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Neutrophil Interaction with Inflamed Postcapillary Venule Endothelium Alters Annexin 1 Expression
Abstract
Annexin 1 (ANX-A1) exerts antimigratory actions in several models of acute and chronic inflammation. This is related to its ability to mimic the effect of endogenous ANX-A1 that is externalized on neutrophil adhesion to the postcapillary endothelium. In the present study we monitored ANX-A1 expression and localization in intravascular and emigrated neutrophils, using a classical model of rat peritonitis. For this purpose, a pair of antibodies raised against the ANX-A1 N-terminus (ie, able to recognize intact ANX-A1) or the whole protein (ie, able to interact with all ANX-A1 isoforms) was used by immunofluorescence and immunocytochemistry analyses. The majority ( approximately 50%) of ANX-A1 on the plasma membrane of intravascular neutrophils was intact. Extravasation into the subendothelial matrix caused loss of this pool of intact protein (to approximately 6%), concomitant with an increase in total amount of the protein; only approximately 25% of the total protein was now recognized by the antibody raised against the N-terminus (ie, it was intact). In the cytoplasm of these cells, ANX-A1 was predominantly associated with large vacuoles, possibly endosomes. In situ hybridization confirmed de novo synthesis of ANX-A1 in the extravasated cells. In conclusion, biochemical pathways leading to the externalization, proteolysis, and synthesis of ANX-A1 are activated during the process of neutrophil extravasation.
... Annexin A1 (ANXA1) is an endogenous anti-inflammatory molecule expressed by mast cells, neutrophils, eosinophils, monocytes, epithelial T cells that can modulate biological events in cancer progression and metastasis and in various models of inflammatory and autoimmune diseases [20][21][22][23][24][25][26]. ANXA1 is a member of the annexin superfamily of calcium-and phospholipid-binding proteins that participates in chronic inflammation, leukocyte migration, tissue growth and apoptosis [27,28]. ...
... ANXA1 is a member of the annexin superfamily of calcium-and phospholipid-binding proteins that participates in chronic inflammation, leukocyte migration, tissue growth and apoptosis [27,28]. ANXA1 has been shown to be very effective in limiting inflammation and is expressed in different immune cells recruited during infectious processes, particularly mast cells and neutrophils [20,29]. ...
... In our study, the numbers and the density of try + MC were elevated in T2R, which can be associated with vasculitis. Evidence from cancer biology revealed that MCs regulate angiogenesis through the production of vascular endothelial growth factor (VEGF) and release of pro-angiogenic proteases [20]. A previous study from our group searching for plasma markers of leprosy reactions by multiplex approach found a marginally significant difference for VEGF in T2R versus reaction-free controls [45]. ...
... The main mechanism of ANXA1 action is by the inhibition of phospholipase A2 (PLA2), an enzyme involved in the adhesive properties of neutrophils to endothelial cells, preventing the neutrophil transmigration through the endothelium [20,22]. This effect may also be beneficial in ischemia-reperfusion situations [23][24][25]. ...
... Despite the inflammatory severity in Hb SS genotype, this group presented the highest levels of ANXA1 among the SCD genotypes. ANXA1 is abundant in neutrophils, which may externalize large amounts of this protein (50% to 70%), especially in response to cytokines released during the inflammatory propagation and neutrophil transmigration [20,22,40]. Homozygous patients for Hb S usually exhibit more anemic, hemolytic, vaso-occlusive and clinical severity than the double heterozygous genotypes [3] and this may represent a potential stimulus for outsourcing and releasing of ANXA1. ...
... Homozygous patients for Hb S usually exhibit more anemic, hemolytic, vaso-occlusive and clinical severity than the double heterozygous genotypes [3] and this may represent a potential stimulus for outsourcing and releasing of ANXA1. Moreover, one of the main mechanisms that trigger the ANXA1 externalization is the contact neutrophil-endothelium [20,22], which is a common interaction in the vaso-occlusive processes in SCD due to the cells increased adhesive properties, platelets and endothelium. ...
[This corrects the article DOI: 10.1371/journal.pone.0165833.].
... The main mechanism of ANXA1 action is by the inhibition of phospholipase A2 (PLA2), an enzyme involved in the adhesive properties of neutrophils to endothelial cells, preventing the neutrophil transmigration through the endothelium [20,22]. This effect may also be beneficial in ischemia-reperfusion situations [23][24][25]. ...
... Despite the inflammatory severity in Hb SS genotype, this group presented the highest levels of ANXA1 among the SCD genotypes. ANXA1 is abundant in neutrophils, which may externalize large amounts of this protein (50% to 70%), especially in response to cytokines released during the inflammatory propagation and neutrophil transmigration [20,22,40]. Homozygous patients for Hb S usually exhibit more anemic, hemolytic, vaso-occlusive and clinical severity than the double heterozygous genotypes [3] and this may represent a potential stimulus for outsourcing and releasing of ANXA1. ...
... Homozygous patients for Hb S usually exhibit more anemic, hemolytic, vaso-occlusive and clinical severity than the double heterozygous genotypes [3] and this may represent a potential stimulus for outsourcing and releasing of ANXA1. Moreover, one of the main mechanisms that trigger the ANXA1 externalization is the contact neutrophil-endothelium [20,22], which is a common interaction in the vaso-occlusive processes in SCD due to the cells increased adhesive properties, platelets and endothelium. ...
Sickle cell disease (SCD) is an inherited hemolytic anemia whose pathophysiology is driven by polymerization of the hemoglobin S (Hb S), leading to hemolysis and vaso-occlusive events. Inflammation is a fundamental component in these processes and a continuous inflammatory stimulus can lead to tissue damages. Thus, pro-resolving pathways emerge in order to restore the homeostasis. For example there is the annexin A1 (ANXA1), an endogenous anti-inflammatory protein involved in reducing neutrophil-endothelial interactions , accelerating neutrophil apoptosis and stimulating macrophage efferocytosis. We investigated the expression of ANXA1 in plasma of SCD patients and its relation with anemic , hemolytic and inflammatory parameters of the disease. Three SCD genotypes were considered: the homozygous inheritance for Hb S (Hb SS) and the association between Hb S and the hemoglobin variants D-Punjab (Hb SD) and C (Hb SC). ANXA1 and proinflamma-tory cytokines were quantified by ELISA in plasma of SCD patients and control individuals without hemoglobinopathies. Hematological and biochemical parameters were analyzed by flow cytometry and spectrophotometer. The plasma levels of ANXA1 were about threefold lesser in SCD patients compared to the control group, and within the SCD genotypes the most elevated levels were found in Hb SS individuals (approximately threefold higher). Proinflammatory cytokines were higher in SCD groups than in the control individuals. Anemic and hemolytic markers were higher in Hb SS and Hb SD genotypes compared to Hb SC patients. White blood cells and platelets count were higher in Hb SS genotype and were positively correlated to ANXA1 levels. We found that ANXA1 is down-regulated and differentially expressed within the SCD genotypes. Its expression seems to depend on the inflammatory, hemolytic and vaso-occlusive characteristics of the diseased. These data may lead to new biological targets for therapeutic intervention in SCD.
... Under all experimental conditions, nondenervated and denervated stomachs with or without adenocarcinomas were positive for ANXA1 in the epithelium and inflammatory cells, particularly mast cells and neutrophils, confirming previous data [29][30][31]. Myenteric denervation significantly decreased ANXA1 levels in the epithelium compared with nondenervated stomachs. ...
... ANXA1 expression is altered in mast cells in inflammatory diseases and cancer. In the trachea and mesentery of rats, mast cells express more ANXA1 after treatment with dexamethasone, suggesting that glucocorticoids inhibit degranulating mast cells [29,30,35]. Similarly, pretreatment of mice with Ac2-26 (the N-terminal region of ANXA1) increases the number of intact mast cells in the pleural cavity and decreases the release of histamine in a model of ovalbumin-induced allergic inflammation, confirming the protective effect of ANXA1 in their activation [36]. ...
... Neutrophils in nondenervated stomachs with adenocarcinomas (ND+MNNG Group) upregulated ANXA1 compared with those in nondenervated stomachs without lesions (ND Group). ANXA1, on the plasma membrane of adherent neutrophils, is inhibitory, reducing the extent of transmigration across endothelial cells [29]. ANXA1 mediates the apoptosis of neutrophils, an important mechanism of limiting the inflammatory response [38]. ...
... Structurally, the annexins contain a small N-terminal region, which varies in length and composition, and a central domain consisting of four to eight repetitions of a highly conserved amino acid sequence. The N-terminal domain is unique for each member of the superfamily, and several investigations characterized this region as a promoter of anti-inflammatory actions (16,19,20). ...
... Although the anti-inflammatory activities of AnxA1 and its mimetic peptides, including Ac2-26, have been explored in several in vivo and in vitro investigations (16,(18)(19)(20)26), the role of exogenous AnxA1 in ocular inflammatory processes has not been elucidated, and there are no studies on the administration of Ac2-26 in ocular tissues. Given the common side effects of the current therapies used to treat uveitis (4,5,15), we evaluated the effects of endogenous and exogenous AnxA1 protein in rodent ocular tissues in EIU and an in vitro LPS-inflamed RPE human cell system and further evaluated AnxA1's mechanism of action. ...
... Histopathological analysis. The intact right eyes of the rats (n = 7) were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde, sodium cacodylate buffer 0.1 M (pH 7.4) for 24 h at 4°C, dehydrated by graded methanol, and embedded in LR Gold resin (London Resin; Reading, Berkshire, U.K.) (19). After collecting the AqH, the left eyes (n = 7) were fixed in 10% formalin, dehydrated in graded alcohol, and embedded in paraffin for immunohistochemical analysis. ...
Annexin A1 (AnxA1) is a protein that displays potent anti-inflammatory properties, but its expression in eye tissue and its role in ocular inflammatory diseases have not been well studied. We investigated the mechanism of action and potential uses of AnxA1 and its mimetic peptide (Ac2-26) in the endotoxin-induced uveitis (EIU) rodent model and in human ARPE-19 cells activated by LPS. In rats, analysis of untreated EIU after 24 and 48 h or EIU treated with topical applications or with a single s.c. injection of Ac2-26 revealed the anti-inflammatory actions of Ac2-26 on leukocyte infiltration and on the release of inflammatory mediators; the systemic administration of Boc2, a formylated peptide receptor (fpr) antagonist, abrogated the peptide's protective effects. Moreover, AnxA1(-/-) mice exhibited exacerbated EIU compared with wild-type animals. Immunohistochemical studies of ocular tissue showed a specific AnxA1 posttranslational modification in EIU and indicated that the fpr2 receptor mediated the anti-inflammatory actions of AnxA1. In vitro studies confirmed the roles of AnxA1 and fpr2 and the protective effects of Ac2-26 on the release of chemical mediators in ARPE-19 cells. Molecular analysis of NF-κB translocation and IL-6, IL-8, and cyclooxygenase-2 gene expression indicated that the protective effects of AnxA1 occur independently of the NF-κB signaling pathway and possibly in a posttranscriptional manner. Together, our data highlight the role of AnxA1 in ocular inflammation, especially uveitis, and suggest the use of AnxA1 or its mimetic peptide Ac2-26 as a therapeutic approach.
... Under all experimental conditions, nondenervated and denervated stomachs with or without adenocarcinomas were positive for ANXA1 in the epithelium and inflammatory cells, particularly mast cells and neutrophils, confirming previous data [29][30][31]. Myenteric denervation significantly decreased ANXA1 levels in the epithelium compared with nondenervated stomachs. ...
... ANXA1 expression is altered in mast cells in inflammatory diseases and cancer. In the trachea and mesentery of rats, mast cells express more ANXA1 after treatment with dexamethasone, suggesting that glucocorticoids inhibit degranulating mast cells [29,30,35]. Similarly, pretreatment of mice with Ac2-26 (the N-terminal region of ANXA1) increases the number of intact mast cells in the pleural cavity and decreases the release of histamine in a model of ovalbumin-induced allergic inflammation, confirming the protective effect of ANXA1 in their activation [36]. ...
... Neutrophils in nondenervated stomachs with adenocarcinomas (ND+MNNG Group) upregulated ANXA1 compared with those in nondenervated stomachs without lesions (ND Group). ANXA1, on the plasma membrane of adherent neutrophils, is inhibitory, reducing the extent of transmigration across endothelial cells [29]. ANXA1 mediates the apoptosis of neutrophils, an important mechanism of limiting the inflammatory response [38]. ...
This study evaluated the properties of endogenous nitric oxide synthases (NOS) and annexin-A1 (ANXA1) and determined how they can be exploited in the N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis and myenteric denervation model. Male Wistar rats were treated with MNNG and/or aminoguanidine (AG) for 20 weeks. In another set of experiments, rats with nondenervated and denervated stomachs were treated with MNNG or water for 28 weeks. Fragments of the pyloric region were processed for histopathology, NOS activity, and immunohistochemistry to explore the activity and expression of constitutive (cNOS) and inducible (iNOS) NO synthase and their relationship with annexin-A1 (ANXA1) expression. NO inhibition by AG increased the percentage of animals with adenocarcinomas (~29%) compared with the untreated MNNG group (~4%). Myenteric denervation did not alter NOS activity. cNOS activity was significantly greater in nondernervated and denervated stomachs with or without lesions (P<0.001) than iNOS activity (P<0.01), as confirmed by immunohistochemistry. Further, cNOS activity in normal stomachs and outside the lesion area was considerably higher than inside it (P<0.01). By densitometric analysis of nondenervated and denervated stomachs, ANXA1 expression was modulated in epithelial and inflammatory cells (mast cells and neutrophils), wherein significant alterations were induced by lesion development and myenteric denervation. In conclusion, NO protects against the development of gastric adenocarcinomas. The pattern of ANXA1 expression was not associated with NOS activity or expression, suggesting that NO and ANXA1 act in gastric tumors in disparate pathways.
... The expression of ANXA1 was detected in several tissues and cell types, including neutrophils [14,15], monocytes [16], mast cells [17,18], and epithelial cells [19]. However, the increased expression of this protein may occur during a systemic inflammatory reaction, such as that observed in an experimental model of lipopolysaccharide (LPS)-induced endotoxaemia [20]. ...
... ANXA1, a protein of 37 kDa, was originally identified as a mediator for several antiinflammatory actions of glucocorticoids [10]. Pharmacological studies with ANXA1 demonstrated its effects on inflammatory cells, including mast cells [17,18], neutrophils [14] and eosinophils [34]. Furthermore, the development of knockout mice for ANXA1 [35] and ultrastructural immunocytochemical analysis [14,36] have allowed for the better definition of roles played by endogenous ANXA1 in several cellular functions, including phagocytosis, migration, and synthesis of mediators [37,38]. ...
... Pharmacological studies with ANXA1 demonstrated its effects on inflammatory cells, including mast cells [17,18], neutrophils [14] and eosinophils [34]. Furthermore, the development of knockout mice for ANXA1 [35] and ultrastructural immunocytochemical analysis [14,36] have allowed for the better definition of roles played by endogenous ANXA1 in several cellular functions, including phagocytosis, migration, and synthesis of mediators [37,38]. ...
The aim of this study was to evaluate the expression of the protein annexin A1 (ANXA1), a potent endogenous regulator of the inflammatory process, in ocular toxoplasmosis.
C57BL/6 female mice were infected using intravitreal injections of either 10(6) tachyzoites of Toxoplasma gondii (RH strain; T. gondii) or PBS only (control groups). After 24, 48, and 72 h, animals were sacrificed and their eyes were harvested for histopathological, immunohistochemical, and ultrastructural immunocytochemical analysis of ANXA1. Human retinal pigment epithelial (RPE) cells (ARPE-19) were infected in vitro with T. gondii and collected after 60, 120, 240 min, and 24 h.
Compared with non-infected eyes, an intense inflammatory response was observed in the anterior (24 h after infection) and posterior segments (72 h after infection) of the infected eye, characterized by neutrophil infiltration and by the presence of tachyzoites and their consequent destruction along with disorganization of normal retina architecture and RPE vacuolization. T. gondii infection was associated with a significant increase of ANXA1 expression in the neutrophils at 24, 48, and 72 h, and in the RPE at 48 and 72 h. In vitro studies confirmed an upregulation of ANXA1 levels in RPE cells, after 60 and 120 min of infection with T. gondii.
The positive modulation of endogenous ANXA1 in the inflammatory and RPE cells during T. gondii infection suggests that this protein may serve as a therapeutic target in ocular toxoplasmosis.
... It is synthetized in some immune cells, mainly myeloid cells including macrophages, MCs, eosinophils, and neutrophils, beyond the neuroendocrine system (15)(16)(17). Glucocorticoids not only stimulate AnxA1 transcription, but also induce the release into the cytoplasm of its pre-existing forms via a receptor-dependent, non-genomic pathway, preceded by phosphorylation at key sites in the N-terminus and other sites (18). ...
... Also, pro-inflammatory cytokines such as IL-6 and TNF-a may induced the AnxA1 protein expression (28). Our research group showed that the induction of peritonitis promotes both reorganization of cytoplasmic granules and de novo synthesis of AnxA1, with important effects in the regulation of the inflammatory infiltrate and cytokine production in the mesentery (16). ...
Mast cells (MCs) are main effector cells in allergic inflammation and after activation, they release stored (histamine, heparin, proteases) and newly synthesized (lipid mediators and cytokines) substances. In the gastrointestinal tract the largest MC population is located in the lamina propria and submucosa whereas several signals such as the cytokine IL-4, seem to increase the granule content and to stimulate a remarkable expansion of intestinal MCs. The broad range of MC-derived bioactive molecules may explain their involvement in many different allergic disorders of the gastrointestinal tract. Annexin A1 (AnxA1) is a 37 KDa glucocorticoid induced monomeric protein selectively distributed in certain tissues. Its activity can be reproduced by mimetic peptides of the N-terminal portion, such as Ac2-26, that share the same receptor FPR-L1. Although previous reports demonstrated that AnxA1 inhibits MC degranulation in murine models, the effects of exogenous peptide Ac2-26 on intestinal MCs or the biological functions of the Ac2-26/FPR2 system in human MCs have been poorly studied. To determine the effects of Ac2-26 on the function of MCs toward the possibility of AnxA1-based therapeutics, we treated WT and IL-4 knockout mice with peptide Ac2-26, and we examined the spontaneous and compound 48/80 stimulated colonic MC degranulation and cytokine production. Moreover, in vitro, using human mast cell line HMC-1 we demonstrated that exogenous AnxA1 peptide is capable of interfering with the HMC-1 degranulation in a direct pathway through formyl peptide receptors (FPRs). We envisage that our results can provide therapeutic strategies to reduce the release of MC mediators in inflammatory allergic processes.
... Annexin A1 (AnxA1) is a glucocorticoid-regulated protein that possesses antiinflammatory and tissue protective actions leading to efficient resolution of inflammation (17)(18)(19). The intact form of AnxA1 is the biologically active form of this protein (20) and when neutrophils migrate into sites of inflammation, it is cleaved by elastase and proteinase-3 to its inactive form (33 kDa) (20,21). At 24 h after S. pneumoniae infection (10 5 CFU), at the peak of neutrophil recruitment into the lungs, the intact AnxA1 band (37kDa) disappeared giving rise to a strong band of cleaved AnxA1 (Fig. 7A). ...
... Annexin A1 (AnxA1) is a glucocorticoid-regulated protein that possesses antiinflammatory and tissue protective actions leading to efficient resolution of inflammation (17)(18)(19). The intact form of AnxA1 is the biologically active form of this protein (20) and when neutrophils migrate into sites of inflammation, it is cleaved by elastase and proteinase-3 to its inactive form (33 kDa) (20,21). At 24 h after S. pneumoniae infection (10 5 CFU), at the peak of neutrophil recruitment into the lungs, the intact AnxA1 band (37kDa) disappeared giving rise to a strong band of cleaved AnxA1 (Fig. 7A). ...
Rationale:
Pneumococcal pneumonia is a leading cause of mortality worldwide. The inflammatory response to bacteria is necessary to control infection but may also contribute to tissue damage. Phosphodiesterase-4 (PDE4) inhibitors, such as rolipram (ROL), effectively reduce inflammation. Here, we examined the impact of rolipram in a pneumococcal pneumonia murine model.
Methods:
Mice were infected intranasally with 105-106CFU of S. pneumoniae, treated with rolipram (ROL) in a prophylactic or therapeutic schedule in combination or not with the antibiotic ceftriaxone. Inflammation and bacteria counts were assessed and ex vivo phagocytosis assays were performed.
Results:
ROL treatment during S. pneumoniae infection decreased neutrophil recruitment into lungs and airways and reduced lung injury. Prophylactic ROL treatment also decreased cytokines levels in the airways. Although modulation of inflammation by ROL ameliorated pneumonia, bacteria burden was not reduced. On the other hand, antibiotic therapy reduced bacteria without reducing neutrophil infiltration, cytokine level and lung injury. Combined ROL and ceftriaxone treatment decreased lethality rates and was more efficient in reducing inflammation, by increasing pro-resolving protein Annexin A1 expression, and bacterial burden by enhancing phagocytosis. Lack of Annexin A1 increased inflammation and lethality induced by pneumococcal infection.
Conclusion:
These data show that immunomodulatory effects of PDE4 inhibitors are useful during severe pneumococcal pneumonia and suggest their potential benefit as adjunctive therapy during infectious diseases.
... During the inflammatory response, macrophages release mediators, endothelial cells become activated and leukocytes transmigrate into the tissues [10]. The inflammatory response is modulated by the action of anti-inflammatory mediators, such as annexin A1 (AnxA1), a 37 kDa calcium and phospholipid binding protein that is an inhibitor of glucocorticoid-induced eicosanoid synthesis and PLA 2 [11]. In addition, AnxA1 induces shedding of L-selectin in tissues which inhibits the adhesion, migration and recruitment of neutrophils to the inflamed site and accelerates neutrophil apoptosis [12,13]. ...
... CV-induced peritonitis caused an intense inflammatory response at 4 hours characterized by increased numbers of blood, peritoneal and mesenteric neutrophils, which was absent at 24 hours. Our findings are in agreement with rodent models of carrageenan and zymosan peritonitis [11,18] which are characterized by the high number of neutrophils that are circulating and transmigrating to the peritoneal cavity in the first 4-6 hours. Previous investigations with Bothrops venoms also observed leukocyte infiltration, predominantly composed of neutrophils, at the site of injury in the first hours after venom application [22,23]. ...
Annexin A1 (AnxA1) is an endogenous glucocorticoid regulated protein that modulates anti-inflammatory process and its therapeutic potential has recently been recognized in a range of systemic inflammatory disorders. The effect of the N-terminal peptide Ac2-26 of AnxA1 on the toxic activities of Bothrops moojeni crude venom (CV) and its myotoxin II (MjTX-II) were evaluated using a peritonitis rat model. Peritonitis was induced by the intraperitoneal injection of either CV or MjTX-II, a Lys-49 phospholipase A2. Fifteen minutes after the injection, the rats were treated with either Ac2-26 or PBS. Four hours later, the CV and MjTX-II-induced peritonitis were characterized by neutrophilia (in the peritoneal exudate, blood and mesentery) and increased number of mesenteric degranulated mast cells and macrophages. At 24 hours post-injection, the local inflammatory response was attenuated in the CV-induced peritonitis while the MjTX-II group exhibited neutrophilia (peritoneal exudates and blood). Ac2-26 treatment prevented the influx of neutrophils in MjTX-II-induced peritonitis and diminished the proportion of mesenteric degranulated mast cells and macrophages in CV-induced peritonitis. Additionally, CV and MjTX-II promoted increased levels of IL-1β and IL-6 in the peritoneal exudates which were significantly reduced after Ac2-26 treatment. At 4 and 24 hours, the endogenous expression of AnxA1 was upregulated in the mesenteric neutrophils (CV and MjTX-II groups) and mast cells (CV group). In the kidneys, CV and MjTX-II administrations were associated with an increased number of macrophages and morphological alterations in the juxtamedullary nephrons in proximal and distal tubules. Ac2-26 promoted significant recovery of the juxtamedullary structures, decreased the number of macrophages and diminished the AnxA1 in epithelial cells from distal tubules and renal capsules. Our results show that Ac2-26 treatment significantly attenuates local and systemic inflammatory processes and indicate this peptide as a potential target for the development of new therapeutic strategies for the snakebite envenomation treatment.
... Thus, it is important to employ new approaches in order to understand activation and sequestration of neutrophils in the intestinal ischemic events [7]. The anti-inflammatory protein annexin-A1 (ANXA1) is a potent mediator of inflammation resolution and a 37-kDa member of the annexin family of calcium and phospholipid-binding proteins, expressed constitutively in many cells, including neutrophil gelatinase granules [8]. Exogenous AnxA1 or its N-terminal peptidomimetic (Ac2-26) administration has been shown to elicit protective anti-inflammatory actions via both in vitro and in vivo anti-neutrophil migration mechanisms89101112. ...
... The anti-inflammatory protein annexin-A1 (ANXA1) is a potent mediator of inflammation resolution and a 37-kDa member of the annexin family of calcium and phospholipid-binding proteins, expressed constitutively in many cells, including neutrophil gelatinase granules [8]. Exogenous AnxA1 or its N-terminal peptidomimetic (Ac2-26) administration has been shown to elicit protective anti-inflammatory actions via both in vitro and in vivo anti-neutrophil migration mechanisms89101112. Moreover, AnxA1 has been shown to have cardio protective effects against myocardial ischemia and reperfusion injury in rats and mice, at least in part due to its inhibitory actions on neutrophils [10,13]. ...
Background
Intestinal ischemia/reperfusion (IR) injury is a serious and triggering event in the development of remote organ dysfunction, from which the lung is the main target. This condition is characterized by intense neutrophil recruitment, increased microvascular permeability. Intestinal IR is also responsible for induction of adult respiratory distress syndrome, the most serious and life-threatening form of acute lung injury. The purpose of this study was to investigate the effect of annexin-A1 protein as an endogenous regulator of the organ remote injury induced by intestinal ischemia/reperfusion. Male C57bl/6 mice were subjected to intestinal ischemia, induced by 45 min occlusion of the superior mesenteric artery, followed by reperfusion.
Results
The intestinal ischemia/reperfusion evoked a high intensity lung inflammation as indicated by the number of neutrophils as compared to control group. Treatment with annexin-A1 peptidomimetic Ac2-26, reduced the number of neutrophils in the lung tissue and increased its number in the blood vessels, which suggests a regulatory effect of the peptide Ac2-26 in the neutrophil migration. Moreover, the peptide Ac2-26 treatment was associated with higher levels of plasma IL-10.
Conclusion
Our data suggest that the annexin-A1 peptidomimetic Ac2-26 treatment has a regulatory and protective effect in the intestinal ischemia/reperfusion by attenuation of the leukocyte migration to the lung and induction of the anti-inflammatory cytokine IL-10 release into the plasma. The anti-inflammatory action of annexin-A1 and its peptidomimetic described here may serve as a basis for future therapeutic approach in mitigating inflammatory processes due to intestinal ischemia/reperfusion.
... In neutrophils, AnxA1 is localized in gelatinase granules and in the cytosol (Perretti et al., 2000) and is released during degranulation and NETosis. After externalization, AnxA1 is subject to proteolytic cleavage by neutrophil elastase and proteinase 3 which results in the release of bioactive, soluble N-terminal protein fragments (Oliani et al., 2001;Rescher et al., 2006;Vong et al., 2007). Both full-length AnxA1 and its fragments have been shown to contribute to the antiinflammatory actions of the protein (Walther et al., 2000;Bode et al., 2019). ...
Non-resolving inflammation plays a critical role during the transition from renal injury towards end-stage renal disease. The glucocorticoid-inducible protein annexin A1 has been shown to function as key regulator in the resolution phase of inflammation, but its role in immune-mediated crescentic glomerulonephritis has not been studied so far.
Methods: Acute crescentic glomerulonephritis was induced in annexin A1-deficient and wildtype mice using a sheep serum against rat glomerular basement membrane constituents. Animals were sacrificed at d5 and d10 after nephritis induction. Renal leukocyte abundance was studied by immunofluorescence and flow cytometry. Alterations in gene expression were determined by RNA-Seq and gene ontology analysis. Renal levels of eicosanoids and related lipid products were measured using lipid mass spectrometry.
Results: Histological analysis revealed an increased number of sclerotic glomeruli and aggravated tubulointerstitial damage in the kidneys of annexin A1-deficient mice compared to the wildtype controls. Flow cytometry analysis confirmed an increased number of CD45 ⁺ leukocytes and neutrophil granulocytes in the absence of annexin A1. Lipid mass spectrometry showed elevated levels of prostaglandins PGE2 and PGD2 and reduced levels of antiinflammatory epoxydocosapentaenoic acid regioisomers. RNA-Seq with subsequent gene ontology analysis revealed induction of gene products related to leukocyte activation and chemotaxis as well as regulation of cytokine production and secretion.
Conclusion: Intrinsic annexin A1 reduces proinflammatory signals and infiltration of neutrophil granulocytes and thereby protects the kidney during crescentic glomerulonephritis. The annexin A1 signaling cascade may therefore provide novel targets for the treatment of inflammatory kidney disease.
... However, what causes the dysregulated activity of these T cells is currently unknown. Several groups have reported an increased level of ANXA1 in an inflammatory setting, in both animal models of disease [68,69] and in human samples [70]. Experimental autoimmune encephalomyelitis (EAE) mouse models of inflammation have shown that ANXA1 levels correlated with disease severity. ...
Annexin‐A1 has a well‐defined anti‐inflammatory role in the innate immune system, but its function in adaptive immunity remains controversial. This glucocorticoid‐induced protein has been implicated in a range of inflammatory conditions and cancers, as well as being found to be overexpressed on the T cells of patients with autoimmune disease. Moreover, the formyl peptide family of receptors, through which annexin‐A1 primarily signals, have also been implicated in these diseases. In contrast, treatment with recombinant annexin‐A1 peptides resulted in suppression of inflammatory processes in murine models of inflammation. This review will focus on what is currently known about annexin‐A1 in heath and disease and discuss the potential of this protein as a biomarker and therapeutic target.
... The data showed that the ANXA1 levels found in M-MDSCs were higher in LL patients when compared to the T1R and T2R, while similar levels were observed in G-MDSC at all patients. The literature shows that the ANXA1 is an endogenous regulatory protein expressed at high concentrations in granulocytes, particularly neutrophils [33][34][35][36]. The lower levels of ANXA1 in M-MDSCs of T1R and T2R might be due to drug treatment. ...
Background
Leprosy is a chronic infectious disease caused by Mycobacterium leprae. Patients have distinct clinical forms, and the host´s immunological response regulate those manifestations. In this work, the presence of the myeloid-derived suppressor cell and the regulatory protein annexin A1 is described in patients with multibacillary leprosy and with type 1 and 2 reactions.
Methods
Patients were submitted to skin biopsy for histopathological analysis to obtain a bacilloscopic index. Immunofluorescence was used to detect myeloid-derived suppressor cells and annexin A1.
Results
The data demonstrated that the presence of granulocytic and monocytic myeloid-derived suppressor cells in leprosy patients. A high number of monocytic myeloid-derived suppressor cells were observed in lepromatous leprosy and type 2 reactional patients. The presence of annexin A1 was observed in all myeloid-derived suppressor cells. In particular, the monocytic myeloid-derived suppressor cell in the lepromatous patients has higher levels of this protein when compared to the reactional patients. This data suggest that the higher expression of this protein may be related to regulatory response against a severe infection, contributing to anergic response. In type 1 reactional patients, the expression of annexin A1 was reduced.
Conclusions
Myeloid-derived suppressor cell are present in leprosy patients and annexin A1 might be regulated the host response against Mycobacterium leprae.
... Hence, annexin A1 appears to be the most significant component of endometrial secretomics at the time of sampling in our study. Annexin A1 is a calcium/phospholipid-binding protein that has several functions including promotion of membrane fusion, involvement in exocytosis, and regulation of PLA2 activity [36]. Phospholipase A2 induces the release of arachidonic acid, which is the precursor molecule of prostaglandins, through the action of cyclooxygenases. ...
Intrauterine devices block luteolysis in cyclic mares, but the underlying mechanism is unknown. To clarify the mechanisms, the protein profile of the endometrial secretome was analyzed using two-dimensional difference gel electrophoresis (2D-DIGE). Twenty-seven mares were classified according to whether they were inseminated (AI) or had an intrauterine device (IUD), a water-filled plastic sphere, inserted into the uterus on Day 3 after ovulation. Uterine lavage fluids were collected on Day 15 from pregnant inseminated mares (AI-P; n = 8), non-pregnant inseminated mares (AI-N; n = 4), and mares with IUD (n = 15). The IUD group was further divided into prolonged (IUD-P; n = 7) and normal luteal phase (IUD-N; n = 8) groups on the basis of ultrasound examinations, serum levels of progesterone and PGFM on Days 14 and 15, and COX-2 results on Day 15. Four mares from each group were selected for the 2D-DIGE analyses. Ten proteins had significantly different abundance among the groups, nine of the proteins were identified. Malate dehydrogenase 1, increased sodium tolerance 1, aldehyde dehydrogenase 1A1, prostaglandin reductase 1, albumin and hemoglobin were highest in pregnant mares; T-complex protein 1 was highest in non-pregnant mares; and annexin A1 and 6-phosphogluconolactonase were highest in IUD mares. The results suggest that the mechanism behind the intrauterine devices is likely related to inflammation.
... The data showed that the ANXA1 levels found in M-MDSCs were higher in LL patients when compared to the T1R and T2R, while similar levels were observed in G-MDSC at all patients. The literature shows that the ANXA1 is an endogenous regulatory protein expressed at high concentrations in granulocytes, particularly neutrophils [39][40][41][42]. The lower levels of ANXA1 in M-MDSCs of T1R and T2R might be due to drug treatment. ...
Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae. Patients have distinct clinical forms, and host´s immunological response regulate those manifestations. In this work, the presence of the myeloid-derived suppressor cell and the regulatory protein annexin A1 is described in patients with multibacillary leprosy and with type 1 and 2 reactions.
Methods: Patients were submitted to skin biopsy for histopathological analysis to obtain bacilloscopic index. Immunofluorescence was used to detect myeloid-derived suppressor cells and annexin A1.
Results: The data demonstrated that the presence of granulocytic and monocytic myeloid-derived suppressor cells in leprosy patients. The high number of monocytic myeloid-derived suppressor cells were observed in lepromatous leprosy and type 2 reactional patients with Bacillus Calmette–Guérin (BCG) vaccination scar. The presence of annexin A1 was observed in all myeloid-derived suppressor cells. In particularly, the monocytic myeloid-derived suppressor cell in the lepromatous patients has higher levels of this protein when compared to the reactional patients. This data suggest that the higher expression of this protein may be related to regulatory response against a severe infection, contributing to anergic response. In type 1 reactional patients, the expression of annexin A1 was reduced.
Conclusions: Myeloid-derived suppressor cell are present in leprosy patients and annexin A1 might be regulated the host response against Mycobacterium leprae.
... Indeed, melatonin potentiates cytotoxicity and affects the sensitivity of cells of cervical (HeLa) and colorectal cancer (HT-29) through the MT3 receptor melatonin, promoting chemotherapeutic action [42]. In addition, previous reports indicate that ANXA1 and ANXA1 2−26 induce neutrophil apoptosis [43]. We provide evidence about the apoptotic action of Cis and reveal that the peptide does not cause the death of SiHa cells, nor does it increase the death caused by Cis. ...
Cisplatin (Cis) is a choice chemotherapy approach to cervical cancer by inducing DNA adducts and subsequent apoptosis. We have investigated the effects of Cis on Annexin A1 (ANXA1) and inhibitor of DNA binding 1 (ID1) proteins expression to elucidate further mechanisms of Cis actions. Human cervical tissue samples from twenty-four patients, with Cervical Intraepithelial Neoplasia (CIN, stage I, II and III), were evaluated to quantified ANXA1 and ID1 expressions. In vitro, human epidermoid carcinoma of the cervix (SiHa cell line) were treated with Annexin A1 peptide (ANXA12−26), Cis or Cis + ANXA12−26 to evaluate cell proliferation and migration, cytotoxicity of treatments as well as ANXA1 and ID1 modulations by mRNA and protein expression. Our findings showed expression of ID1 and ANXA1 proteins in tissue samples from Cervical Intraepithelial Neoplasia (CIN) patients, with intense immunological identification of ID1 in the CIN III stage. In SiHa cells, treatments with Cis alone or Cis + ANXA12−26, increase mRNA expressions of the ANXA1 and reduced the ID1. In agreement, Cis + ANXA12−26 enhanced ANXA1 protein expression and Cis or Cis + ANXA12−26 abolished ID1 protein expression. Cell proliferation was reduced after treatment with ANXA12−26 peptide and more significant after Cis or Cis + ANXA12−26 treatments. These two last treatments reduced cell viability, by inducing late apoptosis, and impaired cell migration. Together, our data highlight endogenous ANXA1 is involved in Cis therapy for cervical cancer.
... To detect AnxA1 and NLRP3, ultrathin macrophage sections (~90 nm) were submitted for immunocytochemistry, as previously described [19]. To detect the proteins, the sheep polyclonal antibody anti-AnxA1 (1:200) and rabbit polyclonal antibody anti-NLRP3 (1:200; Cusabio, Houston, TX, USA), following a donkey anti-sheep IgG and goat anti-rabbit IgG antibody (1:50) conjugated to 10-nm and 20-nm colloidal gold (British Biocell, Cardiff, UK), respectively, were used. ...
Annexin A1 (AnxA1) is a potent anti-inflammatory protein that downregulates proinflammatory cytokine release. This study evaluated the role of AnxA1 in the regulation of NLRP3 inflammasome activation and lipid release by starch-elicited murine peritoneal macrophages. C57bl/6 wild-type (WT) and AnxA1-null (AnxA1-/-) mice received an intraperitoneal injection of 1.5% starch solution for macrophage recruitment. NLRP3 was activated by priming cells with lipopolysaccharide for 3 h, followed by nigericin (1 h) or ATP (30 min) incubation. As expected, nigericin and ATP administration decreased elicited peritoneal macrophage viability and induced IL-1β release, more pronounced in the AnxA1-/- cells than in the control peritoneal macrophages. In addition, nigericin-activated AnxA1-/- macrophages showed increased levels of NLRP3, while points of co-localization of the AnxA1 protein and NLRP3 inflammasome were detected in WT cells, as demonstrated by ultrastructural analysis. The lipidomic analysis showed a pronounced release of prostaglandins in nigericin-stimulated WT peritoneal macrophages, while ceramides were detected in AnxA1-/- cell supernatants. Different eicosanoid profiles were detected for both genotypes, and our results suggest that endogenous AnxA1 regulates the NLRP3-derived IL-1β and lipid mediator release in macrophages.
... However, a marked reduction in these cells occurred by Ac2-26 administration. Other investigations demonstrate that smoking increases the production of histamine by alveolar mast cells with subsequent degranulation, which contributes to destruction of the alveolar septa, recruitment of neutrophils and release of cytokines and chemokines [51][52][53]. ...
... Following cell activation, AnxA1 is readily mobilized to the cell surface where it interacts with its receptor named formyl peptide receptor 2 (FPR2, also known as ALX or FPRL1) in a paracrine/autocrine form [9]. However, AnxA1 may be cleaved by proteases in inflammatory conditions, resulting in various fragments, such as the 33-kDa form, which lacks the main pharmacophore core contained in the N-terminal portion of the parent protein [15,16]. The pro-inflammatory nature of AnxA1 cleavage products, including the 33-kDa form, has also been supported by reports of their increased levels in inflammatory diseases, such as airway secretions of patients with cystic fibrosis [17][18][19] and in lung extracts from ovalbumin-sensitized mice (mouse model of asthma) [20]. ...
Background:
Annexin A1 (AnxA1) is a protein involved in inflammation resolution that might be altered in obesity-associated type 2 diabetes mellitus (DM), which is a chronic inflammatory disease. The aim of this study was to evaluate AnxA1 serum levels in individuals with and without DM stratified according to the body mass index (BMI), and the dynamic of AnxA1 expression in adipose tissue from humans with obesity and non-obesity.
Methods:
Serum samples were obtained from 41 patients with DM (lean, overweight and obese) and 40 controls, and adipose tissue samples were obtained from 16 individuals with obesity (with or without DM), and 15 controls.
Results:
DM patients showed similar AnxA1 serum levels when compared to controls. However, when the individuals were stratified according to BMI, AnxA1 levels were higher in individuals with obesity than lean or overweight, and in overweight compared to lean individuals. Moreover, AnxA1 was correlated positively with IL-6 levels. AnxA1 levels were also positively correlated with BMI, waist circumference and waist-to-hip ratio. Furthermore, higher levels of cleaved AnxA1 were observed in adipose tissue from individuals with obesity, independently of DM status.
Conclusions:
Enhanced levels of AnxA1 in serum of individuals with obesity suggest an attempt to counter-regulate the systemic inflammation process in this disease. However, the higher levels of cleaved AnxA1 in the adipose tissue of individuals with obesity could compromise its anti-inflammatory and proresolving actions, locally. Considering our data, AnxA1 cleavage in the adipose tissue, despite increased serum levels of this protein, and consequently the failure in inflammation resolution, suggests an important pathophysiological mechanism involved in inflammatory status observed in obesity.
... They were then w ashed in the same buffer, dehydrated in an increasing series of methanol (Merck, Germany) at 20°C and pre-included in a 100% methanol mixture with LRGold resin (London Resin CO, Reading, Berkshire, UK), (24 h) at 20°C. Afterwards, they were included in pure LRGold resin for 24 h at 20°C and exposed to ultraviolet light (Oliani et al., 2001). 0.5 µm sections were obtained on the Leica RM2265 microtome and then stained with 1% toluidine blue in 1% borax solution (TAAB Laboratories, IL) for mast cell analysis. ...
... It regulates leukocyte detachment from the postcapillary endothelium in both acute and chronic inflammatory states (Gavins et al., 2012), and deletion of the AnxA1 gene is associated with exacerbated inflammatory responses (Hannon et al., 2003). AnxA1 is widely distributed, being detected in, for example, the lung, kidney, bone marrow, intestine, spleen, thymus or brain (Fava et al., 1989;Gavins et al., 2012;Vital et al., 2016), and secreted by a variety of cell types including MCs (Kwon et al., 2012) and neutrophils (Oliani et al., 2001). The importance of AnxA1 in limiting inflammatory responses is also highlighted by findings that silencing AnxA1 (Kao et al., 2014) or using AnxA1 knockout mice (Gavins et al., 2003) exacerbates inflammation. ...
Background and purpose:
In recent years, studies have focused on the resolution of inflammation, which can be achieved by endogenous anti-inflammatory agonists such as Annexin A1 (AnxA1). Here, we investigated the effects of mast cells (MCs) on early lipopolysaccharide (LPS)-induced neutrophil recruitment and the involvement of the AnxA1-Formyl peptide receptor 2/ALX (Fpr2/ALX or lipoxin A4 receptor) pathway.
Experimental approach:
Intravital microscopy (IVM) was used to visualize and quantify the effects of LPS (10 µg per mouse i.p.) on murine mesenteric cellular interactions. Furthermore, the role MCs play in these inflammatory responses was determined in vivo and in vitro and effects of AnxA1 mimetic peptide Ac2-26 were assessed.
Key results:
LPS increased both neutrophil endothelial cell interactions within the mesenteric microcirculation and MC activation (determined by IVM and ruthenium red dye uptake) which in turn lead to the early stages of neutrophil recruitment. MC recruitment of neutrophils could be blocked by preventing the pro-inflammatory activation (using cromolyn sodium) or enhancing an anti-inflammatory phenotype (using Ac2-26) in MCs. Furthermore, MCs induced neutrophil migration in vitro and MC stabilization enhanced the release of AnxA1 from neutrophils. Pharmacological approaches (such as the administration of Fpr pan-antagonist Boc2, or the Fpr2/ALX antagonist WRW4) revealed neutrophil Fpr2/ALX to be important in this process.
Conclusions and implications:
Data presented here provides evidence for a role of MCs, which are ideally positioned in close proximity to the vasculature, to act as sentinel cells in neutrophil extravasation and resolution of inflammation via the AnxA1-Fpr2/ALX pathway.
... 9 ANX1 is an important endogenous anti-inflammatory mediator activated in response to cell or tissue injury. 10,11 Treatment with ANX1 has offered protection in splanchnic, myocardial, renal, liver and brain models of ischemia and reperfusion. 12-14 ANX1 inhibits neutrophil adhesion and migration through the inflamed post-capillary venule endothelium, decreases neutrophil recruitment in the inflamed tissue, detaches endotheliumadhered leukocytes, accelerates polymorphonuclear cell apoptosis, induces L-selectin shedding and reduces phospholipase A2 activity. ...
The aim of this study was to investigate the effect of the anti-inflammatory and anti-fibrotic actions of ANX1 on erectile function (EF). Forty-eight male Wistar rats were randomly distributed into four equal groups: one group (sham operation—control) and three groups (bilateral cavernous nerve (CN) crush injury). Crush injury groups were treated prior to injury with an intravascular injection of either ANX1 (50 or 100 μg kg− 1) or vehicle. EF was assessed by CN electrical stimulation at 2 and 7 days after CN injury with histomorphometric and immunohistochemical analysis. ANX1 demonstrated functional preservation as the increase in intracavernous pressure (ICP). A dose–response relationship regarding the effect on penile tissue was confirmed, and preservation of the penile dorsal nerves and anti-apoptotic effects in the corpus cavernosum (real P-value vs injured control). ANX1 treatment prevented collagen deposition and smooth muscle loss in the penis. ANX1 normalized the expression of vascular endothelial growth factor and decreased tumor necrosis factor-α in the lumen of the blood vessels of the organ. ANX1 proved effective in preserving EF in a rat model of neurogenic erectile dysfunction. ANX1 treatment before CN injury in rats improved erectile recovery, enhanced vascular regeneration and preserved the micro-architecture of the corpus cavernosum. The clinical availability of this compound merits application in penile rehabilitation studies following radical prostatectomy.
... 9 ANX1 is an important endogenous anti-inflammatory mediator activated in response to cell or tissue injury. 10,11 Treatment with ANX1 has offered protection in splanchnic, myocardial, renal, liver and brain models of ischemia and reperfusion. 12-14 ANX1 inhibits neutrophil adhesion and migration through the inflamed post-capillary venule endothelium, decreases neutrophil recruitment in the inflamed tissue, detaches endotheliumadhered leukocytes, accelerates polymorphonuclear cell apoptosis, induces L-selectin shedding and reduces phospholipase A2 activity. ...
The aim of this study was to investigate the effect of the anti-inflammatory and anti-fibrotic actions of ANX1 on erectile function(EF). Forty-eight male Wistar rats were randomly distributed into four equal groups: one group (sham operation—control) and threegroups (bilateral cavernous nerve (CN) crush injury). Crush injury groups were treated prior to injury with an intravascular injectionof either ANX1 (50 or 100μgkg−1) or vehicle. EF was assessed by CN electrical stimulation at 2 and 7 days after CN injury withhistomorphometric and immunohistochemical analysis. ANX1 demonstrated functional preservation as the increase inintracavernous pressure (ICP). A dose–response relationship regarding the effect on penile tissue was confirmed, and preservationof the penile dorsal nerves and anti-apoptotic effects in the corpus cavernosum (realP-value vs injured control). ANX1 treatmentprevented collagen deposition and smooth muscle loss in the penis. ANX1 normalized the expression of vascular endothelialgrowth factor and decreased tumor necrosis factor-αin the lumen of the blood vessels of the organ. ANX1 proved effective inpreserving EF in a rat model of neurogenic erectile dysfunction. ANX1 treatment before CN injury in rats improved erectile recovery,enhanced vascular regeneration and preserved the micro-architecture of the corpus cavernosum. The clinical availability of thiscompound merits application in penile rehabilitation studies following radical prostatectomy.
... 9 ANX1 is an important endogenous anti-inflammatory mediator activated in response to cell or tissue injury. 10,11 Treatment with ANX1 has offered protection in splanchnic, myocardial, renal, liver and brain models of ischemia and reperfusion. 12-14 ANX1 inhibits neutrophil adhesion and migration through the inflamed post-capillary venule endothelium, decreases neutrophil recruitment in the inflamed tissue, detaches endotheliumadhered leukocytes, accelerates polymorphonuclear cell apoptosis, induces L-selectin shedding and reduces phospholipase A2 activity. ...
The aim of this study was to investigate the effect of the anti-inflammatory and anti-fibrotic actions of ANX1 on erectile function(EF). Forty-eight male Wistar rats were randomly distributed into four equal groups: one group (sham operation—control) and three groups (bilateral cavernous nerve (CN) crush injury). Crush injury groups were treated prior to injury with an intravascular injection
of either ANX1 (50 or 100 μg kg − 1
) or vehicle. EF was assessed by CN electrical stimulation at 2 and 7 days after CN injury with
histomorphometric and immunohistochemical analysis. ANX1 demonstrated functional preservation as the increase in intracavernous pressure (ICP). A dose–response relationship regarding the effect on penile tissue was confirmed, and preservation of the penile dorsal nerves and anti-apoptotic effects in the corpus cavernosum (real P-value vs injured control). ANX1 treatment prevented collagen deposition and smooth muscle loss in the penis. ANX1 normalized the expression of vascular endothelial growth factor and decreased tumor necrosis factor-α in the lumen of the blood vessels of the organ. ANX1 proved effective in preserving EF in a rat model of neurogenic erectile dysfunction. ANX1 treatment before CN injury in rats improved erectile recovery,
enhanced vascular regeneration and preserved the micro-architecture of the corpus cavernosum. The clinical availability of this compound merits application in penile rehabilitation studies following radical prostatectomy
... ANXA1 inhibits the expression of other inflammatory mediators such as iNOS or NOS2 in macrophages and inducible cyclooxygenase (COX-2) in activated microglia 28 . Moreover, ANXA1 could inhibit the chemotaxis of neutrophils and monocytes during inflammation 27,29 . Therefore, higher expression of ANXA1 in PgLPS 1435/1449 -treated HGFs may account for the inhibition of cytokines by this specific isoform of PgLPS 3,10 . ...
Periodontal (gum) disease is a highly prevalent infection and inflammation accounting for the majority of tooth loss in adult population worldwide. Porphyromonas gingivalis is a keystone periodontal pathogen and its lipopolysaccharide (PgLPS) acts as a major virulence attribute to the disease. Herein, we deciphered the overall host response of human gingival fibroblasts (HGFs) to two featured isoforms of tetra-acylated PgLPS1435/1449 and penta-acylated PgLPS1690 with reference to E. coli LPS through quantitative proteomics. This study unraveled differentially expressed novel biomarkers of immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs. PgLPS1690 greatly upregulated inflammatory proteins (e.g. cyclophilin, inducible nitric oxide synthase, annexins, galectin, cathepsins and heat shock proteins), whereas the anti-inflammatory proteins (e.g. Annexin A2 and Annexin A6) were significantly upregulated by PgLPS1435/1449. Interestingly, the antioxidants proteins such as mitochondrial manganese-containing superoxide dismutase and peroxiredoxin 5 were only upregulated by PgLPS1690. The cytoskeletal rearrangement-related proteins like myosin were differentially regulated by these PgLPS isoforms. The present study gives new insight into the biological properties of P. gingivalis LPS lipid A moiety that could critically modulate immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs, and thereby enhances our understanding of periodontal pathogenesis.
... The eyes are immunologically privileged sites where inflammatory cells and antibodies are unable to cross BRB due to the presence of RPE, which are able to limit the accumulation of immune effector cells by phagocytosis of the parasite, avoiding tissue destruction but favoring parasite replication (Schnyder, 1994;Roberts et al., 2001;Bhopale, 2003;Tedesco et al., 2004;Sauer et al., 2013). At the same time, the protein Annexin A1 (ANXA1), which expression depends in part on IL-6 and TNF-α; and is in various cells and tissue types (Harricane et al., 1996;Oliani et al., 2000;Oliani et al., 2001;Sena et al., 2006;Oliani et al., 2008;Gastardelo et al., 2009); it is unregulated by T. gondii in the RPE and neutrophils, seeming to allow cell activation and phagocytosis, which shows some protective effect (Mimura et al., 2012). Intracellular phase depends on the ability of the parasite to regulate processes of the host cells by its secreted effectors. ...
Toxoplasmosis is a cosmopolitan infection caused by the apicomplexan parasite Toxoplasma gondii. This infectious disease is widely distributed across the world where cats play an important role in its spread. The symptomatology caused by this parasite is diverse but the ocular affectation emerges as the most important clinical phenotype. Therefore, we conducted a systematic review of the current knowledge of ocular toxoplasmosis from the genetic diversity of the pathogen towards the treatment available for this infection. This review represents an update to the scientific community regarding the genetic diversity of the parasite, the genetic factors of the host, the molecular pathogenesis and its association with disease, the available diagnostic tools and the available treatment of patients undergoing ocular toxoplamosis. This review will be an update for the scientific community in order to encourage researchers to deploy cutting-edge investigation across this field.
... Another gene studied was ANXA1 (Annexin 1), there was increased expression in Hep-2 cells after treatment with E. tirucalli latex. This gene is important for the carcinogenic process such as cell cycle control, regulation of apoptosis, cell proliferation and growth, inflammatory response and calcium ion transport regulation [35]. The annexin family consists of ubiquitous proteins that interact with phospholipids in a Ca 2+ -dependent manner. ...
Background:
Some plants had been used in the treatment of cancer and one of these has attracted scientific interest, the Euphorbia tirucalli (E. tirucalli), used in the treatment of asthma, ulcers, warts has active components with activities scientifically proven as antimutagenic, anti-inflammatory and anticancer.
Methods:
We evaluate the influence of the antitumoral fraction of the E. tirucalli latex in the larynx squamous cell carcinoma (Hep-2), on the morphology, cell proliferation and gene expression. The Hep-2 cells were cultivated in complete medium (MEM 10 %) and treated with E. tirucalli latex for 1, 3, 5 and 7 days. After statistically analyzing the proliferation of the tested cells, the cells were cultivated again for RNA extraction and the Rapid Subtractive Hybridization (RaSH) technique was used to identify genes with altered expression. The genes found using the RaSH technique were analyzed by Gene Ontology (GO) using Ingenuity Systems.
Results:
The five genes found to have differential expression were validated by real-time quantitative PCR. Though treatment with E. tirucalli latex did not change the cell morphology in comparison to control samples, but the cell growth was significantly decreased. The RaSH showed change in the expression of some genes, including ANXA1, TCEA1, NGFRAP1, ITPR1 and CD55, which are associated with inflammatory response, transcriptional regulation, apoptosis, calcium ion transport regulation and complement system, respectively. The E. tirucalli latex treatment down-regulated ITPR1 and up-regulated ANXA1 and CD55 genes, and was validated by real-time quantitative PCR.
Conclusions:
The data indicate the involvement of E. tirucalli latex in the altered expression of genes involved in tumorigenic processes, which could potentially be applied as a therapeutic indicator of larynx cancer.
... The endogenous peptide Ac2-26 is cleaved from annexin A1, but its K d for FPR1 or FPR2 is in the low micromolar range (Migeotte et al., 2006). In acute and chronic inflammation, annexin A1 is activated during the process of neutrophil extravasation (Oliani et al., 2001). It also mediates the rapid anti-inflammatory effects of neutrophil-derived microparticles (Dalli et al., 2008). ...
The family with sequence similarity 3 (FAM3) gene family is a cytokine-like gene family with four members FAM3A, FAM3B, FAM3C, and FAM3D. In this study, we found that FAM3D strongly chemoattracted human peripheral blood neutrophils and monocytes. To identify FAM3D receptor, we used chemotaxis, receptor internalization, calcium flux and radioligand-binding assays in FAM3D-stimulated HEK293 cells that transiently expressed FPR1 or FPR2 to show that FAM3D was a high affinity ligand of formyl peptide receptors (FPR1 and FPR2), both of which were highly expressed on the surface of neutrophils and monocytes/macrophages. After being injected into the mouse peritoneal cavity, FAM3D chemoattracted CD11b+Ly6G+ neutrophils in a short time. In response to FAM3D stimulation, p-ERK and p-p38 were up-regulated in the mouse neutrophils, which could be inhibited by an inhibitor of FPR1 or FPR2. FAM3D was reported to be constitutively expressed in the gastrointestinal tract. We found that FAM3D expression increased significantly in dextran sulfate sodium-induced colitis. Taken together, we propose that FAM3D plays a role in gastrointestinal homeostasis and inflammation through its receptors FPR1 and FPR2.
... In inflammatory conditions intact AnxA1 (37 kDa) can be cleaved by proteinase-3 and neutrophil elastase generating the 33 kDa cleaved isoform, which is believed to be inactive, and peptides derived from the AnxA1 N-terminus [23][24][25]. The main cleavage sites on AnxA1 are located at A 11 , V 22 , and V 36 , as identified by cleavage assays coupled to mass spectrometric analyses [25]. ...
Neutrophils (also named polymorphonuclear leukocytes or PMN) are essential components of the immune system, rapidly recruited to sites of inflammation, providing the first line of defense against invading pathogens. Since neutrophils can also cause tissue damage, their fine-tuned regulation at the inflammatory site is required for proper resolution of inflammation. Annexin A1 (AnxA1), also known as lipocortin-1, is an endogenous glucocorticoid-regulated protein, which is able to counterregulate the inflammatory events restoring homeostasis. AnxA1 and its mimetic peptides inhibit neutrophil tissue accumulation by reducing leukocyte infiltration and activating neutrophil apoptosis. AnxA1 also promotes monocyte recruitment and clearance of apoptotic leukocytes by macrophages. More recently, some evidence has suggested the ability of AnxA1 to induce macrophage reprogramming toward a resolving phenotype, resulting in reduced production of proinflammatory cytokines and increased release of immunosuppressive and proresolving molecules. The combination of these mechanisms results in an effective resolution of inflammation, pointing to AnxA1 as a promising tool for the development of new therapeutic strategies to treat inflammatory diseases.
... 58 In support of these findings, seminal in vivo studies in rodents on the anti-inflammatory actions of ANXA1 in the periphery have shown that administration of recombinant ANXA1, or peptides derived from its N-terminal region, inhibit the process of neutrophil extravasation and consequent tissue damage at sites of vascular injury following myocardial or splanic ischemia reperfusion. [59][60][61][62][63] In situation of peripheral inflammation ANXA1 has been shown to act as a functional antagonist of the chemo-attractant human formyl peptide receptor (FPR) or the closely related FPR-like 1 receptor (FPRL1), both of which are expressed on mononuclear phagocytes 64 and serve to inhibit the migration of these cells into the extravascular tissue and to desensitize them to chemo-attractant challenge. [65][66][67] There is no doubt that ANXA1 represent a good endogenous anti-inflammatory protein. ...
Annexin A1 is a member of the annexin calcium binding protein family that still lacks a clear physiological role. Gene evolution and organization are new factors which may give information about protein and their mechanism of action. We have attempted in this review to give an up-to date of the protein from gene organization and expression to its pathophysiological significance.
... Annexin A1 (AnxA1) is a phospholipid binding protein able to inhibit leukocyte transmigration and activation. Originally described as a glucocorticoid (GC)-induced factor with antiphospholipase activity, later on AnxA1 has been implicated in prevention of inflammation in several disease models, such as peritonitis, air-pouch edema, myocardium infarct, respiratory tract allergy, endotoxemia, kidney ischemia/reperfusion, uveitis, arthritis and others [1][2][3][4][5][6][7][8][9]. AnxA1 binds to formyl peptide receptors (FPR) [10] and supports the resolution of inflammation and the wound healing suggesting a protective role on the intestinal epithelium damage [11][12][13][14]. ...
... As a potential anti-inflammatory agent we highlight the annexin A1 (ANXA1), a 37 kDa protein that downregulates the inflammatory response in experimental models of acute (Gastardelo et al., 2009;Girol et al., 2013), chronic (Oliani et al., 2008;Dalli et al., 2010) and systemic (Damazo et al., 2005) inflammation and is expressed in different immune cells, particularly mast cells and neutrophils (Oliani et al., 2000(Oliani et al., , 2001Gil et al., 2006). However, the relationship between ANXA1 and ocular inflammation has not been well studied. ...
Annexin A1 (ANXA1), a 37 kDa glucocorticoid-regulated protein, is a potent anti-inflammatory mediator effective in terminating acute inflammatory response, and its role in allergic settings has been poorly studied. The aim of this investigation was to evaluate the mechanism of action of ANXA1 in intraocular inflammation using a classical model of ovalbumin (OVA)-induced allergic conjunctivitis (AC). OVA-immunised Balb/c mice, wild-type (WT) and ANXA1-deficient (AnxA1(-/-)), were challenged with eye drops containing OVA on days 14-16 with a subset of WT animals pretreated intraperitoneally with the peptide Ac2-26 (N-terminal region of ANXA1) or dexamethasone (DEX). After 24 hours of the last ocular challenge, WT mice treated with Ac2-26 and DEX had significantly reduced clinical signs of conjunctivitis (chemosis, conjunctival hyperaemia, lid oedema and tearing), plasma IgE levels, leukocyte (eosinophil and neutrophil) influx and mast cell degranulation in the conjunctiva compared to WT controls. These anti-inflammatory effects of DEX were associated with high endogenous levels of ANXA1 in the ocular tissues as detected by immunohistochemistry. Additionally, Ac2-26 administration was effective to reduce IL-2, IL-4, IL-10, IL-13, eotaxin and RANTES in the eye and lymph nodes compared to untreated WT animals. The lack of ANXA1 produced an exacerbated allergic response as detected by the density of the inflammatory cell influx to the conjunctiva and the cytokine/chemokine release. These different effects observed for Ac2-26 were correlated with diminished level of activated ERK at 24h in the ocular tissues compared to untreated OVA group. Our findings demonstrate the protective effect of ANXA1 during the inflammatory allergic response suggesting this protein as a potential target for new ocular inflammation therapies.
Copyright © 2015. Published by Elsevier Ltd.
... The pattern of expression appears to be punctuate with patches of immunoreactivity seen throughout the cell within a specific subset of neutrophil organelles. Other studies conducted with human resting neutrophils in vitro (Perretti, Christian et al. 2000) and with rat neutrophils within an inflamed vascular bed in vivo (Oliani, Paul-Clark et al. 2001) found that a majority (~50%) of intracellular Annexin A1 colocalized with gelatinase granules, whereas a much smaller proportion was detected on the plasma membrane ( Figure 1-3). ...
... In addition, the inflammatory response is controlled by anti-inflammatory mediators that maintain the homeostasis of the immune response and prevent tissue damage. Among these mediators we highlight annexin A1 (ANXA1), a 37 kDa protein with potent anti-inflammatory functions, expressed in different immune cells, particularly mast cells and neutrophils (Oliani et al. 2001;Gil et al. 2006;Gavins and Hickey 2012). However, the relationship between ANXA1 and endometriosis is not well studied. ...
Endometriosis is a continuous and progressive disease with a poorly understood aetiology, pathophysiology and natural history. This study evaluated the histological differences between eutopic and ectopic endometria (abdominal wall endometriosis) and the expression of mast cell proteases (tryptase and chymase), annexin A1 (ANXA1) and formyl peptide receptor 1 (FPR1). Ectopic endometrium from 18 women with abdominal wall endometriosis and eutopic endometrium from 10 women without endometriosis were obtained. The endometrial samples were analysed by histopathology, immunohistochemistry and ultrastructural immunogold labeling to determine mast cell heterogeneity (tryptase and chymase positive cells) and the expression levels of ANXA1 and FPR1. Histopathological analysis of the endometriotic lesions showed a glandular pattern of mixed differentiation and an undifferentiated morphology with a significant influx of inflammatory cells and a change in mast cell heterogeneity, as evidenced by a significant increase in the number of chymase-positive cells and endogenous chymase expression. The undifferentiated glandular pattern of endometriotic lesions was positively associated with a marked increase and co-localization of ANXA1 and FPR1 in the epithelial cells. In conclusion, the co-upregulated expression of mast cell chymase and ANXA1-FPR1 system in ectopic endometrium suggests their involvement in the development of endometriotic lesions.
... A fifth gene that was mildly downregulated on the RNA level versus all non-OLP/OLR epithelium tested was ANXA1, a widely expressed protein that promotes resolution of inflammation through various mechanisms. 34,35 All are known to be expressed by cells that include keratinocytes and professional immune cells, Langerhans, and mast cells in the mucosa, all of which have the ability to recognize microbial invaders and signal an innate immune response. CD14 has already been shown to be elevated on the protein and RNA levels in oral keratinocytes with OLP. ...
Oral lichen planus (OLP) is a disease of the oral mucosa of unknown cause producing lesions with an intense band-like inflammatory infiltrate of T cells to the subepithelium and keratinocyte cell death. We performed gene expression analysis of the oral epithelium of lesions in subjects with OLP and its sister disease, oral lichenoid reaction (OLR), in order to better understand the role of the keratinocytes in these diseases.
Fourteen patients with OLP or OLR were included in the study, along with a control group of 23 subjects with a variety of oral diseases and a normal group of 17 subjects with no clinically visible mucosal abnormalities. Various proteins have been associated with OLP, based on detection of secreted proteins or changes in RNA levels in tissue samples consisting of epithelium, stroma, and immune cells. The mRNA level of twelve of these genes expressed in the epithelium was tested in the three groups.
Four genes showed increased expression in the epithelium of OLP patients: CD14, CXCL1, IL8, and TLR1, and at least two of these proteins, TLR1 and CXCL1, were expressed at substantial levels in oral keratinocytes.
Because of the large accumulation of T cells in lesions of OLP it has long been thought to be an adaptive immunity malfunction. We provide evidence that there is increased expression of innate immune genes in the epithelium with this illness, suggesting a role for this process in the disease and a possible target for treatment.
... Annexin A1, an antiinflammatory factor, is a 37kDa calcium-dependent phospholipid binding protein, originally reported as glucocorticoid-induced protein with anti-phospholipase activity [12][13][14] that has been shown to regulate diverse cellular functions in several cell types. ANXA1 also exhibits profound inhibitory actions on leukocyte transmigration and activation, leading to the resolution of inflammation [15][16][17][18]. Its protective and anti-inflammatory role has been demonstrated in the animal models of endotoxemia, peritonitis, arthritis, and cerebral and myocardial ischemia [19][20][21][22][23][24][25][26]. ...
Development of inflammatory bowel disease (IBD) involves the interplay of environmental and genetic factors with the host immune system. Mechanisms contributing to immune dysregulation in IBD are not fully defined. Development of novel therapeutic strategies is focused on controlling aberrant immune response in IBD. Current IBD therapy utilizes a combination of immunomodulators and biologics to suppress pro-inflammatory effectors of IBD. However, the role of immunomodulatory factors such as annexin A1 (ANXA1) is not well understood. The goal of this study was to examine the association between ANXA1 and IBD, and the effects of anti-TNF-α, Infliximab (IFX), therapy on ANXA1 expression.
ANXA1 and TNF-α transcript levels in PBMC were measured by RT PCR. Clinical follow up included the administration of serial ibdQs. ANXA1 expression in the gut mucosa was measured by IHC. Plasma ANXA1 levels were measured by ELISA.
We found that the reduction in ANXA1 protein levels in plasma coincided with a decrease in the ANXA1 mRNA expression in peripheral blood of IBD patients. ANXA1 expression is upregulated during IFX therapy in patients with a successful intervention but not in clinical non-responders. The IFX therapy also modified the cellular immune activation in the peripheral blood of IBD patients. Decreased expression of ANXA1 was detected in the colonic mucosa of IBD patients with incomplete resolution of inflammation during continuous therapy, which correlated with increased levels of TNF-α transcripts. Gut mucosal epithelial barrier disruption was evident by increased plasma bacterial 16S levels.
Loss of ANXA1 expression may support inflammation during IBD and can serve as a biomarker of disease progression. Changes in ANXA1 levels may be predictive of therapeutic efficacy.
... The regulatory action of AnxA1 has been demonstrated in models of acute (Gastardelo et al., 2009), chronic (Oliani et al., 2008) and systemic inflammation (Damazo et al., 2005). AnxA1 regulates neutrophil and monocyte adhesion and migration through inflamed endothelium (Oliani et al., 2001;Perretti et al., 2002b) and accelerates polymorphonuclear cell apoptosis (Scannell and Maderna 2007). ...
Immunosuppressive drugs have a critical role in inhibiting tissue damage and allograft rejection. Studies have demonstrated the anti-inflammatory effects of the annexin A1 (AnxA1) in the regulation of transmigration and apoptosis of leucocytes. In the present study, an experimental skin allograft model was used to evaluate a potential protective effect of AnxA1 in transplantation survival. Mice were used for the skin allograft model and pharmacological treatments were carried out using either the AnxA1 mimetic peptide Ac2-26, with or without cyclosporine A (CsA), starting 3 days before surgery until rejection. Graft survival, skin histopathology, leucocyte transmigration and expression of AnxA1 and AnxA5 post-transplantation were analysed. Pharmacological treatment with Ac2-26 increased skin allograft survival related with inhibition of neutrophil transmigration and induction of apoptosis, thereby reducing the tissue damage compared with control animals. Moreover, AnxA1 and AnxA5 expression increased after Ac2-26 treatment in neutrophils. Interestingly, the combination of Ac2-26 and cyclosporine A showed similar survival of transplants when compared with the cyclosporine A group, which could be attributed to a synergistic effect of both drugs. Investigations in vitro revealed that cyclosporine A inhibited extracellular-signal-regulated kinase (ERK) phosphorylation induced by Ac2-26 in neutrophils. Overall, the results suggest that AnxA1 has an essential role in augmenting the survival of skin allograft, mainly owing to inhibition of neutrophil transmigration and enhancement of apoptosis. This effect may lead to the development of new therapeutic approaches relevant to transplant rejection. Copyright © 2013 John Wiley & Sons, Ltd.
... Ea.hy926 cells were prepared for electron microscopy by standard methods as previously described. 23 Briefly, adherent cells were removed using cell dissociation solution and washed with PBS. After resuspension in 0.5 ml of 4% paraformaldehyde and 0.5% glutaraldehyde, 0.1% sodium cacodylate buffer (pH 7.4) for 24 hours at 4°C, they were then washed in sodium cacodylate, dehydrated through a graded series of ethanol (70 to 100%), and embedded in LR Gold (London Resin Co., Reading, Berkshire, UK). ...
Galectin-1 (Gal-1), the prototype of a family of β-galactoside-binding proteins, has been shown to attenuate experimental acute and chronic inflammation. In view of the fact that endothelial cells (ECs), but not human polymorphonuclear leukocytes (PMNs), expressed Gal-1 we tested here the hypothesis that the protein could modulate leukocyte-EC interaction in inflammatory settings. In vitro, human recombinant (hr) Gal-1 inhibited PMN chemotaxis and trans-endothelial migration. These actions were specific as they were absent if Gal-1 was boiled or blocked by neutralizing antiserum. In vivo, hrGal-1 (optimum effect at 0.3 μg equivalent to 20 pmol) inhibited interleukin-1β-induced PMN recruitment into the mouse peritoneal cavity. Intravital microscopy analysis showed that leukocyte flux, but not their rolling velocity, was decreased by an anti-inflammatory dose of hrGal-1. Binding of biotinylated Gal-1 to resting and postadherent human PMNs occurred at concentrations inhibitory in the chemotaxis and transmigration assays. In addition, the pattern of Gal-1 binding was differentially modulated by PMN or EC activation. In conclusion, these data suggest the existence of a previously unrecognized function of Gal-1, that is inhibition of leukocyte rolling and extravasation in experimental inflammation. It is possible that endogenous Gal-1 may be part of a novel anti-inflammatory loop in which the endothelium is the source of the protein and the migrating PMNs the target for its anti-inflammatory action.
... We therefore propose that Ac2-26 may target phospholipase and this then interferes with NADPH oxidase assembly to reduce superoxide generation. Interestingly, endogenous annexin has been detected in endothelial cells following an inflammatory insult [52], suggesting that spontaneous local production of annexin may well be a natural cell defense mechanism for the endothelial cells. However, elevating the production of annexin might not always be favorable. ...
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.
... Previous studies have reported that ANXA1 induced in endotoxemia, peritonitis, and arthritis exerts anti-inflammatory properties by suppression of leukocyte activation and transmigration (9)(10)(11)(12). Such anti-inflammatory properties of ANXA1 are mediated by autocrine and paracrine signaling of a 26-amino-acid ANXA1 N-terminus peptide, Ac2-26, that is released from the fulllength protein through regulated proteolysis (2,(13)(14)(15)(16). ...
N-formyl peptide receptors (FPRs) are critical regulators of host defense in phagocytes and are also expressed in epithelia. FPR signaling and function have been extensively studied in phagocytes, yet their functional biology in epithelia is poorly understood. We describe a novel intestinal epithelial FPR signaling pathway that is activated by an endogenous FPR ligand, annexin A1 (ANXA1), and its cleavage product Ac2-26, which mediate activation of ROS by an epithelial NADPH oxidase, NOX1. We show that epithelial cell migration was regulated by this signaling cascade through oxidative inactivation of the regulatory phosphatases PTEN and PTP-PEST, with consequent activation of focal adhesion kinase (FAK) and paxillin. In vivo studies using intestinal epithelial specific Nox1-/-IEC and AnxA1-/- mice demonstrated defects in intestinal mucosal wound repair, while systemic administration of ANXA1 promoted wound recovery in a NOX1-dependent fashion. Additionally, increased ANXA1 expression was observed in the intestinal epithelium and infiltrating leukocytes in the mucosa of ulcerative colitis patients compared with normal intestinal mucosa. Our findings delineate a novel epithelial FPR1/NOX1-dependent redox signaling pathway that promotes mucosal wound repair.
The unbiased approaches of the last decade have enabled the collection of new data on the biology of annexin A1 (ANXA1) in a variety of scientific aspects, creating opportunities for new biomarkers and/or therapeutic purposes. ANXA1 is found in the plasma membrane, cytoplasm, and nucleus, being described at low levels in the nuclear and cytoplasmic compartments of placental cells related to gestational diabetic diseases, and its translocation from the cytoplasm to the nucleus has been associated with a response to DNA damage. The approaches presented here open pathways for reflection upon, and intrinsic clarification of, the modulating action of this protein in the response to genetic material damage, as well as its level of expression and cellular localization. The objective of this study is to arouse interest, with an emphasis on the mechanisms of nuclear translocation of ANXA1, which remain underexplored and may be beneficial in new inflammatory therapies.
Mast cell-activating signals in cold urticaria are not yet well defined and are likely to be heterogeneous. Cold agglutinins and cryoglobulins have been described as factors possibly associated with cold urticaria, but their relevance has not been explained. We performed a single-center prospective cohort study of 35 cold urticaria patients. Cold agglutinin and cryoglobulin test results, demographics, detailed history data, cold stimulation test results, complete blood count values, C-reactive protein, total immunoglobulin E levels, and basal serum tryptase levels were analyzed. Forty six percent (n = 16) of 35 tested patients had a positive cold agglutinin test and 27% (n = 9) of 33 tested patients had a positive cryoglobulin test. Cold agglutinin positive patients, when compared to cold agglutinin negative ones, were mainly female (P = 0.030). No gender-association was found for cryoglobulins. A positive cold agglutinin test, but not a positive cryoglobulin test, was associated with a higher rate of reactions triggered by cold ambient air (P = 0.009) or immersion in cold water (P = 0.041), and aggravated by increased summer humidity (P = 0.007). Additionally, patients with a positive cold agglutinin test had a higher frequency of angioedema triggered by ingestion of cold foods or drinks (P = 0.043), and lower disease control based on Urticaria Control Test (P = 0.023). Cold agglutinin levels correlated with erythrocyte counts (r = −0.372, P = 0.028) and monocyte counts (r = −0.425, P = 0.011). Cryoglobulin concentrations correlated with basal serum tryptase levels (r = 0.733, P = 0.025) and cold urticaria duration (r = 0.683, P = 0.042). Results of our study suggest that cold agglutinins and cryoglobulins, in a subpopulation of cold urticaria patients, are linked to the course and possibly the pathogenesis of their disease.
Chronic obstructive pulmonary disease (COPD) is related to smoking and anti-inflammatory therapy is indicated. Among the mediators with anti-inflammatory properties, we highlight piperlongumine (PL), an alkaloid/amide of Piper longum. Here we evaluated the PL administration on an experimental model of respiratory inflammation resulting from exposure to cigarette smoke. Male Balb/c mice were exposed to burning of 10 commercial cigarettes, 2x/day, for five weeks on specific equipment. PL efficacy was evaluated in control, exposed to smoke without treatment and PL treated (2.0 mg/kg, 3x/week) groups. Animals were weighed and plethysmographic analyses performed at the end of the exposure protocol. Inflammatory cells were evaluated in the bronchoalveolar lavage (BAL) and hemoglobin and glucose in the blood. Lung fragments were processed for histopathological studies and AnxA1, COX-2, NF-kB and neutrophil elastase expressions. Plethysmography revealed that PL maintained pulmonary frequency, volume and ventilation parameters similar to controls, with respiratory volume reduction compared to untreated animals. Final weight was reduced in both exposed groups. PL decreased hemoglobin concentration, attenuated the reduction of glucose levels and reduced influx of lymphocytes, neutrophils and macrophages in BAL. Histopathologically occured infiltration of inflammatory cells, increase of the interalveolar septa and intra-alveolar spaces in untreated animals. But, PL administration recovered lung tissues and, immunohistochemically, promoted increased expression of AnxA1 and reduction of COX-2, NF-kB and neutrophil elastase. Together the results indicate that PL attenuates systemic and pulmonary inflammatory changes, partially by modulating the expression the endogenous AnxA1, and may represent a promising therapy in preventing the inflammation induced by cigarette smoke.
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.
Annexin A1 (AnxA1) is a glucocorticoid-regulated protein endowed with anti-inflammatory and proresolving properties. Intact AnxA1 is a 37-kDa protein that may be cleaved in vivo at the N-terminal region by neutrophil proteases including elastase and proteinase-3, generating the 33-kDa isoform that is largely inactive. In this study, we investigated the dynamics of AnxA1 expression and the effects of synthetic (sivelestat [SIV]; Eglin) and natural (secretory leukocyte protease inhibitor [SLPI]; Elafin) protease inhibitors on the resolution of LPS-induced inflammation. During the settings of LPS inflammation AnxA1 cleavage associated closely with the peak of neutrophil and elastase expression and activity. SLPI expression increased during resolving phase of the pleurisy. Therapeutic treatment of LPS-challenge mice with recombinant human SLPI or Elafin accelerated resolution, an effect associated with increased numbers of apoptotic neutrophils in the pleural exudates, inhibition of elastase, and modulation of the survival-controlling proteins NF-κB and Mcl-1. Similar effects were observed with SIV, which dose-dependently inhibited neutrophil elastase and shortened resolution intervals. Mechanistically, SIV-induced resolution was caspase-dependent, associated to increased levels of intact AnxA1 and decreased expression of NF-κB and Mcl-1. The proresolving effect of antiproteases was also observed in a model of monosodium urate crystals-induced inflammation. SIV skewed macrophages toward resolving phenotypes and enhanced efferocytosis of apoptotic neutrophils. A neutralizing antiserum against AnxA1 and a nonselective antagonist of AnxA1 receptor abolished the accelerated resolution promoted by SIV. Collectively, these results show that elastase inhibition not only inhibits inflammation but actually promotes resolution, and this response is mediated by protection of endogenous intact AnxA1 with ensuing augmentation of neutrophil apoptosis.
Cervical cancer is the second most frequent cancer in women worldwide and is associated with genetic alterations, infection with Human PapillomaVirus (HPV), angiogenesis and inflammatory processes. The idea that inflammation is involved in tumorigenesis is supported by the frequent appearance of cancer in areas of chronic inflammation. On the other hand, the inflammatory response is controlled by the action of anti-inflammatory mediators, among these mediators, annexin A1 (ANXA1), a 37kDa protein was detected act as a modulator of inflammatory processes and is expressed by tumor cells. The study was carried out on the epithelial cancer cell line (SiHa) treated with the peptide of annexin A1 (ANXA1Ac2-26). We combined subtraction hybridization approach, Ingenuity Systems software and quantitative PCR, in order to evaluate gene expression influenced by ANXA1. We observed that ANXA1Ac2-26 inhibited proliferation in SiHa cells after 72hours. In these cells, 55 genes exhibited changes in expression levels in response to peptide treatment. Six genes were selected and the expression results of 5 up-regulated genes (TPT1, LDHA, NCOA3, HIF1A, RAB13) and one down-regulated gene (ID1) were research by real time quantitative PCR. More four genes (BMP4, BMPR1B, SMAD1 and SMAD4) of the ID1 pathway was investigated and only one (BMPR1B) show the same down regulation. The data indicate the involvement of ANXA1Ac2-26 in the altered expression of genes involved in tumorigenic processes, which could potentially be applied as a therapeutic indicator of cervical cancer.
Copyright © 2015. Published by Elsevier B.V.
Stroke is among the most common diseases of advanced age and is becoming a steadily increasing financial healthcare problem in the industrialized world with the increasing longevity and aging of the population. The incidence of ischemic stroke is highest in the elderly population, representing one of the most common causes of disability and mortality worldwide. Over the past decades, a tremendous amount of research has been undertaken into developing effective therapeutic strategies for the treatment of acute stroke. Unfortunately, many neuroprotective agents that have shown successful results in treating animal models of acute stroke have failed to translate into clinical treatments. Only tissue plasminogen activator is currently licensed for use in the treatment of acute ischemic stroke. Increasing evidence shows that the central nervous system and the immune system interact in complex ways, and better insight into these interactions may be relevant to the treatment of patients with stroke and other forms of central nervous system injury. However, during recent years, promising findings suggest that systemic inflammation and neuroinflammation are central features in cerebrovascular disease. Atherosclerosis, autoimmune disease, and physiological stressors, such as infection or surgery, may be a risk factor for the initial development of cerebral ischemia. In addition, the immune system actively participates in the pathophysiological processes occurring during an ischemic stroke. Thrombosis and hypoxia trigger an intravascular inflammatory cascade which elicits an inflammatory response in the injured brain that is accompanied by a marked local inflammatory reaction that is initiated by ischemia- or hematoma-induced expression of cytokines, adhesion molecules, and other inflammatory mediators, including prostanoids, extracellular proteases, reactive oxygen species, and nitric oxide, leading to the accumulation of inflammatory cells, such as leukocytes and microglia, which is further augmented by the innate immune response to cellular damage occurring in the parenchyma. Many of these compounds are known to promote and sustain inflammatory responses at local and systemic level, producing a neuroinflammatory response and a systemic acute-phase response. The acute-phase inflammatory response after stroke is a reflection of an unspecific systemic inflammatory response syndrome. Classic acute-phase reactants and body temperature are also modified in stroke and may be useful in the prediction of events and outcome and as therapeutic targets. The activation of innate immunity after stroke sets the stage for an adaptive immune response directed against brain antigens. The pathogenic significance of adaptive immunity and its long-term effects on the postischemic brain remains unclear, but it cannot be ruled out that a persistent autoimmune response to brain antigens has deleterious and long-lasting consequences, such as the development of poststroke dementia. This immune activation causes secondary tissue injury, but it is unclear whether modulating the acute immune response to stroke can produce clinical benefits. Better understanding of the role of the postischemic-induced inflammatory response and its potential for modulation might have profound implications for patient treatment. Preclinical studies suggest that interventions that are aimed at attenuating such inflammation reduce the progression of brain damage that occurs during the late stages of cerebral ischemia. In particular, strategies that block the activity of inflammation-related enzymes reduce ischemic damage with an extended therapeutic window. Although, clinical trials using anti-inflammatory strategies did not show benefit in patients with ischemic stroke, there is a strong rationale for continuing to explore the efficacy of anti-inflammatory therapies in the treatment of the late stages of cerebral ischemia acting more on the modulation of these later events than targeting of specific steps in the ischemic cascade.
In vivo interactions between neutrophils and endothelial cells (EC) follow a multistep process involving two distinct neutrophil adhesion receptors. L-selectin, constitutively functional on resting neutrophils, mediates an activation-independent primary interaction resulting in rolling along the venular wall. Subsequent activation of rolling neutrophils induces upregulation and functional activation of beta 2-integrins (CD11/CD18) leading to firm attachment. Based on previous findings we hypothesized that, under shear force, rolling may be essential for successful neutrophil-EC recognition. Here we report results of our studies of human neutrophil behavior in interleukin (IL)-1-activated rabbit mesentery venules, an interaction that requires both L-selectin and beta 2-integrins. Rolling of human neutrophils is L-selection mediated; it was strongly reduced by monoclonal antibody inhibition or enzymatic removal of L-selectin. Furthermore, activation induced L-selectin shedding and, in a dose- and time-dependent fashion, rendered neutrophils unable to recognize inflamed EC despite expression of active beta 2-integrins, which promoted adhesion in vitro. Neutrophils activated for 5 min or longer lost most of their ability to roll. However, 1-3 min after activation, rolling was reduced (not abolished), and cells that were still able to roll displayed a significant tendency for a CD18-dependent transition from rolling to sticking. The whole sequence of events, rolling, sticking, and transendothelial migration, could be observed if an extravascular chemotactic stimulus was applied by superfusing mesenteries with leukotriene B4. Under such conditions, sticking and emigration was blocked when rolling was inhibited by enzymatic removal of L-selectin. Our results indicate that primary neutrophil interaction with inflamed EC through the L-selectin is a prerequisite for neutrophil function at physiological shear rates in vivo.
The mechanism and cofactor requirements of exocytotic membrane fusion in neutrophils are unknown. Cytosolic proteins have been implicated in membrane fusion events. We assessed neutrophil cytosol for the presence of fusogenic proteins using a liposome fusion assay (lipid mixing). A fusogenic 36-kD protein containing amino acid sequence homology with human annexin I was purified from the cytosol of human neutrophils. This protein also shared functional characteristics with annexin I: it associated with and promoted lipid mixing of liposomes in a Ca(2+)-dependent manner at micromolar Ca2+ concentrations. The 36-kD protein required diacylglycerol to promote true fusion (contents mixing) at the same Ca2+ concentrations used for lipid mixing. The 36-kD protein exhibited a biphasic dose-response curve, by both promoting and inhibiting Ca(2+)-dependent lipid-mixing between liposomes and a plasma membrane fraction. The 36-kD protein also promoted Ca(2+)-dependent increases in aggregation of a specific granule fraction, as measured by a turbidity increase. Antiannexin I antibodies depleted the 36-kD protein from the cytosol by greater than 70% and diminished its ability to promote lipid mixing. Antiannexin I antibodies also decreased by greater than 75% the ability of neutrophil cytosol to promote Ca(2+)-dependent aggregation of the specific granules. These data suggest that annexin I may be involved in aggregation and fusion events in neutrophils.
Annexins are primarily intracellular proteins as would be predicted from their lack of hydrophobic signal sequences. However, we now report that the human prostate gland selectively secretes high concentrations of annexin 1 (also called lipocortin 1 and p35) and a proteolytic cleavage product, des1-29-annexin 1, into seminal plasma. Secreted annexin 1 had a blocked amino terminus and was structurally indistinguishable from intracellular annexin 1. Although annexin 1 and the structurally related protein, annexin 4, co-localized to many of the same cells of the ductal epithelium of the prostate, annexin 4 was not secreted. Thus, the secretion of annexin 1 appears to involve a highly selective mechanism that does not involve targeting to the endoplasmic reticulum by a hydrophobic signal sequence.
Lipocortins are structurally related, glucocorticoid-inducible proteins that inhibit phospholipase A2 (PLA2), thereby reducing the liberation of arachidonic acid from phospholipids and so limiting the synthesis of eicosanoid inflammatory mediators. This study is the first demonstration of one lipocortin, lipocortin 1 (Lc 1; 37 kDa), in human lung lavage supernatants. In lavage fluid from healthy volunteers, a higher percentage (greater than 70%) of the detected Lc 1 was in its native form, compared to that from patients with abnormal lungs. In patients' lavage fluids, Lc 1 was more likely to be partially degraded (34 kDa). In abnormal bronchoalveolar lavage fluid (BALF), the more polymorphonuclear neutrophils (PMN)/lavage, the lower the proportion of Lc 1 in the native (37 kDa) form (n = 7 pairs, rs = -0.8214, p less than 0.05). Furthermore, when BALF cells were cultured and the harvested conditioned media incubated with pure human recombinant Lc 1, degradation of the 37 kDa form increased with the percentage of PMN (n = 10 pairs, s = -0.7200 after 1 hr; n = 6 pairs, rs = -0.9241 after 6 hr). These results suggest that factors released from the PMN are responsible for Lc 1 degradation in man. When recombinant human Lc 1 was incubated with human neutrophil elastase, the enzyme degraded Lc 1 in a dose-dependent way, suggesting that neutrophil elastase may be one such factor. Since PMNs are ubiquitous at sites of inflammation, it is possible that Lc 1 degradation is a permissive mechanism, which ensures that sufficient inflammation occurs to destroy the provocative stimulus. However, it is equally possible that, in some circumstances, the mechanism may be pathological and that the inactivation of Lc 1 leads to chronic, uncontrolled inflammation.
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FIGURE 4.
FIGURE 4.
The injection of a suspension of a polyacrylamide gel (bio gel) into the dorsal subcutaneous area of mice induced an inflammatory reaction and the migration of neutrophils towards the inflamed site.
The intravenous administration of anti‐inflammatory drugs (dexamethasone, indomethacin and lysine‐acetylsalicylate) to Polyacrylamide gel‐treated mice inhibited the accumulation of neutrophils in the inflamed site.
A similar administration of a 36 K mouse lipocortin, induced a strong dose‐dependent inhibition of neutrophil accumulation in the inflamed site.
Dexamethasone and lipocortin inhibited the production of eicosanoids, prostaglandin E 2 (PGE 2 ) and leukotriene B 4 (LTB 4 ) in the inflamed site of Polyacrylamide gel‐treated mice.
Lipocortin impaired both phospholipase A 2 (PLA 2 ) activity and chemotaxis of isolated inflammatory neutrophils.
The present studies show an in vivo anti‐inflammatory effect of lipocortin similar to that of glucocorticosteroids. In agreement with recent data on the extracellular effects of various lipocortins, these results might implicate lipocortin(s) in the anti‐inflammatory effects of glucocorticosteroids.
Purified placental lipocortin I but not lipocortin II was proteolyzed during A431 cell membrane-catalyzed phosphorylation reactions. Proteolysis was Ca2+-dependent but was not prevented in the presence of a variety of inhibitors of Ca2+-dependent proteases, suggesting that the Ca2+ effect is a property of lipocortin I itself. Proteolysis was inhibited by Triton X-100 or dithiothreitol and was temperature-dependent, occurring at 30 degrees C but not at 0 degrees C. Tyrosine phosphorylation and proteolysis are distinct events as both phosphorylated and nonphosphorylated lipocortins could be cleaved by the membrane protease, but prephosphorylation enhanced the rate of proteolysis 2-fold during the initial reaction and by 60 min almost half of the phosphorylated lipocortin was proteolyzed. Cleavage of the 38-kDa phosphotyrosyl lipocortin I generated a truncated 37-kDa form of lipocortin which retained the phosphate label, indicating that proteolysis occurred at a site N-terminal to the site of tyrosine phosphorylation, possibly at tryptophan 12. Ando, Y., Imamura, S., Hong, Y.-M., Owada, M.K., Kakunaga, T., and Kannagi, R. [1989) J. Biol. Chem. 264, 6948-6955) have recently reported that in vitro cleavage at sites in the N-terminal tail region of lipocortin I by exogenously added proteases dramatically enhanced the Ca2+ sensitivity of phospholipid binding by lipocortin. The demonstrated ability of an endogenous membrane protease to catalyze a similar and specific cleavage in a Ca2+-dependent manner indicates that this event may occur in the cell where it would have important effects on the functional properties of lipocortin I.
We have purified two 35-kDa proteins from rat peritoneal lavages that inhibit phospholipase A2 activity. Both are calcium/phospholipid-dependent membrane binding proteins and share similar structural and biochemical properties with lipocortins I and II. By sequence analysis we confirmed that they are lipocortin-related, and we refer to the two inhibitors as lipocortins III and V. Using partial sequence information obtained from the purified rat proteins, full length cDNA clones for both proteins and for their human counterparts were isolated. As with lipocortins I and II, the amino acid sequences of lipocortins III and V which were deduced from the cDNA clones are highly conserved, sharing 50% identity with other family members. Related proteins were also purified from bovine intestinal mucosa and characterized by peptide mapping, sequence, and immunological analyses. In addition to lipocortins III and V the bovine preparation contained a third 35-kDa inhibitor and a 68-kDa inhibitor, extending the number of known lipocortins to six distinct proteins. While the various lipocortins are structurally similar, distinct differences in their cellular distribution indicate specialized roles for the individual proteins.
We have purified from rat peritoneal exudates a 37-kDa protein that inhibits phospholipase A2 activity. It is the predominant phospholipase inhibitor protein in these preparations and also is detected in a wide variety of cell lines. Levels of expression range from 0 to 0.5% of total protein. In the peritoneal preparations, the inhibitor is partially proteolyzed into a series of lower mass forms, including species at 30, 24, and 15 kDa. These fragments all are immunoreactive with an antibody raised against the 37-kDa protein. The rat protein also is immunoreactive with an antibody developed against a 6-kDa phospholipase inhibitor protein from snake venom. The primary structure of more than half of the rat inhibitor has been deduced by protein microsequence analysis. These sequences are closely related to sequences from its human analogue, which we recently cloned and expressed (Wallner, B. P., Mattaliano, R. J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L. K., Foeller, C., Chow, E. P., Browning, J. L., Ramachandran, K. L., and Pepinsky, R. B. (1986) Nature, in press), and thus we infer that the inhibitor is highly conserved evolutionarily. Properties of the molecule suggest that it is a member of a family of steroid-induced anti-inflammatory proteins collectively referred to as lipocortin.
Although oligonucleotide probes are useful for in situ hybridization, their low sensitivity compared to riboprobes and cDNA remains a problem. We have systematically examined the protocols to provide a general procedure that increases the sensitivity of oligoprobes for light and electron in situ hybridizations by using mixtures of multiple non-overlapping oligonucleotides (multi-oligoprobes). The protocol achieves these improvements with both radioactive and non-radioactive oligoprobes. With 33P-labeled probes in a semiquantitative assay, we found that mixtures of up to six vasopressin-directed multi-oligoprobes, each employed at saturating concentration, led to an additive signal with no significant increase of the background. Using this approach with non-radioactive oligoprobes, we were able to detect in the hypothalamus several low or moderately abundant mRNAs, such as vasopressin heterogeneous nuclear RNA and the galanin, dynorphin, and tyrosine hydroxylase mRNAs. Moreover, we showed that multi-oligoprobes used in a pre-embedding procedure were suitable for studying the ultrastructural compartmentalization of moderately abundant mRNAs. Finally, with the same basic approach we demonstrated that two sets of multi-oligoprobes can be combined for simultaneous detection of two different mRNAs using fluorescent dyes, making this approach suitable for high-resolution confocal analyses. Overall, our data demonstrate that multi-oligoprobes provide a sensitive tool of choice for various applications in which both well-preserved morphology and high sensitivity are needed. In particular, these probes appear ideal for study of the comparative subcellular localization of mRNAs at both the light and the electron microscopic level.
Annexin I is a 36 kilodalton (kD) calcium-dependent phospholipid-binding protein which may have anti-inflammatory properties. Previous investigations which sampled lower respiratory tract epithelial lining fluid (ELF) via bronchoalveolar lavage (BAL) have demonstrated that annexin I can be degraded in inflammatory lung disease. We analyzed BAL fluid from patients with cystic fibrosis (CF) to determine the effects of lung inflammation on the structure and activity of annexin I. Intact annexin I was absent in 17 out of 20 BAL fluid samples from patients with CF, due largely to degradation to a 33 kD protein. The three CF BAL fluids in which annexin I was detectable had very little or no unopposed neutrophil elastase activity in contrast to the 17 in which no annexin I was detectable. Annexin I was present in all BAL fluid samples from 10 normal volunteer (NV) subjects and 12 patients with interstitial lung disease (ILD). The 33 kD annexin I breakdown product was not detectable in samples from NV, but was detectable only in ILD patients with relatively high percentages of neutrophils on BAL differential cell counts. Annexin I appeared to be cleaved by neutrophil elastase at the N-terminal portion between Val-36 and Ser-37 to yield the 33 kD protein. Cleavage of the N-terminal portion of annexin I was accompanied by a marked change in the annexin I isoelectric point (pI) value (from 6.0 to 8.5-9.0) and greatly diminished annexin I functional activity. Our findings demonstrate that annexin I degradation in epithelial lining fluid is closely related to lung inflammation.
We have examined in whole blood the actions of 2 lipoxin A4 (LXA4) stable analogs, 15-R/S-methyl-LXA4 and 16-phenoxy-LXA4, for their impact on the expression of adhesion molecules on human leukocytes and coronary artery endothelial cells (HCAEC) and on neutrophil adhesion to HCAEC in vitro. Both LXA4 analogs in nanomolar to micromolar concentrations prevented shedding of L-selectin and downregulated CD11/CD18 expression on resting neutrophils, monocytes, and lymphocytes. Changes in CD11/CD18 expression were blocked by the mitogen-activated protein kinase kinase inhibitor PD98059. The LXA4 analogs also attenuated changes in L-selectin and CD11/CD18 expression evoked by platelet-activating factor (PAF), interleukin-8, or C-reactive protein-derived peptide 201-206 with IC50 values of 0.2 to 1.9 μmol/L, whereas they did not affect lipopolysaccharide (LPS)– or tumor necrosis factor-–stimulated expression of E-selectin and intercellular adhesion molecule-1 on HCAEC. These LXA4analogs markedly diminished adhesion of neutrophils to LPS-activated HCAEC. Inhibition of adhesion was additive with function blocking anti–E-selectin and anti–L-selectin antibodies, but was not additive with anti-CD18 antibody. Combining LXA4 analogs with dexamethasone (100 nmol/L) almost completely inhibited PAF-induced changes in adhesion molecule expression on leukocytes and gave additive inhibition of neutrophil adhesion to HCAEC. Culture of HCAEC with dexamethasone, but not with LXA4 analogs, also decreased neutrophil attachment. Together, these results indicate that LXA4 stable analogs modulate expression of both L-selectin and CD11/CD18 on resting and immunostimulated leukocytes and inhibit neutrophil adhesion to HCAEC by attenuating CD11/CD18 expression. These actions are additive with those of glucocorticoids and may represent a novel and potent regulatory mechanism by which LXA4 and aspirin-triggered 15-epi-LXA4 modulate leukocyte trafficking.
We have examined in whole blood the actions of 2 lipoxin A4 (LXA4) stable analogs, 15-R/S-methyl-LXA4 and 16-phenoxy-LXA4, for their impact on the expression of adhesion molecules on human leukocytes and coronary artery endothelial cells (HCAEC) and on neutrophil adhesion to HCAEC in vitro. Both LXA4 analogs in nanomolar to micromolar concentrations prevented shedding of L-selectin and downregulated CD11/CD18 expression on resting neutrophils, monocytes, and lymphocytes. Changes in CD11/CD18 expression were blocked by the mitogen-activated protein kinase kinase inhibitor PD98059. The LXA4 analogs also attenuated changes in L-selectin and CD11/CD18 expression evoked by platelet-activating factor (PAF), interleukin-8, or C-reactive protein-derived peptide 201-206 with IC50 values of 0.2 to 1.9 μmol/L, whereas they did not affect lipopolysaccharide (LPS)– or tumor necrosis factor-–stimulated expression of E-selectin and intercellular adhesion molecule-1 on HCAEC. These LXA4analogs markedly diminished adhesion of neutrophils to LPS-activated HCAEC. Inhibition of adhesion was additive with function blocking anti–E-selectin and anti–L-selectin antibodies, but was not additive with anti-CD18 antibody. Combining LXA4 analogs with dexamethasone (100 nmol/L) almost completely inhibited PAF-induced changes in adhesion molecule expression on leukocytes and gave additive inhibition of neutrophil adhesion to HCAEC. Culture of HCAEC with dexamethasone, but not with LXA4 analogs, also decreased neutrophil attachment. Together, these results indicate that LXA4 stable analogs modulate expression of both L-selectin and CD11/CD18 on resting and immunostimulated leukocytes and inhibit neutrophil adhesion to HCAEC by attenuating CD11/CD18 expression. These actions are additive with those of glucocorticoids and may represent a novel and potent regulatory mechanism by which LXA4 and aspirin-triggered 15-epi-LXA4 modulate leukocyte trafficking.
Annexin 1 is a protein induced by glucocorticoids endowed with extracellular anti-inflammatory properties. In this study, the local expression and secretion of annexins 1–6, in rat proximal colon, were studied at different times after intracolonic administration of 30 mg trinltrobenzenesulfonic acid in 50% ethanol. Secretion was identified by incubating colonic tissues in a culture medium. The expression of annexins was detected by immunoblotting in tissue homogenates and incubation media. Inflammatory stages were evaluated by measuring myeloperoxidase activity. Annexin 1 expression in colons increased after trinitrobenzenesulfonic acid treatment and was maximal between days 1 to 9, during the cellular stage of the inflammation that corresponded to maximal myeloperoxidase activity. From 12 h to 9 days after trinitrobenzenesulfonic acid/ethanol treatment, annexin 1 was specifically secreted. Annexin 3 was also overexpressed during the cellular stage, but the expression of annexins 2, 4, 5, and 6 was unchanged; none of these annexins were secreted. Annexin 1 was shown to be physiologically secreted because its release was specific, abundant, and not correlated with cellular lysis. Annexin 1 may be considered as a putative candidate in the control of the gut inflammatory processes.
British Journal of Pharmacology (1999) 126, 537–550; doi:10.1038/sj.bjp.0702328
Annexin I, a member of the calcium- and phospholipid-binding annexin superfamily of proteins, is largely present in human neutrophils. To determine its exact intracellular distribution a combination of flow cytometry, confocal microscopy and electron microscopy analyses were performed on resting human neutrophils as well as on cells which had been activated. In resting neutrophils, annexin I was found to be present in small amounts in the nucleus, in the cytoplasm and partially also associated with the plasma membrane. The cytoplasmic pool of annexin I was predominant, and the protein was co-localized with gelatinase (marker of gelatinase granules), but not with human serum albumin or CD35 (markers of secretory vesicles), or with lysosomes. Electron microscopy showed the presence of annexin I inside the gelatinase granules. Neutrophil adhesion to monolayers of endothelial cells, but not phagocytosis of particles of opsonized zymosan, provoked an intense mobilization of annexin I, with a marked externalization on the outer leaflet of the plasma membrane. Remaining intracellular annexin I was also found in proximity of the plasma membrane. These results provide a novel mechanism for annexin I secretion from human neutrophils, which is via a degranulation event involving gelatinase granules.
A multi-faceted approach was used to investigate the effect of an anti-inflammatory peptide derived from human lipocortin 1 N-terminus region (amino acid 2–26; termed human Ac2–26) on human neutrophil activation in vitro. When incubated with purified human neutrophils. human Ac2–26 produced a concentration-dependent inhibition of elastase release stimulated by formyl-Met-Leu-Phe (fMLP), platelet-activating factor, or leukotriene B4, with an approximate EC50 of 33 μM (100 μg/ml). At this concentration, human Ac2–26 also inhibited (77%) the release of [3H]-arachidonic acid from neutrophils stimulated with fMLP. The peptide, however, did not inhibit the up-regulation of the β2-integrin CD11b and the concomitant shedding of L-selectin from neutrophil plasma membrane induced by fMLP. In adhesion experiments, human Ac2–26 inhibited neutrophil adhesion to endothelial monolayers when this was stimulated with fMLP, but not when this followed endothelial cell activation with histamine or platelet-activating factor. Again, the effect of the peptide was concentration-dependent, and an approximate EC50 of 33 μM was calculated. When a preparation of 125I-labeled human Ac2–26 was incubated with the neutrophils, the peptide was internalised in an energy-dependent fashion. All together, these observations lead us to propose a model in which this peptide derived from the N-terminus of human lipocortin 1 alters a common cellular mechanism producing a selective inhibition of neutrophil activation.
To gain direct access to the secretory machinery and study the regulation, mechanisms, and effectors of Ca2+-dependent neutrophil secretion, we developed an efficient and reproducible method of plasma membrane permeabilization using streptolysin O. We confirmed previous studies that permeabilized neutrophils secrete in response to calcium alone, but we also found that the Ca2+ dose-response is biphasic. Secretion is detectable at <1.0 microM Ca2+ and reaches a plateau between 1.0 and 60 to 80 microM. When stimulated with >80 microM Ca2+, secretion is two- to threefold greater than at lower [Ca2+], suggesting that two distinct mechanisms of Ca2+-dependent secretion that differ in their affinity for Ca2+ exist in neutrophils. Although permeabilization allows 100% leak of lactate dehydrogenase, maximum secretion from permeabilized cells is 80% that of f-met-leu-phe-stimulated intact cells, indicating that the essential components of the Ca2+-dependent secretory apparatus are predominantly, if not entirely, membrane bound. Permeabilization causes leakage of 100% of annexins V and VI, but 41% of annexin I and 12% of annexin III are retained. Immunofluorescence microscopy revealed that retained annexins I and III are associated with granule membranes. Addition of soluble annexins I and III to permeabilized cells increased Ca2+-induced secretion up to 15% and 90%, respectively, implying that both annexins participate in this secretory pathway. While annexin V is not required for secretion, it inhibits the low Ca2+-affinity mechanism of secretion.
Using immunofluorescence, an affinity-purified anti-annexin-1 polyclonal antibody showed both cytoplasmic and nuclear staining, whereas antibodies against annexins 2, 5 and 6 labelled almost exclusively the cytoplasm of cultured endothelial cells. This was further confirmed by immunogold labelling and electron microscopy using a monoclonal antibody, annexin 1 being detected close to the plasma membrane, in the cytoplasm, as well as inside the nucleus. Finally, using immunoblotting, purified nuclei were shown to contain annexin 1, which was not removed by EDTA treatment. These data open some new perspectives in the understanding of annexin function, including possible involvement in nucleoskeleton dynamics and regulation of proliferation through cell signalling.
The mechanisms that regulate inflammatory responses, and their dysfunction in chronic inflammatory diseases, are poorly understood. Lipocortin 1 is a potential mediator of the anti-inflammatory effects of glucocorticoid hormones. Following the discovery of putative receptors on phagocytes for lipocortin 1, Nick Goulding and Paul Guyre here offer one explanation for the self-limitation of inflammation and the impairment of the mechanism in chronic inflammatory disease.
We have developed two monoclonal antibodies to human lipocortin-1 (103 and 105) as reagents for quantitating the protein in biological systems and neutralizing its activity. Lipo 105 is a high affinity antibody that is functional in ELISA and Western blot formats. The antibody recognizes a site between amino acids 30 and 55 in the lipocortin-1 sequence and can be used on native or denatured protein. Lipo 103 is an antibody that neutralizes the phospholipase A2 inhibitory activity of lipocortin-1 by blocking binding of the protein to phospholipid surfaces. The antibody is specific for native human lipocortin-1. Lipo 103 was recently shown to block lipocortin-1-dependent differentiation of a squamous carcinoma cell line, demonstrating its usefulness as a probe for function.
IL-1 and IL-6 activities were measured in the pleural exudate of rats during carrageenin-induced pleurisy to examine the relationship of the local production of cytokines to the inflammatory reaction. Time courses of appearance of the cytokines and inflammatory parameters in the exudate were compared. IL-1 activity and exudate volume started to increase at 1 h after the carrageenin injection, and then slightly later IL-6 activity and leukocyte number began to increase. IL-1 showed peak activity of approximately 700 U at 3 h and IL-6, of 6000 U at 5 h in the exudate, whereas exudate volume and number of polymorphonuclear leukocytes continued to increase thereafter. Furthermore, IL-6 level in the plasma of the carrageenin-injected rats showed a peak at 4 h (30 U/ml), and when rhIL-1 alpha (100,000 U) was intrapleurally injected, the more rapid increase in plasma IL-6 level was demonstrated at 1 h (30 U/ml). This latter rise was neutralized with simultaneous injection of anti-rhIL-1 alpha antibody. These facts indicate the possibility that IL-1 produced in the exudate or injected could rapidly propagate a signal to induce IL-6 production in the circulation. It took several hours to transmit an inflammatory signal that stimulated the liver to synthesize the acute-phase protein, T-kininogen. The time lag from the peak induction of IL-1 to the T-kininogen-increase in the pleurisy corresponded well to the interval for T-kininogen-increase by exogenous rhIL-1 alpha injection. These results strongly suggest that the initial inflammatory stimulus induces sequentially IL-1, IL-6, and T-kininogen production in this carrageenin inflammation.
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.
Lipocortin-1 protein synthesis in resting monocytes is under the control of glucocorticoid steroids. This induction occurs at reasonable dexamethasone concentrations, may require concomitant synthesis of transcriptional factors, appears to be cell type specific, and has been observed only in primary tissues in our hands. Variability in the magnitude of the induction suggests that the regulation is complex, involving either additional factors or particular differentiation states. In addition to the induction of intracellular lipocortin-1, steroids cause the appearance of labelled lipocortin-1 on the outer surface of the cells. Whether cell breakage can account for this effect is unclear. Considerable microheterogeneity was found in preparations of recombinant-lipocortin-1. Aspects of N-terminal post-translational processing, N-terminal proteolysis, conformational states and the existence of an air-denatured form lacking alpha-helical structure contributed to this heterogeneity. We believe that these aspects are responsible for the variable biological potency of different preparations. It remains unclear whether this protein actually plays a physiological role in the regulation of the inflammatory response or achieves its effects through membrane binding and subsequent non-physiological perturbation of the cells.
Human recombinant lipocortin 1 has been tested for anti-inflammatory activity in a conventional model of acute inflammation. Microgram amounts of the protein, locally administered, inhibited edema of the rat paw when induced by subplantar injections of carrageenin: the ED50 was 10-20 micrograms per paw, and inhibition (maximum of 60-70%) was not dependent upon an intact adrenal cortex. Doses of lipocortin that produced approximately 50% inhibition in the carrageenin test were inactive against edema elicited by bradykinin, serotonin, platelet-activating factor-acether, or dextran, whereas edema caused by Naja mocambique venom phospholipase A2 was strongly inhibited by lipocortin. The protein inhibited edema when rats were pretreated with agents that depleted mast-cell amines, kininogen, or polymorphonuclear leukocytes prior to initiation of the carrageenin edema but had no inhibitory action when rats were pretreated with the dual cyclooxygenase/lipoxygenase inhibitor BW 755C. These results demonstrate that human recombinant lipocortin has potent local anti-inflammatory activity, probably through selectively interfering with eicosanoid generation. Lipocortin is relatively ineffective against edema caused by mast-cell degranulation or kinins, except when degranulation is caused by phospholipase A2.
Lipocortin-I (p35) is a unique calcium- and phospholipid-binding protein of the lipocortin/calpactin family. Although several possibilities have been suggested, functions for the individual proteins of this family are not yet known with certainty. As an initial step in the identification of the biological function(s) of p35, we have used immunohistochemical methods to define precisely many of the cellular phenotypes that contain p35 in vivo. In all organs where p35 is found, we have observed a striking distribution of p35-positive cells. Typically it is highly enriched in a limited range of differentiated cell types while apparently totally absent from most others. Our identification of specific p35-positive cell types in vivo will now set limitations on likely possibilities for functions of this protein and thereby permit a more logical approach to the determination of its true function.
This article has no abstract; the first 100 words appear below.
WITH increasing frequency, the human neutrophil is being implicated as a mediator of tissue-destructive events in inflammatory diseases ranging from rheumatoid arthritis and myocardial reperfusion injury to respiratory distress syndromes, blistering skin disorders, and ulcerative colitis.¹,² In each of these diseases, as well as a variety of other acute inflammatory disorders, important components of these pathologic processes are being linked to the neutrophil's ability to release a complex assortment of agents that can destroy normal cells and dissolve connective tissues. Although these toxins normally defend the host against invading microbes, the neutrophil has little intrinsic ability to differentiate between foreign . . .
Supported in part by grants (HL-28024, AI-21301, and AI-23876) from the National Institutes of Health.
I am indebted to Vivek Reddy for helpful discussions.
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From the Division of Hematology and Oncology, Simpson Memorial Research Institute, Department of Medicine, University of Michigan Medical Center, Ann Arbor. Address reprint requests to Dr. Weiss at the Simpson Memorial Institute, 102 Observatory St., Ann Arbor, MI 48109.
The anti-inflammatory action of glucocorticoids has been attributed to the induction of a group of phospholipase A2 inhibitory proteins, collectively called lipocortin. These proteins are thought to control the biosynthesis of the potent mediators of inflammation, prostaglandins and leukotrienes, by inhibiting the release of their common precursor, arachidonic acid, a process that requires phospholipase A2 hydrolysis of phospholipids. Lipocortin-like proteins have been isolated from various cell types, including monocytes, neutrophils and renal medullary cell preparations. The predominant active form is a protein with an apparent relative molecular mass (Mr) of 40,000 (40K). These partially purified preparations of lipocortin mimic the effect of steroids, and mediate anti-inflammatory activity in various in vivo model systems. Using amino-acid sequence information obtained from purified rat lipocortin, we have now cloned human lipocortin complementary DNA and expressed the gene in Escherichia coli. Our studies confirm that lipocortin is a potent inhibitor of phospholipase A2 activity.
Inhibition by trasylol of carrageenin-induced swelling in the rat's paw was studied as well as the ability of carrageenin to release kinin-like substances from plasma substrates. The findings support the hypothesis that carrageenin acts through a proteolytic process with formation of kinin-like mediator(s).
Our recent studies suggest that lipocortin 1 (LC1), a potential mediator of the anti-inflammatory, antiproliferative and anti-fever actions of glucocorticoids in peripheral tissues, may also contribute to the powerful negative feedback actions of the steroids on the hypothalamo-pituitary-adrenal (HPA) axis. In the present study we have used (1) an in vitro model to examine the influence of a specific neutralizing monoclonal anti-LC1 antibody (anti-LC1 mAb) on the capacity of dexamethasone to suppress the cytokine-induced release of the 41-amino acid corticotropin-releasing factor (CRF-41) and arginine vasopressin (AVP) from the rat hypothalamus and (2) a passive immunization protocol to assess the contribution of LC1 to the inhibitory actions of dexamethasone on the HPA responses to immunological (i.p. injection of interleukin 1 beta, IL-1 beta) and surgical (laparotomy under ether anaesthesia) stress. In vitro, Il-1 alpha (0.2 ng/ml), IL-1 beta (0.5 ng/ml), IL-6 (10 ng/ml) and IL-8 (1 ng/ml) each caused significant increases in the release of immunoreactive (ir)-CRF-41 and ir-AVP from hypothalami removed from rats adrenalectomized 10-12 days before autopsy; these responses were readily inhibited by preincubation of the tissue with dexamethasone (10(-7) M). The inhibitory actions of the steroid were attenuated and, in many instances, abolished by inclusion in the medium of a monoclonal anti-LC1 antibody (LC1 mAb, diluted 1:15,000); an isotype-matched control antibody (antispectrin alpha+beta, diluted 1:15,000) was ineffective in this regard. IL-1 alpha (0.2 ng/ml), IL-1 beta (0.5 ng/ml) and IL-6 (10 ng/ml) also initiated similar increases in the release of CRF-41 and AVP from hypothalami from intact rats which were effectively blocked by dexamethasone (10(-7) M). However, although the inhibitory actions of the steroid on the pharmacologically evoked release of CRF-41 were specifically overcome by anti-LC1 mAb (diluted 1:15,000), the steroid blockade of AVP release was not. In vivo, rats pretreated with either a polyclonal anti-LC1 antibody (anti-LC1 pAb, 1 ml/day s.c. for 2 days) or a corresponding volume of a nonimmune sheep serum (NSS) responded to immunological (IL-1 beta, 3 micrograms/kg i.p.) or surgical (laparotomy under ether anaesthesia) trauma with significant increases in the serum ACTH and corticosterone concentrations. In the NSS-treated groups, dexamethasone (100 micrograms/kg), which had no effect on the prestress concentrations of ACTH and corticosterone in the blood, completely prevented the HPA responses to both IL-1 beta and laparotomy.(ABSTRACT TRUNCATED AT 400 WORDS)
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.
Adenosine receptors are present on most cells and organs, yet, although the physiological effects of adenosine were first described over 60 years ago, the potential therapeutic uses of adenosine have only been recognized and realized recently. A decade ago the potent anti-inflammatory effects of adenosine were first described; adenosine, acting at specific A2 receptors, inhibits some, but not all, neutrophil functions. Adenosine inhibits phagocytosis, generation of toxic oxygen metabolites, and adhesion (to some surfaces and to endothelial cells) but does not inhibit degranulation or chemotaxis. Occupancy of adenosine A2 receptors modulates leukocyte function by a novel mechanism. Although adenosine A2 receptors are classically linked to heterotrimeric GS signaling proteins and stimulation of adenylate cyclase, adenosine 3',5'-cyclic monophosphate does not act as the second messenger for inhibition of leukocyte function. By a mechanism that still remains obscure, occupancy of adenosine A2 receptors on neutrophils "uncouples" chemoattractant receptors from their stimulus-transduction proteins. The concentrations of adenosine that inhibit inflammatory cell function are similar to those observed in vivo and suggest a role for adenosine in the modulation of inflammation in vivo. Indeed, recent studies indicate that nonmetabolized adenosine receptor agonists are potent anti-inflammatory agents, and other studies indicate that methotrexate, a commonly used anti-inflammatory agent, diminishes inflammation by increasing adenosine release at inflamed sites. The observations reviewed here suggest that adenosine and agents that act through adenosine are excellent candidates for development as anti-inflammatory agents.
The kinetic expression and potential cellular source of tumor necrosis factor-alpha (TNF-alpha) in lipopolysaccharide-(LPS) induced acute lung inflammation was investigated using a rat model by Northern blot analysis, in situ hybridization and immunohistochemistry. LPS induced a polymorphonuclear leukocyte infiltrate in the lung that peaked between 6 and 24 hours. TNF-alpha messenger (m)RNA was strongly induced by LPS in whole lung tissues shown by Northern analysis. Both alveolar macrophages and polymorphonuclear leukocytes (PMNs), purified from bronchoalveolar lavage fluids of LPS-treated rats, were shown to express TNF-alpha mRNA by Northern analysis. However, PMNs displayed several times more TNF-alpha mRNA, relative to actin mRNA, than alveolar macrophages at 6 and 12 hours. By in situ hybridization, most of the cells positive for TNF-alpha mRNA at 6 and 12 hours seemed to be PMNs located within the tissue near bronchioles or vessels. By immunohistochemistry, TNF-alpha protein was localized mainly to alveolar macrophages at early times (1 to 3 hours) after LPS challenge, and thereafter, PMNs seemed to be the predominant source of TNF-alpha protein as more than 90% of total intraalveolar positive cells at 6 and 12 hours were PMN. Thus, our data provide the first in vivo evidence that PMNs can serve as a significant source of TNF-alpha at sites of acute inflammation.
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.
We have studied the occurrence, distribution and disposition of lipocortins (annexins) 1, 2 and 5 in mixed peritoneal leucocytes obtained from rats in which glucocorticoid levels were altered by adrenalectomy, administration of the glucocorticoid antagonist, RU486, or by injection of dexamethasone or hydrocortisone, as well as from rats in which the peritoneal cells were elicited by inflammatory stimuli.
In cells obtained from untreated rats with an intact adrenal cortex, lipocortins 1, 2 and 5 were readily detectable: the majority of each of the proteins was apparently located intracellularly with much smaller amounts in the membrane. Lipocortin 1 and to a lesser extent lipocortin 5 were also seen in a Ca ²⁺ ‐dependent association with the external plasma membrane. Following administration of RU486 (2 × 20 mg kg ⁻¹ ) the amounts of lipocortin 1 and 2 in cells were greatly reduced. Conversely, injection of hydrocortisone (1 mg kg ⁻¹ ) or dexamethasone (0.08 mg kg ⁻¹ ) caused an increase in the amount of lipocortin 1 and 2 in peritoneal cells within 30 min. Lipocortin 5 was unchanged by any manipulation of glucocorticoid levels.
Lipocortins 1 and 2 were elevated in both intracellular and membrane‐associated fractions of macrophages elicited by intraperitoneal injection in inflammogens. This phenomenon also occurred in adrenalectomized animals.
Our data indicate that glucocorticoids control the synthesis of some members of the lipocortin family in rat mixed peritoneal cells but also suggest the existence of a separate system for controlling the generation of this protein. The significance of these observations is considered in relation to the mechanism of glucocorticoid hormone action on eicosanoid production.
Annexin I is an abundant protein in U937 cells differentiated towards a macrophagic phenotype. These cells become able to kill Escherichia coli, however, the intracellular pathogen Brucella suis, known to interfere with phagosome maturation, multiply in these differentiated cells. We have analysed by confocal and electron microscopy the cellular localization of annexin I during phagocytosis of yeast, non-pathogenic E. coli and the intracellular pathogen B. suis. Using immunocytochemical detections annexin I was found mainly as patches in the cytoplasm of uninfected cells. Upon phagocytosis of yeast or E. coli organisms, annexin I rapidly translocated and concentrated around phagosomes. On the other hand, annexin I was never detected around live B. suis-containing phagosomes. However, when dead brucellae were used, annexin I did translocate to the periphagosomal region. Our results suggest that annexin I could play a role in the molecular mechanism of phagosome maturation, which is impaired by some intracellular pathogens.
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.
Annexin I is a member of a multigene family of Ca2+/phospholipid-binding proteins and a major substrate for the epidermal growth factor (EGF) receptor kinase, which has been implicated in membrane-related events along the endocytotic pathway, in particular in the sorting of internalized EGF receptors occurring in the multivesicular body. We analyzed in detail the intracellular distribution of this annexin by cell fractionation and immunoelectron microscopy. These studies used polyclonal as well as a set of species-specific monoclonal antibodies, whose epitopes were mapped to the lateral surface of the molecule next to a region thought to be involved in vesicle aggregation. Unexpectedly, the majority of annexin I was identified on early and not on multivesicular endosomes in a form that required micromolar levels of Ca2+ for the association. The specific cofractionation with early endosomes was also observed in transfected baby hamster kidney cells when the intracellular fate of ectopically expressed porcine annexin I was analyzed by using the species-specific monoclonal antibodies in Western blots of subcellular fractions. Interestingly, a truncation of the N-terminal 26, but not the N-terminal 13 residues of annexin I altered its intracellular distribution, shifting it from fractions containing early to those containing late and multivesicular endosomes. These findings underscore the regulatory importance of the N-terminal domain and provide evidence for an involvement of annexin I in early endocytotic processes.
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.
The effect of dexamethasone, lipocorton-12–26 and an antiserum to lipocortin-12–26 (LCPS1) upon the hyperalgesic activities in rats of carrageenin, bradykinin, tumour necrosis factor α (TNFα), interleukin-12, interleukin-6 (IL-6), interleukin-8 (IL-8), prostaglandin Eβ (PGE2) and dopamine were investigated in a model of mechanical hyperalgesia.
Hyperalgesic responses to intraplantar (i.pl.) injections of carrageenin (100 μg), bradykinin (500 ng), TNFα (2.5 pg), IL-1β (0.5 pg), and IL-6 (1.0 ng), but not responses to IL-8 (0.1 ng), PGE2 (100 ng) and dopamine (10 μg), were inhibited by pretreatment with dexamethasone (0.5 mg kg−1, subcutaneously, s.c., or 0.04–5.0 μg/paw).
Inhibition of hyperalgesic responses to injections (i.pl.) of bradykinin (500 ng) and IL-1β (0.5 pg) by dexamethasone (0.5 mg kg−1, s.c.) was reversed by LCPS1 (0.5 ml kg−1, injected s.c., 24 h and 1 h before hyperalgesic substances) and hyperalgesic responses to injections (i.pl.) of bradykinin (500 ng), TNFα (2.5 pg) and IL-1β (0.5 pg), but not responses to PGE2 (100 ng), were inhibited by pretreatment with lipocortin-12–26 (100 μg/paw). Also, lipocortin-12–26 (30 and 100 μg ml−1) and dexamethasone (10 μg ml−1) inhibited TNFα release by cells of the J774 (murine macrophage-like) cell-line stimulated with LPS (3 μg ml−1), and LCPS1 partially reversed the inhibition by dexamethasone. These data are consistent with an important role for endogenous lipocortin-12–26 in mediating the anti-hyperalgesic effect of dexamethasone, with inhibiton of TNFα production by lipocortin-12–26 contributing, in part, to this role.
Although arachidonic acid by itself was not hyperalgesic, the hyperalgesic response to IL-1β (0.25 pg, i.pl.) was potentiated by arachidonic acid (50 μg) and the potentiated response was inhibited by dexamethasone (50 μg, i.pl.) and lipocortin-12–26 (100 μg, i.pl.). Also, lipocortin-12–26 (30 and 100 μg ml−1) inhibited/abolished PGE2 release by J774 cells stimulated with LPS (3 μg ml−1). These data suggest that, in inflammatory hyperalgesia, inhibition of the induction of cyclo-oxygenase 2 (COX-2), rather than phospholipase A2, by dexamethasone and lipocortin-12–26 accounts for the anti-hyperalgesic effects of these agents.
The above data support the notion that induction of lipocortin by dexamethasone plays a major role in the inhibition by dexamethasone of inflammatory hyperalgesia evoked by carrageenin, bradykinin and the cytokines TNFα, IL-1β and IL-6, and provides additional evidence that the biological activity of lipocortin resides within the peptide lipocortin-12–26. Further, the data suggest that inhibition of lipocortin-12–26 of eicosanoid production by COX-2 also contributes to the anti-hyperalgesic effect of lipocortin-1.
Lipocortin 1 is considered a mediator of the anti-inflammatory actions of glucocorticoids. We have shown that this protein is overexpressed and secreted during an experimental colitis induced by intraluminal injection of trinitrobenzenesulfonic acid (TNBS) in rats. We studied here the in vivo regulation of lipocortin 1 expression and secretion in this model, either by glucocorticoids using adrenalectomized or dexamethasone-treated (3 mg/24 h) animals or by pituitary factors using hypophysectomized animals. Inflammation was evaluated by measuring myeloperoxidase activity and by histological scoring of the damage. Lipocortin 1 was detected by immunoblotting, and its secretion was studied by incubating colonic specimens in-culture medium. In the colon of TNBS-injected animals, cumulative histological damage scores were increased in adrenalectomized and decreased in dexamethasone-treated animals compared with control and hypophysectomized animals. The colons of all TNBS-injected animals (controls, adrenalectomized, dexamethasone treated, hypophysectomized) overexpressed and secreted lipocortin 1. In conclusion, the induction of lipocortin 1 overexpression and secretion during this colitis occurs independently of glucocorticoids or pituitary factors.
Leukocyte extravasation occurs in many pathophysiological conditions, including inflammation, neoplasia and asthma. In recent years many studies have elucidated the steps that promote the initial interaction between extravasating cells and endothelium of the post-capillary venule; the sequential role of several classes of adhesion molecules (cell-specific chemokines) and activators (multipotent cytokines) is well established. In this review, Mauro Perretti focuses on a less well investigated mechanism by which the host downregulates extravasation at the leukocyte-endothelium interface. The neutrophilic polymorphonuclear leukocyte is used as the prime example of a leukocyte that interacts with the endothelium, and particular emphasis is given to the possibility that novel anti-inflammatory therapies might be developed from a better understanding of the inhibitory mechanisms activated by endogenous mediators such as adenosine, lipocortin 1, NO, prostacyclin and cathepsin G.
The glucocorticoid-induced antiinflammatory protein lipocortin 1 is present in arthritic synovium but its ability to regulate joint inflammation has not previously been studied. We investigated the role of lipocortin 1 in the antiinflammatory activity of glucocorticoids in an acute arthritis model induced by intraarticular injection of carrageenan. Compared to control joints (0.09 +/- 0.08 x 10(6) synovial fluid cell count), carrageenan injected joints exhibited marked infiltration of PMN (10.2 +/- 0.7 x 10(6), p < 0.001). Both intraperitoneal (1.0 mg/kg) and intraarticular administration (5 micrograms) of dexamethasone (DEX) significantly suppressed arthritis severity (p < 0.001 and 0.005, respectively), and the effects of DEX were significantly prevented by intra-articular injection of antilipocortin 1 mAb (p < 0.05). Carrageenan arthritis was also significantly inhibited by intraarticular administration of the N-terminal lipocortin 1 peptide Ac2-26 at doses of 1 or 2 mg/kg (p < 0.01). Intraarticular injection antilipocortin 1 mAb in the absence of DEX also significantly exacerbated arthritis severity (p < 0.005). In vitro treatment of PMN with DEX was associated with significant inhibition of phagocytosis (p < 0.005) and reactive oxygen species (ROS) generation (p < 0.001). Antilipocortin 1 mAb significantly reduced the inhibitory effects of DEX (p < 0.01 and 0.005, respectively). These results demonstrate that lipocortin 1 mediates the effects of exogenous glucocorticoids on neutrophil migration in carrageenan-induced acute arthritis, exerts an endogenous antiinflammatory influence, and mediates glucocorticoid inhibition of neutrophil activation.