context of viral infections [35,46]. ...
Chikungunya (CHIKV) is an arthritogenic alphavirus that causes a self-limiting disease usually accompanied by joint pain and/or polyarthralgia with disabling characteristics. Immune responses developed during the acute phase of CHIKV infection determine the rate of disease progression and resolution. Annexin A1 (AnxA1) is involved in both initiating inflammation and preventing over-response, being essential for a balanced end of inflammation. In this study, we investigated the role of the AnxA1-FPR2/ALX pathway during CHIKV infection. Genetic deletion of AnxA1 or its receptor enhanced inflammatory responses driven by CHIKV. These knockout mice showed increased neutrophil accumulation and augmented tissue damage at the site of infection compared with control mice. Conversely, treatment of wild-type animals with the AnxA1 mimetic peptide (Ac2–26) reduced neutrophil accumulation, decreased local concentration of inflammatory mediators and diminished mechanical hypernociception and paw edema induced by CHIKV-infection. Alterations in viral load were mild both in genetic deletion or with treatment. Combined, our data suggest that the AnxA1-FPR2/ALX pathway is a potential therapeutic strategy to control CHIKV-induced acute inflammation and polyarthralgia.
... Tout comme pour les neutrophiles, l'ANXA1 est également capable de moduler l'afflux des monocytes au niveau du tissu lésé ainsi que leur apoptose. Effectivement, l'administration intraveineuse répétée du peptide Ac2-26 aux souris développant une péritonite suite à l'injection de zymosan, prévient l'infiltration de monocytes dans la cavité péritonéale [89]. De surcroit, les cellules monocytaires U937 qui surexpriment l'ANXA1 présentent une MTE réduite [90]. ...
L’annexine A1 (ANXA1) appartient à la vaste superfamille des annexines qui englobe des protéines capables de se lier aux phospholipides membranaires de façon calcium dépendante. C’est une protéine multifonctionnelle initialement décrite pour ses propriétés anti-inflammatoires. Elle est présente dans le noyau, le cytosol et les membranes cellulaires et peut également être sécrétée et clivée dans le milieu environnant. L’ANXA1 présente un important intérêt en oncologie du fait de sa dérégulation dans de nombreux cancers. En fonction des types de cancers, l’ANXA1 est surexprimée ou sous-exprimée par rapport au tissu sain. Elle est surexprimée dans le cancer du sein triple négatif par rapport aux autres sous-types de tumeurs mammaires et dans les mélanomes par rapport aux mélanocytes. Dans ces deux pathologies, bien que l’ANXA1 soit associée aux processus de prolifération, migration et invasion, les mécanismes précis de son rôle au sein d’une tumeur restent mal connus, notamment ceux impliquant la fraction extracellulaire. Les études menées sur des souris n’exprimant pas l’Anxa1 ont également montré que l’ANXA1 stromale est impliquée dans le développement et la progression tumorale.Le premier objectif a donc été de tester la possibilité de bloquer la protéine extracellulaire via un anticorps monoclonal breveté, le VJ4B6, pour diminuer la migration (in vitro) et la dissémination (in vivo). Nos données montrent que le VJ4B6 ne permet pas de limiter la migration cellulaire des lignées de cancer du sein triple négatif (MDA-MB-231-luc) et de mélanome (SK-MEL-28 et A375-MA2) présentant de l’ANXA1 extracellulaire. Cet anticorps ne permet pas non plus de limiter le développement et la dissémination tumorale du mélanome B16Bl6 in vivo. Le second objectif a été d’étudier le rôle de l’ANXA1 tumorale et stromale dans le développement et la dissémination du mélanome. Nos résultats ont montré que l’ANXA1 tumorale est impliquée dans la prolifération des cellules A375-MA2 et SK-MEL-28 in vitro. L’utilisation de souris invalidées pour l’Anxa1 a également permis de mettre en évidence que l’ANXA1 stromale favorise le développement tumoral et la formation de métastases des cellules murines B16Bl6 in vivo. De façon intéressante, les études réalisées sur les tumeurs ont montré que l’absence d’ANXA1 stromale limite la prolifération des cellules tumorales ainsi que l’angiogenèse. Ceci peut donc expliquer la progression limitée des tumeurs chez les souris invalidées. De plus, les tumeurs développées chez ces dernières présentent une surexpression des marqueurs lymphocytaires (CD3, CD4, FoxP3, CD8a, NKp46) par rapport à celles développées chez les souris sauvages. Cet afflux lymphocytaire concerne à la fois les lymphocytes pro- et anti-tumoraux. Nous avons donc émis l’hypothèse que ce dernier soit une conséquence d’une perméabilité accrue des vaisseaux tumoraux au niveau des tumeurs des souris invalidées pour l’Anxa1.
... We found that treatment with flunisolide reduced the number of F4/80 positive cells and levels of lung cytokines (TNF-α and TGF-β) and chemokines (MIP-1α/CCL-3 and MIP-2/CXCL-2), suggesting that mediators released by macrophages were inhibited by flunisolide. Down-regulation of macrophage functionality can be associated with the effect of glucocorticoids on the cytoskeleton, leading to reduction of cell adherence and/or migration to the inflammatory site (39)(40)(41)(42)(43) or even induction of apoptosis (44)(45)(46), thus contributing to the inhibition of fibrosis by flunisolide. Alternatively, macrophages that have taken up silica particles could be carried off the lungs by the lymphatics (15). ...
Silicosis is an occupational disease triggered by the inhalation of fine particles of crystalline silica and characterized by inflammation and scarring in the form of nodular lesions in the lungs. In spite of the therapeutic arsenal currently available, there is no specific treatment for the disease. Flunisolide is a potent corticosteroid shown to be effective for controlling chronic lung inflammatory diseases. In this study, the effect of flunisolide on silica-induced lung pathological changes in mice was investigated. Swiss-Webster mice were injected intranasally with silica particles and further treated with flunisolide from day 21 to 27 post-silica challenge. Lung function was assessed by whole body invasive plethysmography. Granuloma formation was evaluated morphometrically, collagen deposition by Picrus sirius staining and quantitated by Sircol. Chemokines and cytokines were evaluated using enzyme-linked immunosorbent assay. The sensitivity of lung fibroblasts was also examined in in vitro assays. Silica challenge led to increased leukocyte numbers (mononuclear cells and neutrophils) as well as production of the chemokine KC/CXCL-1 and the cytokines TNF-α and TGF-β in the bronchoalveolar lavage. These alterations paralleled to progressive granuloma formation, collagen deposition and impairment of lung function. Therapeutic administration of intranasal flunisolide inhibited granuloma and fibrotic responses, noted 28 days after silica challenge. The upregulation of MIP-1α/CCL-3 and MIP-2/CXCL-2 and the cytokines TNF-α and TGF-β, as well as deposition of collagen and airway hyper-reactivity to methacholine were shown to be clearly sensitive to flunisolide, as compared to silica-challenge untreated mice. Additionally, flunisolide effectively suppressed the responses of proliferation and MCP-1/CCL-2 production from IL-13 stimulated lung fibroblasts from silica- or saline-challenged mice. In conclusion, we report that intranasal treatment with the corticosteroid flunisolide showed protective properties on pathological features triggered by silica particles in mice, suggesting that the compound may constitute a promising strategy for the treatment of silicosis.
... ANXA1 is best known for its anti-inflammatory and proresolving properties. Known mechanisms of action span from the inhibition of pro-inflammatory mediators release (PGE 2 and leukotrines) (33), to tissue repair (34), and to the blockade of leukocyte migration through an inflamed endothelium (35,36). ANXA1 induces L-selectin shedding on neutrophils and the detachment of monocytic cells from the endothelium by reducing α4β1 integrin clustering and activation (12). ...
Annexin-A1 (ANXA1) was first discovered in the early 1980's as a protein, which mediates (some of the) anti-inflammatory effects of glucocorticoids. Subsequently, the role of ANXA1 in inflammation has been extensively studied. The biology of ANXA1 is complex and it has many different roles in both health and disease. Its effects as a potent endogenous anti-inflammatory mediator are well-described in both acute and chronic inflammation and its role in activating the pro-resolution phase receptor, FPR2, has been described and is now being exploited for therapeutic benefit. In the present mini review, we will endeavor to give an overview of ANXA1 biology in relation to inflammation and functions that mediate pro-resolution that are independent of glucocorticoid induction. We will focus on the role of ANXA1 in diseases with a large inflammatory component focusing on diabetes and microvascular disease. Finally, we will explore the possibility of exploiting ANXA1 as a novel therapeutic target in diabetes and the treatment of microvascular disease.
... 18 Initially, Annexin A1, also named as macrocortin, 19 renocortin, 20 lipomodulin, 21 and lipocortin I, 22 was identified as the inhibitor of phospholipase A2 (PLA2). 18 Thus, it was mainly used as an inhibitor of pro-inflammatory factors prostaglandins (PGs) to study the inhibition of leukocyte aggregation in an inflammatory model [23][24][25][26] for a long time. Annexin A1 is particularly abundant in neutrophils, 27 but not abundant in lymphocytes. ...
Annexin A1 is a Ca²⁺-dependent phospholipid binding protein involved in a variety of pathophysiological processes. Accumulated evidence has indicated that Annexin A1 has important functions in cell proliferation, apoptosis, differentiation, metastasis, and inflammatory response. Moreover, the abnormal expression of Annexin A1 is closely related to the occurrence and development of tumors. In this review article, we focus on the structure and function of Annexin A1 protein, especially the recent evidence of Annexin A1 in the pathophysiological role of inflammatory and cancer. This summary will be very important for further investigation of the pathophysiological role of Annexin A1 and for the development of novel therapeutics of inflammatory and cancer based on targeting Annexin A1 protein.
... Perretti and Flower (1993) demonstrated that AnxA1 attenuated IL-1 and IL-8 induced neutrophil migration into the murine air pouch [24]. Additionally, Getting et al. showed that both endogenous and exogenous AnxA1 were able to inhibit the neutrophil and monocyte recruitment in murine peritoneal cavity [25]. These findings suggest that AnxA1 retains its antiinflammatory action irrespective of stimuli. ...
Cardiovascular disease (CVD) continues to be the leading cause of death in the world. Increased inflammation and an enhanced thrombotic milieu represent two major complications of CVD, which can culminate into an ischemic event. Treatment for these life-threatening complications remains reperfusion and restoration of blood flow. However, reperfusion strategies may result in ischemia–reperfusion injury (I/RI) secondary to various cardiovascular pathologies, including myocardial infarction and stroke, by furthering the inflammatory and thrombotic responses and delivering inflammatory mediators to the affected tissue. Annexin A1 (AnxA1) and its mimetic peptides are endogenous anti-inflammatory and pro-resolving mediators, known to have significant effects in resolving inflammation in a variety of disease models. Mounting evidence suggests that AnxA1, which interacts with the formyl peptide receptor (FPR) family, may have a significant role in mitigating I/RI associated complications. In this review article, we focus on how AnxA1 plays a protective role in the I/R based vascular pathologies.
... The main benefits attributed to annexin-A1 peptide mimetics include cardioprotection based on their anti-inflammatory effect to preserve myocardial viability after MI but also other inflammation-independent properties that directly protect cardiomyocytes viability and contractile function (Qin et al., 2015). The subcutaneous administration of annexin-A1 N-terminal derived peptide Ac2-26 has been shown to confer protection against ischemia-reperfusion injury by reducing myeloperoxidase activity and IL-1β levels in the infarcted heart, as well as down-regulate monocyte accumulation and inhibit phagocytic activity of macrophages in different rodent experimental models (Getting et al., 1997;La et al., 2001). Another annexin-A1 mimetic is CGEN-855A, a 21 amino acid peptide displays anti-inflammatory effects by inhibition of polymorphonuclear neutrophils recruitment and also provides protection against ischemia-reperfusion-mediated injury to the myocardium after being injected intravenously in mice (Hecht et al., 2009). ...
Cardiovascular disease (CVD) remains a leading cause of mortality and morbidity worldwide. Numerous therapies are currently under investigation to improve pathological cardiovascular complications, but yet, there have been very few new medications approved for intervention/treatment. Therefore, new approaches to treat CVD are urgently required. Attempts to prevent vascular complications usually involve amelioration of contributing risk factors and underlying processes such as inflammation, obesity, hyperglycaemia, or hypercholesterolemia. Historically, the development of peptides as therapeutic agents has been avoided by the Pharmaceutical industry due to their low stability, size, rate of degradation, and poor delivery. However, more recently, resurgence has taken place in developing peptides and their mimetics for therapeutic intervention. As a result, increased attention has been placed upon using peptides that mimic the function of mediators involved in pathologic processes during vascular damage. This review will provide an overview on novel targets and experimental therapeutic approaches based on peptidomimetics for modulation in CVD. We aim to specifically examine apolipoprotein A-I (apoA-I) and apoE mimetic peptides and their role in cholesterol transport during atherosclerosis, suppressors of cytokine signaling (SOCS)1-derived peptides and annexin-A1 as potent inhibitors of inflammation, incretin mimetics and their function in glucose-insulin tolerance, among others. With improvements in technology and synthesis platforms the future looks promising for the development of novel peptides and mimetics for therapeutic use. However, within the area of CVD much more work is required to identify and improve our understanding of peptide structure, interaction, and function in order to select the best targets to take forward for treatment.
... In addition to naturally occurring ANXA1 in EVs, hydrogels containing ANXA1 peptide mimetic Ac2-26 have been generated and shown to promote wound repair (Del Gaudio et al., 2015). ANXA1 facilitates resolution of inflammation and repair by a number of mechanisms that include inhibition of leukocytes recruitment by decreasing their adhesion/recruitment and transmigration (Chatterjee et al., 2005;Getting et al., 1997;Mancuso et al., 1995). ...
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, ANXA1 is a virus-inducible protein in vivo and in vitro, which indicates that the induction of ANXA1 Our in vivo results show that high levels of ANXA1 is detrimental to the host, resulting in more weight loss and IAV-induced morbidity, due to higher viral titers and viral propagation. ANXA1 has been shown to be an antiinflammatory mediator, modulating cytokine production in response to zymosan 24,25 or LPS 24 and in an asthma mouse model. [26][27][28] However, ANXA1 may also promote anti-viral cytokine production in response to poly(I:C), 29 dendritic cells exhibit a mature phenotype and produced less pro-inflammatory cytokines. ...
The influenza virus infects millions of people each year and can result in severe complications. Understanding virus recognition and host responses to influenza infection will enable future development of more effective anti-viral therapies. Previous research has revealed diverse yet important roles for the annexin family of proteins in modulating the course of influenza A virus (IAV) infection. However, the role of Annexin-A1 (ANXA1) in IAV infection has not been addressed. Here, we show that ANXA1 deficient mice exhibit a survival advantage, and lower viral titers after infection. This was accompanied with enhanced inflammatory cell infiltration during IAV infection. ANXA1 expression is increased during influenza infection clinically, in vivo and in vitro. The presence of ANXA1 enhances viral replication, influences virus binding, and enhances endosomal trafficking of the virus to the nucleus. ANXA1 colocalizes with early and late endosomes near the nucleus, and enhances nuclear accumulation of viral nucleoprotein. In addition, ANXA1 enhances IAV-mediated apoptosis. Overall, our study demonstrates that ANXA1 plays an important role in influenza virus replication and propagation through various mechanisms and that we predict that the regulation of ANXA1 expression during IAV infection may be a viral strategy to enhance its infectivity.Cell Death and Differentiation advance online publication, 4 March 2016; doi:10.1038/cdd.2016.19.
... As targeting estrogen signaling is an unlikely route for drug development as a result of the obvious hormone-imbalance effect, this study sought to explore further male-female differences in the inflammatory response. We investigated sex differences in the sources of recruited leukocytes by examining BM and spleen storage pools and probed further into the inflammatory response by also focusing on other leukocyte subsets that use the wellcharacterized murine zymosan-induced peritonitis model of resolving inflammation [4][5][6]. ...
Sexual dimorphisms exist in the incidence and severity of many diseases, with females demonstrating relative protection from inflammatory conditions. The extent and mechanisms by which excessive leukocyte recruitment underlies these differences are not well established, and better understanding is essential for the development of targeted therapies. Here, we set out to compare the male and female inflammatory response in a murine zymosan-induced peritonitis model and to understand how leukocyte subsets are mobilized from storage pools in both sexes. This work shows that female C57BL/6 mice recruit fewer classical monocytes and neutrophils during zymosan-induced peritonitis. In addition, sex differences were evident in the circulation, as female mice showed reduced neutrophilia and monocytosis vs. male counterparts, despite having similar mobilization from BM stores. Importantly, we show that storage and trafficking of splenic leukocytes during acute inflammation are distinct between the sexes. Male mice have greater splenic stores of neutrophils and classical and nonclassical monocytes, despite similar spleen sizes, signifying another source of potential pathogenic leukocytes. This work demonstrates that males and females have distinct leukocyte-trafficking profiles in acute inflammation and suggests that the spleen, not the BM, plays a role in determining sex differences in the available pool of immune cells. Such dimorphisms demonstrate the importance of considering gender in assay development, drug design, and clinical trials.
© Society for Leukocyte Biology.
... In this way, treatment with the glucocorticoid dexamethasone was used as a control in the experiments conducted in vivo. Getting et al. (1997) demonstrated that dexamethasone inhibited the infiltration of neutrophils and monocytes in an acute inflammatory model, induced by zymosan, through a mechanism mediated by the ANXA1 protein. ...
Endogenous glucocorticoids are pro-resolving mediators, an example of which is the endogenous glucocorticoid-regulated protein Annexin A1 (AnxA1). Since silicosis is an occupational lung disease characterized by unabated inflammation and fibrosis, in this study we tested the therapeutic properties of the N-terminal AnxA1-derived peptide Ac2-26 on experimental silicosis.
Swiss-Webster mice received silica particles intranasally and were subsequently treated with intranasal peptide Ac2-26 (200 μg.mouse(-1) ) or dexamethasone (25 μg.mouse(-1) ) for 7 days, starting 6 h post-challenge. Peptide Ac2-26 abolished leukocyte infiltration, collagen deposition, granuloma formation and the generation of pro-inflammatory cytokines following silica provocation, readouts only partially inhibited by dexamethasone.
A clear exacerbation of these pathological changes was observed in AnxA1 knockout mice as compared to the wild-type littermate controls. Lung fibroblasts from WT mice, but not from formyl peptide receptor (Fpr) type 1 knockout, had the IL-13 or TGFβ-induced production of CCL2/MCP-1 and collagen reduced after incubation with peptide Ac2-26 in vitro. This compound also inhibited the production of CCL2/MCP-1 from fibroblasts of Fpr2 knockout mice.
Collectively, our findings unveil novel protective properties of the AnxA1 derivative peptide Ac2-26 on the inflammatory and fibrotic responses promoted by silica, and suggest that AnxA1 mimetic agents might be a promising strategy in innovative anti-fibrotic approaches for treatment of silicosis.
This article is protected by copyright. All rights reserved.
... Annexin1 inhibits PLA 2 , COX-2, and iNOS expression and activates formyl peptide and lipoxin A 4 receptors as well as inducing IL-10 and clearance of apoptotic cells (44). Such antiinflammatory effects of annexin1 have been shown to ameliorate diseases such as endotoxemia and peritonitis by suppressing leukocyte activation and transmigration (45,46). Furthermore, it has been shown that activation of T cells leads to secretion of annexin1, which binds to its receptor (formyl peptide receptor [FPR]) in an autocrine/paracrine manner, promoting activation and differentiation of T cells. ...
The phagocytosis of apoptotic cells and associated vesicles (efferocytosis) by DCs is an important mechanism for both self tolerance and host defense. Although some of the engulfment ligands involved in efferocytosis have been identified and studied in vitro, the contributions of these ligands in vivo remain ill defined. Here, we determined that during Mycobacterium tuberculosis (Mtb) infection, the engulfment ligand annexin1 is an important mediator in DC cross-presentation that increases efferocytosis in DCs and intrinsically enhances the capacity of the DC antigen-presenting machinery. Annexin1-deficient mice were highly susceptible to Mtb infection and showed an impaired Mtb antigen-specific CD8+ T cell response. Importantly, annexin1 expression was greatly downregulated in Mtb-infected human blood monocyte-derived DCs, indicating that reduction of annexin1 is a critical mechanism for immune evasion by Mtb. Collectively, these data indicate that annexin1 is essential in immunity to Mtb infection and mediates the power of DC efferocytosis and cross-presentation.
... There are a plethora of examples of these anti-inflammatory actions observed with administration of either exogenous ANX-A1 or its commonly-studied, biologically active, Nterminal peptide N-terminal-derived annexin-A1 peptide (acetyl-AMVSEFLKQAWIENEEQEYVVQTVK), Ac2-26, in a broad range of animal models of inflammation. These include inhibition of: (i) cytokineinduced leukocyte migration (mouse air-pouch model ); (ii) carrageenan-induced acute inflammation (rat paw edema, murine peritonitis and arthritis (Cirino et al., 1993;Getting et al., 1997;Yang et al., 1997)); (iii) acute I-R injury (rat heart, kidney, murine mesenteric, brain, and intestinal microcirculation (La et al., 2001a,b;Gavins et al., 2003;Gavins et al., 2007;Souza et al., 2007;Facio et al., 2011)); and (iv) allergen (ovalbumin)-provoked inflammatory responses (rat pleurisy, including mast cell degranulation and plasma protein leakage (Bandeira-Melo et al., 2005)). Ac2-26 also enhances healing of acetic acid-induced gastric ulcers (Martin et al., 2008). ...
... Analogie zu der enzymatischen Inaktivierung von Chemokinen und Hormonen (Struyf et al. 2003;Scholzen & Luger 2004) (Getting et al. 1997;Harris et al. 1995 ...
... And although dexamethasone treatment might reduce LPL expression, suppressed ingress of both monocytes and macrophages are known sources of LPL into the cochlea. Dexamethasone has been reported to reduce monocyte recruitment in the rat [18] and importantly has been demonstrated to reduce macrophage presence following electrode insertion in the cochlea of guinea pig [19]. ...
A cochlear implant is an indispensable apparatus for a profound hearing loss patient. But insertion of the electrode entails a great deal of stress to the cochlea, and may cause irreversible damage to hair cells and related nerve structure. Although damage prevention effects of dexamethasone have been reported, long-term administration is difficult. In this study, we used a dexamethasone-eluting electrode in the guinea pig cochlea, and compared the gene expression after 7 days insertion with that of a normal electrode and non-surgically treated control by microarray. 40 genes were up-regulated 2-fold or more in the normal electrode group compared to the non-surgically treated group. Most of the up-regulated genes were associated with immune response and inflammation. In the dexamethasone-eluting group, compared to the normal electrode group, 7 of the 40 genes were further up-regulated, while 12 of them were down-regulated and there was a tendency to return to the non-surgical condition. 9 genes were down-regulated 2-fold or less with normal electrode insertion, and 4 of the 9 tended to return to the non-surgical condition in the dexamethasone-eluting group. These genes are certainly involved in the maintenance of the physiological functions of the cochlea. Our results indicate that the dexamethasone-eluting electrode will have an effect on the normalization of homeostasis in the cochlea.
... In vivo the Ac2-26 peptide has been shown to exert an anti-inflammatory effect in models of myocardial ischaemia reperfusion (I/R) (La, D'Amico et al. 2001), mesentery I/R (Gavins, Yona et al. 2003), glycogen peritonitis (Teixeira, Das et al. 1998) and IL1β airpouch (Perretti, Ahluwalia et al. 1993), where it was reported to significantly reduce the recruitment of neutrophils to the site of injury/inflammation (Perretti and Flower 1993). In other more complex acute models such as zymosan peritonitis apart from a reduction in PMN recruitment at an early time point (4h) a reduction of monocyte recruitment into the peritoneal cavity after 24h was also reported (Getting, Flower et al. 1997). ...
... 7 At the cellular level, Anx-A1 is implicated in the control of cell growth 8 and differentiation, 9 signal transduction and arachidonic acid release, 10,3 as well as vesicle trafficking. [11][12][13] In vivo, Anx-A1 is implicated in the glucocorticoid regulation of leukocyte migration, 14 acute 15 and chronic 16 inflammation, ischaemic damage, [17][18][19] pain, 20 fever, 21 HPA activity 22 and other aspects of neuroendocrine function, notably the control of growth hormone and prolactin. 23,24 There have been suggestions that Anx-A1 is involved in human disease. ...
... Inhibition of leukocyte migration was due to diminished macrophage and lymphocyte recruitment whereas no effect was seen in the neutrophil subset. Weak effect on neutrophils might be due to late time point analyzed (18h after induction of inflammation) as neutrophil numbers have been shown to peak as early as 4 hours after zymosan induced peritonitis (Getting et al., 1997). Determination of which leukocytes will eventually infiltrate target tissue can be affected by any step of the leukocyte extravasation process although transmigration forms the final obstacle. ...
... Annexin A1 (AnxA1), a 37-kDa protein, the first-characterized member of the family, was originally reported for its antiphospholipase activity after glucocorticoid induction (4,5). Subsequent studies showed that both recombinant AnxA1 and AnxA1-derived N-terminal peptides possess a wide range of anti-inflammatory properties (6)(7)(8). In addition to the regulatory region, the Nterminus also contains the sites for phosphorylation (3). ...
Unlabelled:
Annexin A1 (AnxA1), a phospholipid-binding protein and regulator of glucocorticoid-induced inflammatory signaling, has implications in cancer. Here, a role for AnxA1 in prostate adenocarcinoma was determined using primary cultures and a tumor cell line (cE1), all derived from the conditional Pten deletion mouse model of prostate cancer. AnxA1 secretion by prostate-derived cancer-associated fibroblasts (CAF) was significantly higher than by normal prostate fibroblasts (NPF). Prostate tumor cells were sorted to enrich for epithelial subpopulations based on nonhematopoietic lineage, high SCA-1, and high or medium levels of CD49f. Compared with controls, AnxA1 enhanced stem cell-like properties in high- and medium-expression subpopulations of sorted cE1 and primary cells, in vitro, through formation of greater number of spheroids with increased complexity, and in vivo, through generation of more, larger, and histologically complex glandular structures, along with increased expression of p63, a basal/progenitor marker. The differentiated medium-expression subpopulations from cE1 and primary cells were most susceptible to gain stem cell-like properties as shown by increased spheroid and glandular formation. Further supporting this increased plasticity, AnxA1 was shown to regulate epithelial-to-mesenchymal transition in cE1 cells. These results suggest that CAF-secreted AnxA1 contributes to tumor stem cell dynamics via two separate but complementary pathways: induction of a dedifferentiation process leading to generation of stem-like cells from a subpopulation of cancer epithelial cells and stimulation of proliferation and differentiation of the cancer stem-like cells.
Implications:
AnxA1 participates in a paradigm in which malignant prostate epithelial cells that are not cancer stem cells are induced to gain cancer stem cell-like properties.
... Since its discovery, our work, together with that from other laboratories, has implicated Anx-1 in control of cell growth (30) and differentiation (31), signal transduction and arachidonic acid release (32,33), as well as intracellular vesicle trafficking (34)(35)(36). In mammals, glucocorticoids regulate the synthesis, phosphorylation and cellular disposition of Anx-1 and work from our laboratory, as well as from others, has provided further evidence for the involvement of Anx-1 in the regulation by these drugs of leukocyte migration (37), acute (38) and chronic (39) inflammation, ischaemic damage (40)(41)(42), pain (43) and fever (44). ...
... Annexin 1 and its derived N-terminus peptide Ac2-26 are well known to exert anti-inflammatory activities in inflammatory cells such as neutrophils and macrophages [30,31,32]. Information about their actions in other cells that are important to inflammation particularly endothelial cells is limited [33]. ...
The anti-inflammatory peptide annexin-1 binds to formyl peptide receptors (FPR) but little is known about its mechanism of action in the vasculature. Here we investigate the effect of annexin peptide Ac2-26 on NADPH oxidase activity induced by tumour necrosis factor alpha (TNFα) in human endothelial cells. Superoxide release and intracellular reactive oxygen species (ROS) production from NADPH oxidase was measured with lucigenin-enhanced chemiluminescence and 2',7'-dichlorodihydrofluorescein diacetate, respectively. Expression of NADPH oxidase subunits and intracellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) were determined by real-time PCR and Western blot analysis. Promoter activity of nuclear factor kappa B (NFκB) was measured by luciferase activity assay. TNFα stimulated NADPH-dependent superoxide release, total ROS formation and expression of ICAM-1and VCAM-1. Pre-treatment with N-terminal peptide of annexin-1 (Ac2-26, 0.5-1.5 µM) reduced all these effects, and the inhibition was blocked by the FPRL-1 antagonist WRW4. Furthermore, TNFα-induced NFκB promoter activity was attenuated by both Ac2-26 and NADPH oxidase inhibitor diphenyliodonium (DPI). Surprisingly, Nox4 gene expression was reduced by TNFα whilst expression of Nox2, p22phox and p67phox remained unchanged. Inhibition of NADPH oxidase activity by either dominant negative Rac1 (N17Rac1) or DPI significantly attenuated TNFα-induced ICAM-1and VCAM-1 expression. Ac2-26 failed to suppress further TNFα-induced expression of ICAM-1 and VCAM-1 in N17Rac1-transfected cells. Thus, Ac2-26 peptide inhibits TNFα-activated, Rac1-dependent NADPH oxidase derived ROS formation, attenuates NFκB pathways and ICAM-1 and VCAM-1 expression in endothelial cells. This suggests that Ac2-26 peptide blocks NADPH oxidase activity and has anti-inflammatory properties in the vasculature which contributes to modulate in reperfusion injury inflammation and vascular disease.
... AnxA1 (also known as lipocortin-1) is a 37-kDa calcium-dependent phospholipid-binding protein from the annexin superfamily that binds to and activates FPR2/ALXR, a receptor also shared with LXs (Perretti & D'Acquisto, 2009). AnxA1 is a known anti-inflammatory molecule, with its activities studied predominantly in the context of inhibition of pro-inflammatory PGs and leukocyte recruitment in various models of inflammation ( Getting et al., 1997;Bandeira-Melo et al., 2005;Babbin et al., 2008). AnxA1 knockout mice show exacerbated inflammatory responses and are resistant to some of the antiinflammatory effects of GCs ( Hannon et al., 2002;Yang et al., 2004). ...
... In inflammatory models, exogenous ANX1 inhibited recruitment of polymorphonuclear (PMN) leukocytes in vivo (5,6). Moreover, treatments with Ab to ANX1 blocked the anti-inflammatory effect of dexamethasone in animal models of acute inflammation (7). In these models, the inhibition of cell migration to inflammatory sites appeared to be one of the mechanisms by which GCs exert their anti-inflammatory effect. ...
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 a4 integrin,
and a direct interaction between ANX1 and the a4 integrin could be documented by immunoprecipitation experiments. Moreover,
ANX1 competed with the endothelial integrin counterreceptor, VCAM-1, for binding to a4 integrin. These results indicate that ANX1 plays an important physiological role in modulating monocyte firm adhesion to the endothelium. The Journal of Immunology,
2000, 165: 1573–1581.
Background
Neutrophil depletion strategies remain fraught with the inability to achieve deep neutrophil clearance from the circulation and issues surrounding accurate neutrophil detection. Boivin et al. have demonstrated an isotype switch method of achieving significantly profound neutrophil clearance using a combination of anti-Ly6G and anti-rat κ Ig light chain antibodies in adult C57/Bl6 mice, proven by extra- and intracellular Ly6G detection by flow cytometry. We adapted this technique to neonatal mice to test four neutrophil depletion strategies.
Methods
Four protocols were tested: P3 Ly6G and P1-3 Ly6G (anti-Ly6G antibody on postnatal days (P) 3 and 1-3 respectively), and P3 Dual and P1-3 Dual (anti-Ly6G and anti-rat κ Ig light chain antibodies on P3 and P1-3 respectively). Both intracellular and extracellular Ly6G presence was detected using flow cytometry. Isotype control antibodies were used for control experiments.
Results
P1-3 Dual achieved significantly better neutrophil depletion than the P1-3 Ly6G or P3 Ly6G protocols (97% vs 74% and 97% vs 50%, respectively). The P3 Dual protocol alone was enough to achieve significantly better neutrophil clearance (93%) than any of the Ly6G alone protocols. The Ly6G alone protocols led to near-total elimination of extracellular Ly6G, but there was a significant presence of intracellular Ly6G in the CD45+ cell population, which would evade detection by conventional fluorophore-conjugated Ly6G antibody-based detection methods.
Discussion
Anti-Ly6G antibody alone is ineffective in producing effective circulating neutrophil clearance in neonatal mice. Dual antibody-based neutrophil depletion strategies achieve>90% clearance in postnatal day four mice. A single day of dual antibody treatment can achieve 93% neutrophil depletion in neonatal mice and be a better alternative to daily anti-Ly6g antibody injections.
Annexin A1 (ANXA1) is an endogenous protein, which plays a central function in the modulation of inflammation. While the functions of ANXA1 and its exogenous peptidomimetics, N-Acetyl 2-26 ANXA1-derived peptide (ANXA1Ac2-26), in the modulation of immunological responses of neutrophils and monocytes have been investigated in detail, their effects on the modulation of platelet reactivity, haemostasis, thrombosis, and platelet-mediated inflammation remain largely unknown. Here, we demonstrate that the deletion of Anxa1 in mice upregulates the expression of its receptor, formyl peptide receptor 2/3 (Fpr2/3, orthologue of human FPR2/ALX). As a result, the addition of ANXA1Ac2-26 to platelets exerts an activatory role in platelets, as characterised by its ability to increase the levels of fibrinogen binding and the exposure of P-selectin on the surface. Moreover, ANXA1Ac2-26 increased the development of platelet-leukocyte aggregates in whole blood. The experiments carried out using a pharmacological inhibitor (WRW4) for FPR2/ALX, and platelets isolated from Fpr2/3-deficient mice ascertained that the actions of ANXA1Ac2-26 are largely mediated through Fpr2/3 in platelets. Together, this study demonstrates that in addition to its ability to modulate inflammatory responses via leukocytes, ANXA1 modulates platelet function, which may influence thrombosis, haemostasis, and platelet-mediated inflammation under various pathophysiological settings.
Identification of differentially expressed proteins in antiphospholipid syndrome (APS) is a developing area of research for unique profiles of this pathology. Advances in technologies of mass spectrometry brings improvements in proteomics and results in assessment of soluble or cellular proteins which could be candidates for clinical biomarkers of primary APS. The use of blood as a source of proteins ease the acquisition of samples for proteomics analyses and later for disease diagnosis. We performed a systematic review to explore the proteomics studies carried out in circulating released proteins (serum, plasma) or cellular proteins (monocytes and platelets) of APS patients. The study groups differentiate among clinical APS cases with the aim to translate molecular findings to disease stratification and to improve APS diagnosis and prognosis. These studies also include the unravelling of new autoantibodies in non-criteria APS or how post-translational protein modifications provides clues about the pathological mechanisms of antigen-autoantibody recognition. Herein, we identified 82 proteins that were dysregulated in APS across eleven studies. Enrichment analysis revealed its connection to cellular activation and degranulation that eventually leads to thrombosis as the main biological process highlighted by these studies. Validation of APS-relevant proteins by functional and mechanistic studies will be essential for patient stratification and the development of targeted therapies for every clinical subtype of APS.
Uncontrolled inflammation is a leading cause of many clinically relevant diseases. Current therapeutic strategies focus mainly on immunosuppression rather than on the mechanisms of inflammatory resolution. Glucocorticoids (GCs) are still the most widely used anti-inflammatory drugs. GCs affect most immune cells but there is growing evidence for cell type specific mechanisms. Different subtypes of monocytes and macrophages play a pivotal role both in generation as well as resolution of inflammation. Activation of these cells by microbial products or endogenous danger signals results in production of pro-inflammatory mediators and initiation of an inflammatory response. GCs efficiently inhibit these processes by down-regulating pro-inflammatory mediators from macrophages and monocytes. On the other hand, GCs act on “naïve” monocytes and macrophages and induce anti-inflammatory mediators and differentiation of anti-inflammatory phenotypes. GC-induced anti-inflammatory monocytes have an increased ability to migrate toward inflammatory stimuli. They remove endo- and exogenous danger signals by an increased phagocytic capacity, produce anti-inflammatory mediators and limit T-cell activation. Thus, GCs limit amplification of inflammation by repressing pro-inflammatory macrophage activation and additionally induce anti-inflammatory monocyte and macrophage populations actively promoting resolution of inflammation. Further investigation of these mechanisms should lead to the development of novel therapeutic strategies to modulate undesirable inflammation with fewer side effects via induction of inflammatory resolution rather than non-specific immunosuppression.
Annexin A1 (ANXA1) has long been classed as an anti-inflammatory protein due to its control over leukocyte-mediated immune responses. However, it is now recognized that ANXA1 has widespread effects beyond the immune system with implications in maintaining the homeostatic environment within the entire body due to its ability to affect cellular signalling, hormonal secretion, foetal development, the aging process and development of disease. In this review, we aim to provide a global overview of the role of ANXA1 covering aspects of peripheral and central inflammation, immune repair and endocrine control with focus on the prognostic, diagnostic and therapeutic potential of the molecule in cancer, neurodegeneration and inflammatory-based disorders.
Annexin A1 (AnxA1) is a glucocorticoidregulated protein known for its antiinflammatory and proresolving effects. We have previously shown that cAMP enhancing compounds rolipram (ROL - a PDE4 inhibitor) and db-cAMP (cAMP mimetic) drive caspasedependent resolution of neutrophilic inflammation. In this follow up study, we investigated whether AnxA1 could be involved in the proresolving properties of these compounds using a model of LPS-induced inflammation in BALB/c mice. The treatment with ROL or db-cAMP at the peak of inflammation shortened resolution intervals, improved resolution indices and increased AnxA1 expression. In vitro studies showed that ROL and db-cAMP induced AnxA1 expression and phosphorylation and this effect was prevented by PKA inhibitors, suggesting the involvement of PKA on ROL-induced AnxA1 expression. Akin to these in vitro findings, H89 prevented ROL and db-cAMP-induced resolution of inflammation, and it was associated with decreased levels of intact AnxA1. Moreover, two different strategies to block the AnxA1 pathway - by using BOC-1 a nonselective AnxA1 receptor antagonist or by using an anti-AnxA1 neutralizing antiserum -prevented ROL and db-cAMP-induced resolution and neutrophil apoptosis. Likewise, the ability of ROL or db-cAMP to induce neutrophil apoptosis was impaired in AnxA knockout mice. Finally, in in vitro settings ROL and db-cAMP overrode the survival-inducing effect of LPS in human neutrophils in an AnxA1dependent manner. Our results show that AnxA1 is at least one of the endogenous determinants mediating the proresolving properties of cAMP elevating agents and cAMPmimetic drugs.
These anti-inflammatory drugs that are used to treat acute gout are discussed in detail. Their administration, pharmacology, and toxicity are considered. Then, urate-lowering therapy is thoroughly described, again considering the administration, pharmacology, and toxicity of these agents. The widespread mismanagement of gout in general and even specialty medical practice makes this information important for patients and their physicians.
Significance
Reduced bioavailable nitric oxide generated within the vascular wall is a key pathogenic mechanism involved in the formation and rupture of an atheromatous plaque, the latter leading to myocardial infarction or stroke. We show that inorganic nitrate supplementation through delivery of nitric oxide reduces systemic leukocyte rolling and adherence, circulating neutrophil numbers, and monocyte activation through direct repression of neutrophil activation and up-regulation of interleukin-10–dependent antiinflammatory pathways in athero-prone mice. These antiinflammatory effects of inorganic nitrate result in reduced macrophage content of atherosclerotic plaques coupled with an elevation of smooth muscle content, resulting in a stable plaque phenotype. Our data suggest that inorganic nitrate supplementation has antiinflammatory effects, which may have clinical utility in the prophylaxis of atheroma development.
Im Rahmen der Embryonalentwicklung oder der Gewebshomöostase entstehen kontinuierlich apoptotische Zellen, die effizient von professionellen Phagozyten, aber auch von nicht-professionellen Phagozyten aufgenommen werden. Im Gegensatz zur Aufnahme von Pathogenen ist die Phagozytose apoptotischer Zellen (Efferozytose) ein anti-inflammatorischer Prozess. Somit trägt die Efferozytose essentiell zur Erhaltung der peripheren Toleranz bei, indem sie Immunreaktionen gegen Autoantigene verhindert. Die molekularen Mechanismen des immunsuppressiven Effekts der Efferozytose sind bis heute nur unzureichend aufgeklärt. Annexin A1 (AnxA1), ein intrazelluläres, Phospholipid-bindendes Protein, gehört zu den beschriebenen eat me-Signalen und transloziert an die Membran früh-apoptotischer Zellen. Darüber hinaus ist AnxA1 in der Lage, die Toll like Rezeptor (TLR)-vermittelte, pro-inflammatorische Antwort in dendritischen Zellen (DC) zu supprimieren. Die Schlüsselfrage nach dem AnxA1-Rezeptor, der diese Immunsuppression auf DC vermittelt, blieb bislang jedoch unbeantwortet. Im Rahmen der vorliegenden Arbeit wurde deswegen eine neue UV-Kreuzvernetzungsmethode etabliert, um die Bindung von AnxA1 an seinen putativen Rezeptor mittels einer kovalenten Bindung zu stabilisieren. Dadurch konnte das Low density lipoprotein receptor-related protein 1 (LRP1) als potentieller AnxA1-Rezeptor identifiziert werden. Die Interaktion von LRP1 mit AnxA1, sowie mit weiteren Annexinen, wurde anhand verschiedenster Bindungsstudien in vitro validiert. Des Weiteren ermittelten quartz crystal mircobalance-Studien eine hohe, Calcium-abhängige Bindungsaffinität von LRP1 an verschiedene Annexine. Anhand DC- und Makrophagen-spezifischer LRP1 knock out-Mäuse wurde die Rolle von LRP1 auf die AnxA1-vermittelte Immunsuppression untersucht. In dieser Studie konnte erstmalig gezeigt werden, dass AnxA1, neben der Suppression in DC, auch die TLR-induzierte Antwort in Makrophagen supprimiert. Diese Immunsuppression erfolgte sowohl in Makrophagen als auch in DC unabhängig von LRP1. Da LRP1 und AnxA1 im Kontext der Efferozytose beschrieben sind, wurde anschließend die Relevanz der Interaktion für die Phagozytose apoptotischer Zellen untersucht. Durch die putative Redundanz von eat me-Signalen und deren Rezeptoren konnte kein Einfluss des AnxA1-LRP1-Komplexes auf die Efferozytose nachgewiesen werden. Jedoch konnte in einem System, in dem einzelne eat me-Signale isoliert betrachtet wurden, gezeigt werden, dass LRP1 die Phagozytose von AnxA1-gekoppelten Beads in DC und nicht-professionellen Phagozyten beeinflusst. Zusammengefasst konnte in der vorliegenden Arbeit erstmalig LRP1 als neuer Rezeptor für Annexine identifiziert und charakterisiert werden. Die AnxA1-LRP1-Interaktion vermittelt dabei nicht die Toleranzinduktion apoptotischer Zellen, sondern spielt eine wichtige Rolle bei Phagozytose-Prozessen. Durch die gezielte Manipulation der Interaktion von LRP1 mit verschiedenen Annexinen in Patienten mit Autoimmunerkrankungen, die auf einer defekten Efferozytose beruhen, könnte der AnxA1-LRP1-Komplex im therapeutischen Kontext von großem Interesse sein.
The role of Annexin A1 (ANXA1) in counter-regulating the activities of innate immune cells, such as the migration of neutrophils and monocytes, and the generation of pro-inflammatory mediators in various models of inflammatory and autoimmune diseases is well documented. However, while ANXA1 has been proposed as an important mediator of the adaptive immune response, its involvement in this respect has been less studied. Furthermore, while there have been numerous studies on the role of ANXA1 in inflammatory diseases, less has been reported on its influence in immunity against infection. A recent study reported a link between ANXA1 and tuberculosis, and proposed a model in which Mycobacterium tuberculosis exerts its virulence by manipulating the ANXA1-mediated host apoptotic response. This has prompted us to further investigate the role of ANXA1 in the pathogenesis of tuberculosis in vivo. Here, we show that ANXA1(-/-) mice are more susceptible to M. tuberculosis infection, as evidenced by a transient increase in the pulmonary bacterial burden, and exacerbated and disorganized granulomatous inflammation. These pathological manifestations correlated with an impaired ability of ANXA1(-/-) dendritic cells to activate naïve T cells, thereby supporting a role for ANXA1 in shaping the adaptive immunity against M. tuberculosis.
Copyright © 2014. Published by Elsevier GmbH.
Inflammation is a physiological response of the immune system to injury or infection but may become chronic. In general, inflammation is self-limiting and resolves by activating a termination program named resolution of inflammation. It has been argued that unresolved inflammation may be the basis of a variety of chronic inflammatory diseases. Resolution of inflammation is an active process that is fine-tuned by the production of proresolving mediators and the shutdown of intracellular signaling molecules associated with cytokine production and leukocyte survival. Apoptosis of leukocytes (especially granulocytes) is a key element in the resolution of inflammation and several signaling molecules are thought to be involved in this process. Here, we explore key signaling molecules and some mediators that are crucial regulators of leukocyte survival
in vivo
and that may be targeted for therapeutic purposes in the context of chronic inflammatory diseases.
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.
Most anti-inflammatory agents used in the treatment of joint diseases exert inhibitory effects on leukocyte infiltration. Methotrexate, a disease-modifying drug, and corticosteroids also inhibit leukocyte accumulation during inflammation. However, the mechanisms of action of these different compounds on leukocytes vary and in the case of non-steroidal anti-inflammatory drugs (NSAIDs) the mechanism(s) may be indirect. No current drug for inflammatory or degenerative joint disease has been proposed to act specifically by an inhibitory action on neutrophilic leukocytes. Oxaceprol is an amino acid derivative that has been used for several years for the treatment of osteoarthritis and rheumatoid arthritis, ameliorating pain and stiffness and showing good gastrointestinal safety, particularly in comparison with NSAIDs. Recent experimental studies have shown that oxaceprol does not inhibit the synthesis of prostaglandins in vitro, but markedly inhibits neutrophil infiltration into the joints of rats with adjuvant arthritis. These results support earlier screening data showing inhibition by oxaceprol of leukocyte infiltration into sites of acute inflammation. In studies on surgical ischemia reperfusion in hamsters in vivo, oxaceprol was an effective inhibitor of leukocyte adhesion and extravasation. It is proposed that oxaceprol represents a therapeutic agent for degenerative and inflammatory joint diseases, which acts predominantly by inhibiting leukocyte adhesion and migration.
The effects of glucocorticoids on biochemical functions of macrophages from man, mouse, rabbit, and guinea pig were examined. Secretion of plasminogen activator by human peripheral blood monocytes was decreased by 50% with 1 nM dexamethasone. Differentiation of murine monocytic and granulocytic colonies in agar from bone marrow precursors was decreased by 50% at 7 days with 20 nM dexamethasone. Secretion of elastase, collagenase, and plasminogen activator by resident and thioglycollate-elicited mouse peritoneal macrophages was decreased by dexamethasone, cortisol, and triamcinolone acetonide (1--1,000 nM), but not by progesterone, estradiol, and dihydrotestosterone (1,000 nM); in contrast, secretion of lysozyme was not affected by glucocorticoids or other steroids. The inhibition of macrophage secretion by dexamethasone was both time and dose dependent. Effects were detected within 1--6 h after addition of the glucocorticoids, became maximum by 24 h, and were reversed during a similar time period after removal of the hormones. The extent of inhibition of macrophage secretion increased with increasing glucocorticoid concentration. Half-maximum inhibition of secretion of elastase, collagenase, and plasminogen activator was seen at dexamethasone concentrations (1--10 nM) similar to those that half-saturated the specific glucocorticoid receptors in these cells. At high concentrations of dexamethasone (100--1,000 nM) the secretion of plasminogen activator was inhibited to a greater extent (greater than 95%) than the secretion of elastase (60--80%). Progesterone alone had no effect on secretion, but it blocked the inhibitory effects of dexamethasone and cortisol. Secretion of collagenase, neutral proteinases, and plasminogen activator by elicited rabbit alveolar macrophages was inhibited with glucocorticoids (0.1--100 nM) but not with progesterone or sex steroids. Secretion of a neutral elastinolytic proteinase by guinea pig alveolar macrophages was also inhibited by dexamethasone. These data support the regulatory role of glucocorticoids on macrophage functions at physiological concentrations.
Glucocorticoids inhibit superoxide (O2-) generation by phagocytes through a mechanism that remains unclear. We investigated this effect by using dexamethasone on guinea pig alveolar macrophages. O2- generation was induced either by the calcium ionophore A23187, a potent stimulus of phospholipase A2, or by the protein kinase C activator, phorbol myristate acetate (PMA). Dexamethasone inhibited O2- generation initiated by A23187 by 50-55%. This inhibition required: (a) more than 45 min incubation and was maximal after 2 h; (b) glucocorticoid receptor occupancy; and (c) protein synthesis. The inhibitory effect of dexamethasone could not be explained by an interaction with the respiratory burst enzyme NADPH oxidase since O2- generation was only weakly affected upon PMA stimulation. Lipocortin I, a glucocorticoid inducible and phospholipase A2 inhibitory protein, inhibited O2- generation initiated by A23187 but failed to modulate the respiratory burst activated by PMA. These results were obtained with lipocortin I purified from mouse lungs, human blood mononuclear cells, and with human recombinant lipocortin I. We propose that lipocortin I is capable of inhibiting the activation of NADPH oxidase only when membrane signal transduction involves phospholipase A2. By mimicking the effect of dexamethasone, lipocortin I may extend its potential anti-inflammatory action to the partial control of the formation of oxygen reactive species by phagocytes.
Recombinant human annexin I and a monoclonal antibody specific for this protein (mAb 1B) were used to investigate surface binding of this member of the annexin family of proteins to peripheral blood monocytes. Flow cytometric analysis demonstrated trypsin-sensitive, saturable binding of annexin I to human peripheral blood monocytes but not to admixed lymphocytes. A monoclonal antibody that blocks the anti-phospholipase activity of annexin I also blocked its binding to monocytes. These findings suggest the presence of specific binding sites on monocytes. Furthermore, surface iodination, immunoprecipitation and SDS/PAGE analysis were used to identify two annexin I-binding proteins on the surface of monocytes with molecular masses of 15 kDa and 18 kDa respectively. The identification and characterization of these annexin I-binding molecules should help us to better understand the specific interactions of annexin I with monocytes that lead to down-regulation of pro-inflammatory cell functions.
1Lipocortin-1 and its N-terminal derivatives exert potent inhibitory actions in various models of acute inflammation. The present study examined the ability of lipocortin (LC)-1 to suppress the release of the acute pro-inflammatory mediators, tumour necrosis factor (TNFα) and prostaglandin E2 (PGE2) from human peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide (LPS) or recombinant human interleukin-1β (rhIL-1β).2LPS (10 μg ml−1)-stimulated release of TNFα and PGE2 from PBMC was significantly inhibited by (4 h) co-incubation of the cells with 10−6m dexamethasone (Dex), but not with 10−9m to 10−7m of a N-terminal fragment (amino acids 1–188) of recombinant human LC-1 (LC-1 fragment). However, Dex suppression of LPS-stimulated TNFα and PGE2 secretion from PBMC was reversed when polyclonal antibody to LC-1 fragment (1:10,000 dilution) was included in the medium. rhIL-1β (5times10−8m)-stimulated release of TNFα and PGE2 from PBMC (after 18 h) was abolished by co-incubation of the cells with 10−7m LC-1 fragment.3After incubation with Dex (4 h), cellular proteins from PBMC were immunoblotted using anti-LC-1 fragment antibody (which showed no cross-reactivity with human annexins 2 to 6). Dex caused no increase in immunoreactive (ir)LC-1 content of PBMC, although there was a three fold increase in the amount of a lower mass species with LC-1-like immunoreactivity. This was accompanied by the appearance of irLC-1 in the extracellular medium.4The results of the present study implicate endogenous LC-1 in glucocorticoid suppression of TNFα and PGE2 release from human PBMC and suggest an extracellular site of action for LC-1. LC-1 may also inhibit rhIL-1β-stimulated TNFα and PGE2 secretion from PBMC.
Macrophages fulfill such functions as (i) housekeeping and scavenging, (ii) protective and defense, and (iii) memory. Glucocorticoids are hormones also used as anti-inflammatory and immuno-suppressive drugs. They act on the many functions of macrophages, mainly by interfering with functions (ii) and (iii). Glucocorticoids interfere with these macrophage functions by modulating the production of inflammatory mediators such as cytokines, phospholipid-derived mediators, proteases, oxygen metabolites. For inducing these effects, glucocorticoids interact with their receptors, transcription factors that recognize specific genomic sequences, glucocorticoid responsive elements (GRE). Glucocorticoids modulate the transcription of genes in association with other transcription factors such as Fos, Jun, CREB, These combinatorial associations--differing according to the differentiation and/or activation state of the cell--may therefore produce a fine-tuning of the induction or repression of genes. These mechanisms shed new light for understanding the complexity of glucocorticoid effects on macrophage function in the inflammatory reaction.
As a putative mediator of inflammation interleukin-1 has been implicated in the recruitment of leukocytes during the early stages of the inflammatory reaction. In the present report we have investigated the release of endogenous IL-1 in the rat zymosan pleurisy and in the mouse zymosan peritonitis. In both cases the release of the cytokine was maximal 4 hours after zymosan injection and appeared to be time-related to neutrophil migration into the inflammatory site. The effect of in vivo treatment with dexamethasone in rat pleurisy and with polyclonal anti-murine IL-1 beta antibody in mouse peritonitis was also assessed. The steroid reduced both cell migration and the release of IL-1-like activity as well as the formation of exudate and the release of eicosanoids. The anti-IL-1 beta serum inhibited selectively the number of neutrophil that migrated to the inflamed site (approximately 40%) and the IL-1 activity recovered in (approximately 70%) the exudate. In vitro incubation of the inflammatory exudate with polyclonal anti-murine IL-1 alpha or anti-murine IL-1 beta sera allowed the identification of the IL-1 species present. In the rat pleurisy IL-1 biological activity was mainly due to the alpha species, whereas IL-1 beta was the only species apparently present in the mouse peritoneal exudate.
The effect of dexamethasone on mRNA and protein synthesis of lipocortins (LCT) 1, 2 and 5 has been investigated in U-937 cells. A constitutive expression of both mRNAs and proteins was detected in undifferentiated U-937 cells. This constitutive level was increased time- and dose-dependently by incubation with phorbol myristate acetate (PMA). In U-937 cells differentiated by 24 h incubation with 6 ng/ml PMA, dexamethasone (DEX) (1 microM for 16 h) caused an increased synthesis of the mRNA level of LCT-1 and 2, but not of LCT-5, over the level induced by PMA. DEX had no effect in undifferentiated cells. Moreover, DEX stimulated the extracellular release of LCT-1 and 5, but not of LCT-2, and inhibited the release of PGE2 and TXB2 only in the differentiated U-937 cells. These results suggest that the responsiveness of these cells to glucocorticoids is dependent on the phase of cell differentiation. The selective release of lipocortins by differentiated U-937 cells may explain, at least in part, the inhibition by DEX of the prostanoid release.
The presence and amount of the anti-inflammatory protein lipocortin 1 was determined in plasma and peripheral blood leucocytes by a highly specific, enzyme-linked immunosorbent assay. Within 120 min of a single intravenous dose of 100 mg hydrocortisone, the intracellular concentrations of lipocortin 1 in peripheral monocytes in 7 of 8 healthy men increased by a median of 225% (range 129-507%) compared with pretreatment levels, and mononuclear cell-surface lipocortin increased by a median of 224% (range 76-483%). Placebo injections had no effect. There was no increase at any time in free plasma or polymorph-associated lipocortin. In 3 of 4 subjects, induction of lipocortin was also observed when whole unseparated blood was incubated in vitro after steroid administration, but cells which had first been isolated and purified were refractory to such induction. Thus rapid changes in the concentration of an active anti-inflammatory protein can occur in man after normal therapeutic doses of hydrocortisone.
Anti-inflammatory glucocorticoids inhibit prostaglandin (PG) biosynthesis by preventing arachidonic acid release from phospholipids rather than inhibiting the cyclooxygenase. As in other cells, this steroid action depends on receptor occupation and de novo protein/RNA biosynthesis. We have previously shown in guinea pig perfused lungs and rat peritoneal leukocytes that the effect of steroids in PG generation is mediated by an uncharacterized 'second messenger'. Now, we report that this factor (which we have named 'macrocortin') is an intracellular polypeptide whose release and synthesis are stimulated by steroids. Macrocortin derived from rat peritoneal leukocytes is very similar to that released from guinea pig lungs.
We have studied the ability of the glucocorticoid, dexamethasone, to induce annexin 1 secretion by either human blood monocytes or rat peritoneal leukocytes.
The in vivo treatment of rats with dexamethasone (1.25 mg kg ⁻¹ ) selectively induced secretion of annexin 1 by peritoneal leukocytes, as assessed by incubating these cells in culture medium. Annexin 1 secretion was also induced in human cultured monocytes, in vitro , by 10 ⁻⁶ m dexamethasone.
Annexin 1 secretion was inhibited in the presence of 20 mM NH 4 C1 or by conducting the experiments at 18°C. In contrast, it was not inhibited by monensin, nocodazole or brefeldin A.
The time necessary for annexin 1 synthesis and secretion was less than 15 min.
These data indicate that glucocorticoids induce annexin 1 secretion by monocytes or peritoneal leukocytes. Because it is not inhibited by monensin, nocodazole or brefeldin A and it is rapid, annexin 1 secretion seems to occur by the secretory pathway similar to that used by several cytosolic proteins such as interleukin‐iβ.
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.
Cytokines orchestrate the complex network of cellular interactions that regulate effector cell functions of natural and immune resistance. Although T cells, natural killer (NK) cells and monocytes/macrophages are the main producers of cytokines, a number of reports in the last few years have demonstrated that polymorphonuclear neutrophils (PMN) also have the ability to synthesize and release immunoregulatory cytokines. Here, Marco Cassatella describes novel facets of the regulation of cytokine production by PMN that highlight the involvement of of PMN in cell-cytokine crosstalk.
It has been suggested that the induction of lipocortin-1, a phospholipase A2-inhibitory protein, may mediate the anti-inflammatory action of glucocorticoids. We assessed the production of prostaglandin E2, thromboxane B2, and leukotriene B4 and the expression of lipocortin-1 in different populations of blood leukocytes and in alveolar macrophages (obtained by bronchoalveolar lavage) from patients with inflammatory lung diseases (bronchial asthma, n = 21; interstitial lung disease, n = 6) undergoing glucocorticoid treatment at clinically effective doses. No inhibition of eicosanoid production was observed in either whole blood or single populations of blood leukocytes (granulocytes and monocytes) stimulated with ionophore A-23187, N-formyl-L-methionyl-L-leucyl-L-phenylalanine, or zymosan. Conversely, eicosanoid production from alveolar macrophages (assessed in 9 cases) was significantly inhibited by glucocorticoids. After ionophore stimulation, eicosanoid production was as follows (in ng/ml): prostaglandin E2, 0.19 +/- 0.06 and 0.06 +/- 0.01; thromboxane B2, 2.9 +/- 0.9 and 0.5 +/- 0.1; leukotriene B4, 6.6 +/- 1.1 and 3.6 +/- 1.0, before and after treatment, respectively (P < 0.05 for all differences). Lipocortin-1 expression, determined by Western blot and enzyme immunoassay, was significantly (P < 0.05) stimulated in alveolar macrophages, but not in blood leukocytes, by glucocorticoid treatment. These results indicate that alveolar macrophages, at variance from blood leukocytes, are the most likely cell target for glucocorticoid-induced eicosanoid inhibition and lipocortin expression. We suggest that cell responsiveness to glucocorticoids is acquired during differentiation from monocyte to tissue macrophage.
Lipocortin-1, a 37 kDa member of the annexin superfamily of proteins, originally evoked interest as one of the 'second messengers' of the anti-inflammatory actions of the glucocorticoids. Subsequent research has shown that the protein plays a major regulatory role in systems as diverse as cell-growth regulation and differentiation, neutrophil migration, CNS responses to cytokines, neuroendocrine secretion and neurodegeneration. The role of lipocortin-1 in mediating glucocorticoid-induced effects in these systems has been demonstrated using immunoneutralization strategies and by mimicking steroid actions with highly purified or recombinant lipocortin-1 or its biologically active peptide fragments. Originally the mode of action of lipocortin-1 seemed to be largely through inhibition of prostaglandin formation, but it is now clear that it can modify other aspects of cell function, perhaps pointing to a more fundamental mechanism than was originally envisaged. In this article Rod Flower and Nancy Rothwell review the nature, possible mechanisms and clinical relevance of these diverse actions of lipocortin-1.
The activity of the steroid-inducible protein lipocortin-1 (LC1; with a primary sequence of 346 amino acids; also called annexin 1), a fragment corresponding to amino acids 1-188 and a short peptide from the N-terminus (amino acid 2-26) were tested for anti-inflammatory actions in three models of acute inflammation in the mouse in comparison with a mAb anti-CD11b (αCD11b). In the mouse air-pouch model LC1, fragment 1-188 and peptide Ac2- 26 exhibited powerful inhibitory effects (ED50 ≃ 5.2, 38 and 88 μg/mouse, respectively) on leukocyte migration elicited by IL-1. LC1 was approximately 200 times more potent than Ac2-26 on a molar basis although both gave maximal inhibitions, in contrast fragment 1-188 only produced a partial dose-response curve. LC1 was approximately 20 times more potent on a molar basis in this assay than the αCD11b mAb. Peptide Ac2-26 and the mAb αCD11b also blocked cell migration into the air-pouch induced by IL-8 with approximately the same potency. In the mouse skin edema and zymosan peritonitis assays Ac2-26 was inhibitory (ED50 of 200 μg/mouse) but less so than the αCD11b antibody (ED50 ≃ 0.5 mg/mouse). Both LC1 (10 μg) and Ac2-26 (200 μg) completely blocked FMLP-induced neutropenia in the mouse. Studies using an inactivated LC1 preparation, which binds to the same high affinity binding sites as the biologically active material, indicated that the short peptide acts on the same sites as the native LC1. This study confirms the activity of LC1 in another model of experimental inflammation and suggests that it acts partly through inhibition of leukocyte activation with an overall effect qualitatively comparable to the blocking of CD11b portion of a β2-integrin complex. It also shows that peptides derived from the N-terminal domain of LC1 may mimic the activity of the full length molecule and points the way for a new family of anti-inflammatory substances that inhibit leukocyte trafficking.
IL-1 is a pro-inflammatory cytokine which controls many features of the immune and inflammatory response. When injected into a mouse 6-day-old air-pouch, human rIL-1 (1 to 100 ng) induced in a dose-dependent fashion a migration of PMN that could be reliably assessed 4 h after injection. Both IL-1 alpha and IL-1 beta were active in this model. The effect of the cytokine was inhibited by local administration of actinomycin D (1 to 10 micrograms), alpha-melanocyte-stimulating hormone (200 micrograms), and a mAb recognizing IL-1R type I (10 micrograms). Indomethacin (1 mg/kg), an inhibitor of cyclo-oxygenase, and BW4AC (2 mg/kg), a selective lipoxygenase inhibitor, were without effect but moderate inhibition was seen with the platelet-activating factor antagonist WEB2086 (1 to 10 mg/kg). The glucocorticoid dexamethasone (0.015 to 1.5 mg/kg) potently inhibited the elicitation of neutrophils induced by IL-1 when given systemically 2 h before the cytokine. The steroid-induced anti-inflammatory protein lipocortin 1 (LC1) also produced a dose-dependent inhibition of PMN migration into the pouch with an ED50 of approximately 0.15 to 0.21 mg/kg. The denatured protein was without effect. Passive immunization of mice with a polyclonal sheep antiserum or a mAb raised against LC1 abolished the inhibitory action of dexamethasone whereas preimmune serum or control IgG were without significant effect. These findings provide further evidence that LC1 is involved in the anti-inflammatory action of glucocorticosteroids and suggest that this protein may act as an endogenous regulator of IL-1 action.
The role of proteases and reactive oxygen species (ROS) in polymorphonuclear neutrophil (PMN) induced cartilage degradation in vitro were studied. ONO-5046, a novel synthetic elastase inhibitor, significantly and dose dependently protected cartilage from degradation induced by PMNs stimulated with phorbol myristate acetate (PMA), opsonized zymosan, N-formyl-methionyl-leucyl-phenylalanine plus cytochalasin-B, or A-23187. The degradation by PMA-stimulated PMNs was unaffected by protease inhibitors which lack anti-elastase activity. However, the hydrogen peroxide (H2O2) reducing agent catalase afforded significant protection. Measurement of elastase activity following PMN activation by PMA showed that antioxidants which reduce H2O2 and/or hypochlorous acid decreased elastase activity. Thus, it is suggested that an indirect interaction between ROS and elastase activity may exist in PMN induced cartilage degradation. Furthermore, the possible implication of an endogenous elastase inhibitor(s) is discussed.
Treatment of mice with rolipram, a phosphodiesterase type 4 inhibitor, selectively modified the acute inflammatory reaction elicited by zymosan administration in 6-day-old mouse air-pouches. Rolipram (1-10 mg kg-1, i.p.) prevented the rise of endogenous tumor necrosis factor-alpha (TNF-alpha) in the lavage fluids (approximately 60% inhibition) induced by zymosan, with no effect upon interleukin-1 alpha levels. This action was not accompanied by changes in neutrophil accumulation, but the amount of elastase released in the lavage fluids was significantly reduced (approximately 50%). Dexamethasone (1.5 mg kg-1, i.v.), used for comparative purposes, significantly reduced the release of TNF-alpha (> 50%), interleukin-1 alpha (> 70%) and cellular infiltration (approximately 50%), but had only a marginal effect on the release of elastase activity. In conclusion, in this murine model of acute inflammation induced by zymosan, rolipram inhibited the endogenous TNF-alpha production at a local site of inflammation, such as the subcutaneous air-pouch, and prevented the full activation of migrated cells.
The ability of macrophages and neutrophils to defend tissue homeostasis and participate in inflammatory responses depends on their ability to mobilize granule-membrane proteins and granule content into their external milieu and into phagosomes by regulated secretory processes. Many laboratories have invested much time and effort into furthering our understanding of vesicular transport and secretion. A surge of interest in phagocytosis and phagosomal maturation is also apparent (e.g., the March 1995 issue of Trends in Cell Biology was entirely devoted to phagocytosis). The signaling and the regulation of the secretory response are most likely different for secretion into phagosomes than for secretion into the external milieu. However, these differentially targeted secretory processes rely both upon proteins in vesicular membranes, plasma membrane/phagosomal membrane, and cytosol and upon their interactions with cytoskeletal structures. It is the complex molecular interactions between these components that form the basis for regulation and control of secretion. In the following, the signaling role of granular and cytosolic pH in phagocyte lysosomal secretion is discussed and the current literature on regulated secretion by macrophages and neutrophils is reviewed.
Lipocortin 1 (LC1) immunoreactivity in murine peripheral blood leukocytes was quantified by use of a flow cytometric technique associated with a permeabilisation protocol with saponin. Using specific antisera raised against the whole protein or against its N‐terminus peptide, cell‐associated LC1‐like immunoreactivity was easily detected in circulating neutrophils and monocytes, whereas very low levels were found in lymphocytes. Of the total protein measured 17.6% and 36% were associated with the external plasma membrane in neutrophils and monocytes, as assessed in the absence of cell permeabilisation, whereas no signal was detected on lymphocyte plasma membrane.
Treatment of mice with dexamethasone (Dex; 0.5‐5 μg per mouse corresponding to ∼ 0.015‐1.5 mg kg ⁻¹ ) increased LC1 levels in neutrophils and monocytes. The 2–3 fold increase in LC1 levels was time‐dependent with a peak at 2 h. Treatment of mice with the steroid antagonist, RU486 (two doses of 20 mg kg ⁻¹ orally) decreased LC1‐like immunoreactivity in all three types of circulating leukocytes by ≥50%.
Extravasation of blood neutrophils into inflamed tissue sites resulted in a consistent reduction (≥50%) in LC1 levels compared with circulating neutrophils. A high LC1‐like immunoreactivity was also measured in resident macrophages, of which approximately one third was membrane‐associated. Induction of an acute inflammatory response in the murine peritoneal cavity did not modify total LC1 levels measured in macrophages, but reduced membrane‐associated LC1 to a significant extent, i.e. up to 70%.
In conclusion, flow cytometric analysis is a rapid and convenient method for detecting and measuring LC1 in murine leukocytes. We confirmed that LC1 protein expression is controlled by exogenous and endogenous glucocorticoids. Amongst other factor(s) influencing protein concentrations, extravasation was found to be associated with a reduced LC1 expression in the emigrated cells.
1. An immuno-neutralization strategy was employed to investigate the role of endogenous lipocortin 1 (LC1) in acute inflammation in the mouse. 2. Mice were treated subcutaneously with phosphate-buffered solution (PBS), non-immune sheep serum (NSS) or with one of two sheep antisera raised against LC1 (LCS3), or its N-terminal peptide (LCPS1), three times over a period of seven days. Twenty four hours after the last injection several parameters of acute inflammation were measured including zymosan-induced inflammation in 6-day-old air-pouches, zymosan-activated serum (ZAS)-induced oedema in the skin, platelet-activating factor (PAF)-induced neutrophilia and interleukin-1 beta (IL-1 beta)-induced corticosterone (CCS) release. 3. At the 4 h time-point of the zymosan inflamed air-pouch model, treatment with LCS3 did not modify the number of polymorphonuclear leucocytes (PMN) recruited: 7.84 +/- 1.01 and 7.00 +/- 0.77 x 10(6) PMN per mouse for NSS- and LCS3 group, n = 7. However, several other parameters of cell activation including myeloperoxidase (MPO) and elastase activities were increased (2.2 fold, P < 0.05, and 6.5 fold, P < 0.05, respectively) in the lavage fluids of these mice. Similarly, a significant increase in the amount of immunoreactive prostaglandin E2 (PGE2; 1.81 fold, P < 0.05) and IL-1 alpha (2.75 fold, P < 0.05), but not tumour necrosis factor-alpha (TNF-alpha), was also observed in LCS3-treated mice. 4. The recruitment of PMN into the zymosan inflamed air-pouches by 24 h had declined substantially (4.13 +/- 0.61 x 10(6) PMN per mouse, n = 12) in the NSS-treated mice, whereas high values were still measured in those treated with LCS3 (9.35 +/- 1.20 x 10(6) PMN per mouse, n = 12, P < 0.05). A similar effect was also found following sub-chronic treatment of mice with LCPS1: 6.48 +/- 0.10 x 10(6) PMN per mouse, vs. 2.77 +/- 1.20 and 2.64 +/- 0.49 x 10(6) PMN per mouse for PBS- and NSS-treated groups (n = 7, P < 0.05). Most markers of inflammation were also increased in the lavage fluids of LCS3-treated mice: MPO and elastase showed a 2.47 fold and 17 fold increase, respectively (P < 0.05 in both cases); TNF-alpha showed a 11.1 fold increase (P < 0.05) whereas the IL-1 alpha levels were not significantly modified. PGE2 was still detectable in most (5 out of 7) of the mice treated with LCS3 but only in 2 out of 7 of the NSS-treated mice. 5. Intradermal injection of 50% ZAS caused a significant increase in the 2 hoedema formation in the skin of LCS3-treated mice in comparison to PBS- and NSS-treated animals: 16.7 +/- 1.5 microliters vs. 10.8 +/- 1.2 microliters and 10.2 +/- 1.0 microliters, respectively (n = 14 mice per group, P < 0.05). ZAS-induced oedema had subsided by 24 h in control animals but a residual significant amount of extravasation was still detectable in LCS3-treated mice: 4.4 +/- 0.8 microliters (P < 0.05). 6. A recently described model driven by endogenous glucocorticoids is the blood neutrophilia observed following administration of PAF. In our experimental conditions, a single bolus of PAF (100 ng, i.v.) provoked a marked neutrophilia at 2 h (2.43 and 2.01 fold) in NSS- and PBS-treated mice (n = 11), respectively, which was significantly attenuated in the animals treated with LCS3: 1.26 fold increase in circulating PMN (n = 11, P < 0.01 vs. NSS- and PBS-groups). 7. Intraperitoneal injection of IL-1 beta (5 micrograms kg-1) caused a marked increase in circulating plasma CCS by 2 h, to a similar extent in all experimental groups. In contrast, measurement of CCS levels in the plasma of mice bearing air-pouches inflamed with zymosan revealed significant differences between LCS3 and NSS-treated mice at the 4 h time-point: 198 +/- 26 ng ml-1 vs. 110 +/- 31 ng ml-1 (n = 8, P < 0.05). 8. In conclusion, we found a remarkable exacerbation of the inflammatory process with respect to both humoral and cellular components in mice passively immunised agains
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.
Mobilisation of lipocortin 1 in adherent human polymorphonuclear leukocytes negatively modulates their transmigration
- Perretti
Biochemical actions of glucocorticoids on macrophage in culture. Specific inhibition of elastase, collage‐nase, and plasminogen activator secretion and effects on other metabolic functions
- Webb Z.