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Mobilizing lipocortin 1 in adherent human leukocytes downregulates their transmigration
Abstract
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.
... Ce processus comprend différentes étapes : la capture correspondant à la fixation faible des neutrophiles à l'endothélium, le roulement de ces derniers le long des cellules endothéliales puis l'adhésion ferme et le passage à travers la barrière endothéliale. Ces processus sont régulés dans un premier temps par des protéines de type sélectine au niveau des neutrophiles ( [69]. On estime qu'entre 50 et 70% de l'ANXA1 se retrouve à la surface cellulaire dans ces conditions [10]. ...
... De plus le traitement avec un anticorps dirigé contre l'ANXA1 s'est montré efficace pour augmenter la MTE des PMN sans modifier leur adhésion. Ceci s'est traduit in vivo par une augmentation du nombre de PMN recrutés dans la cavité péritonéale de souris présentant une péritonite suite à l'injection intra-péritonéale de zymosan [69]. L'utilisation de souris génétiquement inactivées pour l'Anxa1 a confirmé l'augmentation du recrutement des PMN au site inflammatoire dans ce même modèle de péritonite. ...
... L'action extracellulaire de l'ANXA1 a notamment été décrite dans l'inflammation. En effet, lors de l'adhésion des neutrophiles avec l'endothélium par exemple, la protéine est externalisée et sécrétée [69] afin de limiter leur transmigration [74]. Cette action de l'ANXA1 passe par son interaction avec les récepteurs FPR1 et FPR2 [80], [81]. ...
L’annexine A1 (ANXA1) appartient à la vaste superfamille des annexines qui englobe des protéines capables de se lier aux phospholipides membranaires de façon calcium dépendante. C’est une protéine multifonctionnelle initialement décrite pour ses propriétés anti-inflammatoires. Elle est présente dans le noyau, le cytosol et les membranes cellulaires et peut également être sécrétée et clivée dans le milieu environnant. L’ANXA1 présente un important intérêt en oncologie du fait de sa dérégulation dans de nombreux cancers. En fonction des types de cancers, l’ANXA1 est surexprimée ou sous-exprimée par rapport au tissu sain. Elle est surexprimée dans le cancer du sein triple négatif par rapport aux autres sous-types de tumeurs mammaires et dans les mélanomes par rapport aux mélanocytes. Dans ces deux pathologies, bien que l’ANXA1 soit associée aux processus de prolifération, migration et invasion, les mécanismes précis de son rôle au sein d’une tumeur restent mal connus, notamment ceux impliquant la fraction extracellulaire. Les études menées sur des souris n’exprimant pas l’Anxa1 ont également montré que l’ANXA1 stromale est impliquée dans le développement et la progression tumorale.Le premier objectif a donc été de tester la possibilité de bloquer la protéine extracellulaire via un anticorps monoclonal breveté, le VJ4B6, pour diminuer la migration (in vitro) et la dissémination (in vivo). Nos données montrent que le VJ4B6 ne permet pas de limiter la migration cellulaire des lignées de cancer du sein triple négatif (MDA-MB-231-luc) et de mélanome (SK-MEL-28 et A375-MA2) présentant de l’ANXA1 extracellulaire. Cet anticorps ne permet pas non plus de limiter le développement et la dissémination tumorale du mélanome B16Bl6 in vivo. Le second objectif a été d’étudier le rôle de l’ANXA1 tumorale et stromale dans le développement et la dissémination du mélanome. Nos résultats ont montré que l’ANXA1 tumorale est impliquée dans la prolifération des cellules A375-MA2 et SK-MEL-28 in vitro. L’utilisation de souris invalidées pour l’Anxa1 a également permis de mettre en évidence que l’ANXA1 stromale favorise le développement tumoral et la formation de métastases des cellules murines B16Bl6 in vivo. De façon intéressante, les études réalisées sur les tumeurs ont montré que l’absence d’ANXA1 stromale limite la prolifération des cellules tumorales ainsi que l’angiogenèse. Ceci peut donc expliquer la progression limitée des tumeurs chez les souris invalidées. De plus, les tumeurs développées chez ces dernières présentent une surexpression des marqueurs lymphocytaires (CD3, CD4, FoxP3, CD8a, NKp46) par rapport à celles développées chez les souris sauvages. Cet afflux lymphocytaire concerne à la fois les lymphocytes pro- et anti-tumoraux. Nous avons donc émis l’hypothèse que ce dernier soit une conséquence d’une perméabilité accrue des vaisseaux tumoraux au niveau des tumeurs des souris invalidées pour l’Anxa1.
... 18 Thus, it was mainly used as an inhibitor of pro-inflammatory factors prostaglandins (PGs) to study the inhibition of leukocyte aggregation in an inflammatory model [23][24][25][26] for a long time. Annexin A1 is particularly abundant in neutrophils, 27 but not abundant in lymphocytes. [28][29][30] Annexin A1 is mainly distributed in the cytoplasm and accounts for about 2-4% of the cytoplasmic protein. ...
... 31 A small amount of Annexin A1 is also found in the nucleus, 32 but the Annexin A1 protein is mobilized to the cell surface when the cell is activated. 27 Furthermore, Annexin A1 can also stably or reversibly bind to cytoskeletal proteins, regulating the interaction of cells with extracellular matrix. 31 In this review, we mainly focus on the pathological role of Annexin A1 in inflammation and tumorigenesis. ...
... 36 The N-terminal domain of Annexin A1 is composed of 44 amino acids, in which the first 26 amino acids form two α-helices of Ala 2 -Asn 16 and Glu 18 -Lys 26 with 60°reverse tilt on Glu. 17 The non-structural peptide Ser 27 -Asn 43 plays a crucial role in linking the N-terminal region to the core region. 36 Hall et al 40 found that Thr, 24 Ser, 27 Ser, 28 and Thr 41 have phosphorylation sites of protein kinase C (PKC) by MS/MS. Tyr 21 is the phosphorylation site of epidermal growth factor receptor kinase, Ser 5 is the phosphorylation site of TRMP7, and there are glycosylation, acetylation, acrylation, and proteolytic sites on other amino residues. ...
Annexin A1 is a Ca²⁺-dependent phospholipid binding protein involved in a variety of pathophysiological processes. Accumulated evidence has indicated that Annexin A1 has important functions in cell proliferation, apoptosis, differentiation, metastasis, and inflammatory response. Moreover, the abnormal expression of Annexin A1 is closely related to the occurrence and development of tumors. In this review article, we focus on the structure and function of Annexin A1 protein, especially the recent evidence of Annexin A1 in the pathophysiological role of inflammatory and cancer. This summary will be very important for further investigation of the pathophysiological role of Annexin A1 and for the development of novel therapeutics of inflammatory and cancer based on targeting Annexin A1 protein.
... 31 Circulating neutrophils showed a higher expression of intracellular ANXA1 compare with transmigrated neutrophils upon acute inflammation indicating a loss of ANXA1 during neutrophil cell adhesion and transmigration. 32,33 In vitro, human neutrophils interacted with the endothelium, and more specifically with Intercellular Adhesion Molecule 1 (ICAM-1) and Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1). This interaction provoked the externalization of ANXA1 toward the outer leaflet of the plasma membrane. ...
... This interaction provoked the externalization of ANXA1 toward the outer leaflet of the plasma membrane. 31,32 Moreover, other factors have been shown to provoke ANXA1 mobilization and externalization in neutrophils. 34 Examples include, pro-resolving lipid lipoxin A4 (LXA4) and pro-inflammatory ligand N-FormyL-Met-Leu-Phe (fMLF), which induced cytosolic (but not granular) ANXA1 to mobilize to the cell surface. ...
... Use of an ANXA1 blocking antibody increased the ability of neutrophils to transmigrate through an endothelial monolayer indicating that ANXA1 is able to impair neutrophil transmigration. 32,62 The ability of inhibiting neutrophil adhesion and emigration by ANXA1 and its derived peptide Ac2-26 was further supported by intravital microscopy analysis of the mesenteric postcapillary venules, which showed no change in rolling, but reduced adhesion and emigration upon inflammation. 63,64 Interestingly, ANXA1 and Ac2-26 detached adherent neutrophils within 2 minutes after administration during ongoing inflammation proving a tremendous effect of those proteins. ...
The inflammatory response protects the human body against infection and injury. However, uncontrolled and unresolved inflammation can lead to tissue damage and chronic inflammatory diseases. Therefore, active resolution of inflammation is essential to restore tissue homeostasis. This review focuses on the pro-resolving molecule annexin A1 (ANXA1) and its derived peptides. Mechanisms instructed by ANXA1 are multidisciplinary and affect leukocytes as well as endothelial cells and tissue resident cells like macrophages and mast cells. ANXA1 has an outstanding role in limiting leukocyte recruitment and different aspects of ANXA1 as modulator of the leukocyte adhesion cascade are discussed here. Additionally, this review details the therapeutic relevance of ANXA1 and its derived peptides in cardiovascular diseases since atherosclerosis stands out as a chronic inflammatory disease with impaired resolution and continuous leukocyte recruitment.
... AnxA1 can also be cleaved at the N-terminal domain by different proteases including metalloproteases and elastases among others Vong et al., 2007;Blume et al., 2012). AnxA1 exerts anti-inflammatory actions outside the cells (Perretti et al., 1996;Wein et al., 2004). Exogenous AnxA1 can also be cleaved by proteolytic enzymes leading to AnxA1-derived peptides such as Ac2-26 . ...
... In resting conditions, neutrophils, monocytes and macrophages constitutively contain a large amount of AnxA1 in their cytoplasm (Morand et al., 1995). Upon leukocyte activation, AnxA1 is mobilized to the cell surface and secreted (Perretti et al., 1996). In particular, in macrophages, the interaction of ABCA1 with AnxA1 promotes the translocation of AnxA1 from the cytoplasm to the cell membrane, thus mediating AnxA1 secretion (Wein et al., 2004). ...
Cardiovascular diseases (CVD) remain the leading cause of mortality worldwide. The main cause underlying CVD is associated with the pathological remodeling of the vascular wall, involving several cell types, including endothelial cells, vascular smooth muscle cells, and leukocytes. Vascular remodeling is often related with the development of atherosclerotic plaques leading to narrowing of the arteries and reduced blood flow. Atherosclerosis is known to be triggered by high blood cholesterol levels, which in the presence of a dysfunctional endothelium, results in the retention of lipoproteins in the artery wall, leading to an immune-inflammatory response. Continued hypercholesterolemia and inflammation aggravate the progression of atherosclerotic plaque over time, which is often complicated by thrombus development, leading to the possibility of CV events such as myocardial infarction or stroke. Annexins are a family of proteins with high structural homology that bind phospholipids in a calcium-dependent manner. These proteins are involved in several biological functions, from cell structural organization to growth regulation and vesicle trafficking. In vitro gain- or loss-of-function experiments have demonstrated the implication of annexins with a wide variety of cellular processes independent of calcium signaling such as immune-inflammatory response, cell proliferation, migration, differentiation, apoptosis, and membrane repair. In the last years, the use of mice deficient for different annexins has provided insight into additional functions of these proteins in vivo , and their involvement in different pathologies. This review will focus in the role of annexins in CVD, highlighting the mechanisms involved and the potential therapeutic effects of these proteins.
... was lost during neutrophil adhesion and migration. 43 In vitro, neutrophils interact with endothelial cells to stimulate Annexin A1 externalization through intercellular adhesion molecules and platelet adhesion molecules. 43 Externalized Annexin A1 binds to formyl peptide receptors to exert anti-inflammatory effects, 44 In our study, it was revealed that miRNA-196a2 rs11614913 T>C may not be associated with Kawasaki disease susceptibility in a southern Chinese population and that there was no significant association in the stratified analysis. ...
... 43 In vitro, neutrophils interact with endothelial cells to stimulate Annexin A1 externalization through intercellular adhesion molecules and platelet adhesion molecules. 43 Externalized Annexin A1 binds to formyl peptide receptors to exert anti-inflammatory effects, 44 In our study, it was revealed that miRNA-196a2 rs11614913 T>C may not be associated with Kawasaki disease susceptibility in a southern Chinese population and that there was no significant association in the stratified analysis. The advantage of this study is that it is the first report involving miRNA-196a2 rs11614913 T>C and Kawasaki disease susceptibility. ...
Background
miRNAs play important roles in a variety of diseases. Thus, the association between miRNA‐196a2 rs11614913 T>C polymorphism and Kawasaki disease susceptibility is still unknown.
Methods
We included 532 children with Kawasaki disease and 623 healthy children from South China, and their DNA was extracted for genotyping by TaqMan methodology. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the strength of association.
Results
No significant associations were observed between the miRNA‐196a2 rs11614913 T>C polymorphisms and Kawasaki disease risk (TC vs TT: adjusted OR = 1.04, 95% CI = 0.79‐1.37; CC vs TT: adjusted OR = 0.87, 95% CI = 0.63‐1.21; dominant model: adjusted OR = 0.99, 95% CI = 0.76‐1.27; and recessive model: adjusted OR = 0.85, 95% CI = 0.64‐1.13). There was also no significant correlation found in stratified analyses.
Conclusion
This study suggests that miRNA‐196a2 rs11614913 T>C may not be associated with Kawasaki disease susceptibility in a southern Chinese population. Larger, multicenter studies are needed to confirm our conclusions.
... The role of neutrophils in regulating acute inflammatory pain and resolution of inflammation is well established, but neutrophil-mediated modulation of nociceptive processing beyond the inflammatory resolution phase has not been explored (37). Neutrophils have been shown to play a role in the resolution of inflammatory pain (38), and neutrophil-derived products such as annexin A1 and resolvins are key proresolving mediators (39,40). In contrast, several findings in animal studies also point toward a pronociceptive role of neutrophils ever since the first report of proalgesic effects of articular neutrophils in joints of dogs, promoted by administration of lipopolysaccharides (41). ...
Fibromyalgia is a debilitating widespread chronic pain syndrome that occurs in 2 to 4% of the population. The prevailing view that fibromyalgia results from central nervous system dysfunction has recently been challenged with data showing changes in peripheral nervous system activity. Using a mouse model of chronic widespread pain through hyperalgesic priming of muscle, we show that neutrophils invade sensory ganglia and confer mechanical hypersensitivity on recipient mice, while adoptive transfer of immunoglobulin, serum, lymphocytes, or monocytes has no effect on pain behavior. Neutrophil depletion abolishes the establishment of chronic widespread pain in mice. Neutrophils from patients with fibromyalgia also confer pain on mice. A link between neutrophil-derived mediators and peripheral nerve sensitization is already established. Our observations suggest approaches for targeting fibromyalgia pain via mechanisms that cause altered neutrophil activity and interactions with sensory neurons.
... ANXA1 was first identified as an endogenous mediator of the anti-inflammatory effects of the glucocorticoids. Initially, ANXA1 was studied in neutrophils, eosinophils and monocytes where it is highly expressed (Perretti et al., 1996;Hannon et al., 2003;Perretti and Solito, 2004), and thereafter, it was widely detected in different tissues and involved in multiple cell processes, including cell survival, proliferation, apoptosis, differentiation and migration (Lim and Pervaiz, 2007). ...
Annexins are an extensive superfamily of structurally related calcium- and phospholipid-binding proteins, largely conserved and widely distributed among species. Twelve human annexins have been identified, referred to as Annexin A1-13 (A12 remains as of yet unassigned), whose genes are spread throughout the genome on eight different chromosomes. According to their distinct tissue distribution and subcellular localization, annexins have been functionally implicated in a variety of biological processes relevant to both physiological and pathological conditions. Dysregulation of annexin expression patterns and functions has been revealed as a common feature in multiple cancers, thereby emerging as potential biomarkers and molecular targets for clinical application. Nevertheless, translation of this knowledge to the clinic requires in-depth functional and mechanistic characterization of dysregulated annexins for each individual cancer type, since each protein exhibits varying expression levels and phenotypic specificity depending on the tumor types. This review specifically and thoroughly examines the current knowledge on annexin dysfunctions in carcinogenesis. Hence, available data on expression levels, mechanism of action and pathophysiological effects of Annexin A1-13 among different cancers will be dissected, also further discussing future perspectives for potential applications as biomarkers for early diagnosis, prognosis and molecular-targeted therapies. Special attention is devoted to head and neck cancers (HNC), a complex and heterogeneous group of aggressive malignancies, often lately diagnosed, with high mortality, and scarce therapeutic options.
... Annexin A1 (ANXA1) is an endogenous glucocorticoid-induced protein, which is known to modulate a number of systemic anti-inflammatory processes including reducing eicosanoid synthesis, leukocyte transmigration and in the resolution of inflammation via macrophage mediated phagocytosis of neutrophils [8][9][10]. The vast range of antiinflammatory/pro-resolving effects of ANXA1 and its peptides have shown to have significant effects in resolving inflammation in several disease models including, inflammatory bowel disease and arthritis [11,12]. ...
Obesity is a multifactorial disease and is associated with an increased risk of developing metabolic syndrome and co-morbidities. Dysregulated expansion of the adipose tissue during obesity induces local tissue hypoxia, altered secretory profile of adipokines, cytokines and chemokines, altered profile of local tissue inflammatory cells leading to the development of low-grade chronic inflammation. Low grade chronic inflammation is considered to be the underlying mechanism that increases the risk of developing obesity associated comorbidities. The glucocorticoid induced protein annexin A1 and its N-terminal peptides are anti-inflammatory mediators involved in resolving inflammation. The aim of the current study was to investigate the role of annexin A1 in obesity and associated inflammation. To achieve this aim, the current study analysed data from two feasibility studies in clinical populations: (1) bariatric surgery patients (Pre- and 3 months post-surgery) and (2) Lipodystrophy patients. Plasma annexin A1 levels were increased at 3-months post-surgery compared to pre-surgery (1.2 ± 0.1 ng/mL, n = 19 vs. 1.6 ± 0.1 ng/mL, n = 9, p = 0.009) and positively correlated with adiponectin (p = 0.009, r = 0.468, n = 25). Plasma annexin A1 levels were decreased in patients with lipodystrophy compared to BMI matched controls (0.2 ± 0.1 ng/mL, n = 9 vs. 0.97 ± 0.1 ng/mL, n = 30, p = 0.008), whereas CRP levels were significantly elevated (3.3 ± 1.0 µg/mL, n = 9 vs. 1.4 ± 0.3 µg/mL, n = 31, p = 0.0074). The roles of annexin A1 were explored using an in vitro cell based model (SGBS cells) mimicking the inflammatory status that is observed in obesity. Acute treatment with the annexin A1 N-terminal peptide, AC2-26 differentially regulated gene expression (including PPARA (2.8 ± 0.7-fold, p = 0.0303, n = 3), ADIPOQ (2.0 ± 0.3-fold, p = 0.0073, n = 3), LEP (0.6 ± 0.2-fold, p = 0.0400, n = 3), NAMPT (0.4 ± 0.1-fold, p = 0.0039, n = 3) and RETN (0.1 ± 0.03-fold, p < 0.0001, n = 3) in mature obesogenic adipocytes indicating that annexin A1 may play a protective role in obesity and inflammation. However, this effect may be overshadowed by the continued increase in systemic inflammation associated with rapid tissue expansion in obesity.
... Glucocorticoids are likely anti-inflammatory by up-regulating annexin expression (Hannon et al., 2003). Formyl peptide receptors (f-Met-Leu-Phe receptors and f-Met-Leu-Phe receptor like 1) bind the N-terminus of exogenous annexin-1 to cause cell detachment, inhibit leukocyte transmigration, and block the inflammatory response (Perretti et al., 1996;Lim et al., 1998;Rescher et al., 2002). Annexin-1 affects the activities of innate and adaptive immune cells including macrophages and T lymphocytes. ...
... ANXA1 is mainly produced by a variety of immune cells, including monocytes, macrophages, and neutrophils. 22,23 DN is characterized by the infiltration of macrophages and the increased production of cytokines in kidneys. The decrease of circulating ANXA1 in DN may be caused by the increase of ANXA1 in the target inflammatory sites (i.e., kidneys). ...
Since failed resolution of inflammation is a major contributor to the progression of diabetic nephropathy, identifying endogenously generated molecules that promote the physiological resolution of inflammation may be a promising therapeutic approach for this disease. Annexin A1 (ANXA1), as an endogenous mediator, plays an important role in resolving inflammation. Whether ANXA1 could affect established diabetic nephropathy through modulating inflammatory states remains largely unknown. In the current study, we found that in patients with diabetic nephropathy, the levels of ANXA1 were upregulated in kidneys, and correlated with kidney function as well as kidney outcomes. Therefore, the role of endogenous ANXA1 in mouse models of diabetic nephropathy was further evaluated. ANXA1 deficiency exacerbated kidney injuries, exhibiting more severe albuminuria, mesangial matrix expansion, tubulointerstitial lesions, kidney inflammation and fibrosis in high fat diet/streptozotocin-induced-diabetic mice. Consistently, ANXA1 overexpression ameliorated kidney injuries in mice with diabetic nephropathy. Additionally, we found Ac2-26 (an ANXA1 mimetic peptide) had therapeutic potential for alleviating kidney injuries in db/db mice and diabetic Anxa1 knockout mice. Mechanistic studies demonstrated that intracellular ANXA1 bound to the transcription factor NF-κB p65 subunit, inhibiting its activation thereby modulating the inflammatory state. Thus, our data indicate that ANXA1 may be a promising therapeutic approach to treating and reversing diabetic nephropathy.
... Neutrophils are key players in the innate immune system [21] and are a potent source of the anti-inflammatory AnxA1 protein [22][23][24]. Our study evaluated the role of AnxA1 in NLRP3 inflammasome-derived IL-1β production by LPS-primed neutrophils following nigericin or ATP stimulation. ...
This study evaluated the role of endogenous and exogenous annexin A1 (AnxA1) in the activation of the NLRP3 inflammasome in isolated peritoneal neutrophils. C57BL/6 wild-type (WT) and AnxA1 knockout mice (AnxA1-/-) received 0.3% carrageenan intraperitoneally and, after 3 h, the peritoneal exudate was collected. WT and AnxA1-/- neutrophils were then stimulated with lipopolysaccharide, followed by the NLRP3 agonists nigericin or ATP. To determine the exogenous effect of AnxA1, the neutrophils were pretreated with the AnxA1-derived peptide Ac2-26 followed by the NLRP3 agonists. Ac2-26 administration reduced NLRP3-derived IL-1β production by WT neutrophils after nigericin and ATP stimulation. However, IL-1β release was impaired in AnxA1-/- neutrophils stimulated by both agonists, and there was no further impairment in IL-1β release with Ac2-26 treatment before stimulation. Despite this, ATP- and nigericin-stimulated AnxA1-/- neutrophils had increased levels of cleaved caspase-1. The lipidomics of supernatants from nigericin-stimulated WT and AnxA1-/- neutrophils showed potential lipid biomarkers of cell stress and activation, including specific sphingolipids and glycerophospholipids. AnxA1 peptidomimetic treatment also increased the concentration of phosphatidylserines and oxidized phosphocholines, which are lipid biomarkers related to the inflammatory resolution pathway. Together, our results indicate that exogenous AnxA1 negatively regulates NLRP3-derived IL-1β production by neutrophils, while endogenous AnxA1 is required for the activation of the NLRP3 machinery.
... The peritoneal-covering skin was removed, 5 ml PBS injected into the Nuclei were meta-chromatically stained red/purple and cytoplasm pink/yellow (See Supplementary Materials Fig. 9). before separation through a double-density gradient as described 52 . ...
Neutrophils must navigate accurately towards pathogens in order to destroy invaders and thus defend our bodies against infection. Here we show that hydrogen peroxide, a potent neutrophil chemoattractant, guides chemotaxis by activating calcium permeable TRPM2 ion channels and thus generating an intracellular leading-edge calcium 'pulse'. The thermal sensitivity of TRPM2 activation means that chemotaxis towards hydrogen peroxide is strongly promoted by small temperature elevations, suggesting that an important function of fever may be to enhance neutrophil chemotaxis by facilitating calcium influx through TRPM2. Chemotaxis towards conventional chemoattractants such as LPS, CXCL2 and C5a does not depend on TRPM2 but is driven in a similar way by leading-edge calcium pulses. Other proposed initiators of neutrophil movement, such as PI3K, Rac and lyn, influence chemotaxis by modulating the amplitude of calcium pulses. We propose that intracellular leading-edge calcium pulses are universal drivers of the motile machinery involved in neutrophil chemotaxis.
... This protein was previously recognised as renocortin, lipomodulin, lipocortin-1 and phospholipase A2 (PLA2) inhibitor [4]. It is widely distributed in the body, which includes biological fluids, lymphocytes, monocytes and granulocytes, whereby T cells and neutrophils are the predominant in the lymphocyte and granulocyte subgroups, respectively [5][6][7]. It possesses anti-inflammatory effects and also mimics the effects of glucocorticoids on leukocytes such as the inhibition of superoxide generation, chemotaxis and eicosanoids production [8]. ...
Purpose: To review systematically the involvement of Annexin A1 (ANXA1) in various leukemia cells in order to advance the understanding of ANXA1 role in leukemia. Methods: The systematic review was carried out via a comprehensive search of electronic databases for all relevant articles published up to September 2017. Specific key words were used to retrieve the articles. All articles were imported into EndNote software while duplicates were removed from the list. The retrieved articles were selected using inclusion and exclusion criteria. Results: FK228, a novel HDACi and FR235222, increased expression of ANXA1 in Kasumi-1, SKNO-1 and U937 cells, respectively, and induced apoptosis. The study also neutralized ANXA1 in the same cells, which caused a complete blockage of the FK228-induced apoptosis. Resveratrol was reported to markedly increase ANXA1 levels which led to caspase 3-mediated apoptosis on HL-60 cells. Dexamethasone, 17β-estradiol (E2β), all-trans retinoic acid and okadaic acid enhanced ANXA1 mRNA expression in U937, human CCRF-CEM, ATRA-NB4 and HL-60 cell lines. Rp-8-Br-cAMPs prevented dexamethasone-, E2β-and dBcAMP-induced ANXA1 synthesis via the activation of cAMP-respond-element binding protein (CREB). ANXA1 levels were reduced dramatically in K562/ADR cells as compared to K562 cells. When ANXA1 was upregulated by transfection in these cells, the cells exhibited a decrease in resistance to ADR and vincristine. Conclusion: ANXA1 expression is induced by different drugs which leads to apoptosis in different types of cell. ANXA1 plays a role in the drug resistance of leukemic cells. This is an Open Access article that uses a funding model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) and the Budapest
... Glucocorticoids are known to delay this process, and it has been shown that annexin Al downregulates leukocyte transmigration when externalised by an as yet unknown mechanism from endothelium-adherent leukocytes [126]. This inhibitory effect has also been described for exogenously applied annexin A l, as well as for its N-terminal domain, in several in vitro systems and animal models [127,128]. ...
Annexin A11 is a ubiquitously expressed member of the annexin family of Ca2+-dependent phospholipid binding proteins, which lies at the root of the vertebrate annexin evolutionary tree. Annexin A11 is known to be localised to the nuclei of cells in culture, and to interact Ca2+-dependently in vitro with the S100 protein S100A6 and the penta EF-hand protein ALG-2 through its long glycine, proline and tyrosine-rich N-terminal domain, but very little is known about the physiological functions performed by this annexin in vivo. An investigation into the cellular roles of annexin A11 both in interphase and during cell division is presented in this thesis. Using various molecular and cell biology approaches, annexin A11 was identified in the nucleus and in cytoplasmic vesicles that appear to be transported along the microtubular network, linking annexin A11 function with intracellular membrane trafficking processes. Annexin A11 has also been identified in membrane domains involved in signal transduction at the basolateral membrane of A431 cells. A role for this annexin in membrane remodelling steps during cell growth is proposed in view of the accumulation of annexin A11-containing vesicles in the cytoplasm of non-contact inhibited cells and at the leading edge of monolayer outgrowths, together with the concentration of annexin A11 at the cell-cell contact sites in the plasma membrane of confluent monolayers. Annexin A11 relocates to the nuclear envelope and is tyrosine-phosphorylated in the presence of calcium in interphase cells, and invades the nuclear envelope at the invaginations created during nuclear envelope breakdown at the onset of mitosis, and is also detected at the nuclear envelope during nuclear envelope reassembly after cell division, making the nuclear envelope a specific target for annexin A11. The study of the function of annexin A11 during cell division led to the identification of an essential role for annexin A11 in the terminal phase of cytokinesis. Annexin 11 was observed to translocate from the nucleus to the spindle poles in metaphase, and then to the spindle midzone in anaphase, and to be recruited to the midbody in late telophase where it co-localises and associates with the mitotic kinesin- like protein CHO1. Depletion of annexin A11 by RNA interference has shown that the absence of this annexin leads to failure to establish a functional midbody, incomplete daughter cell separation, and ultimately cell death by apoptosis.
... Various research reports have shown that dexamethasone and Anx-1 can inhibit the expression of COX-2 and iNOS along with the release of PGE 2 . Similar to GCs, Anx-1 inhibits eicosanoid synthesis, obstructs leukocyte migration [75] and induces inflammatory cells death [76]. It has been proposed that the above described effects of Anx-1 may be attributed to release of IL-10 and a concurrent reduction of IL-12 [77]. ...
Inflammation is a physiological intrinsic host response to injury meant for removal of noxious stimuli and maintenance of homeostasis. It is a defensive body mechanism that involves immune cells, blood vessels and molecular mediators of inflammation. Glucocorticoids (GCs) are steroidal hormones responsible for regulation of homeostatic and metabolic functions of body. Synthetic GCs are the most useful anti-inflammatory drugs used for the treatment of chronic inflammatory diseases such as asthma, chronic obstructive pulmonary disease (COPD), allergies, multiple sclerosis, tendinitis, lupus, atopic dermatitis, ulcerative colitis, rheumatoid arthritis and osteoarthritis whereas, the long term use of GCs are associated with many side effects. The anti-inflammatory and immunosuppressive (desired) effects of GCs are usually mediated by transrepression mechanism whereas; the metabolic and toxic (undesired) effects are usually manifested by transactivation mechanism. Though GCs are most potent anti-inflammatory and immunosuppressive drugs, the common problem associated with their use is GC resistance. Several research studies are rising to comprehend these mechanisms, which would be helpful in improving the GC resistance in asthma and COPD patients. This review aims to focus on identification of new drug targets in inflammation which will be helpful in the resolution of inflammation. The ample understanding of GC mechanisms of action helps in the development of novel anti-inflammatory drugs for the treatment of inflammatory and autoimmune disease with reduced side effects and minimal toxicity.
... An imbalance between pro-and anti-inflammatory actions is believed to be important in the development and progression of MS (44). ANXA1, a glucocorticoid-inducible protein abundantly expressed in neutrophils and innate immune cells, has long been known as a potent anti-inflammatory mediator (11,45). Several studies suggest that its mechanism of action is mainly related to a direct action on monocytes, involving both transcriptional changes as well as rapid posttranslational effects (28,29,46). ...
Chronic neuroinflammation is a key pathological hallmark of multiple sclerosis (MS) that suggests that resolution of inflammation by specialized proresolving molecules is dysregulated in the disease. Annexin A1 (ANXA1) is a protein induced by glucocorticoids that facilitates resolution of inflammation through several mechanisms that include an inhibition of leukocyte recruitment and activation. In this study, we investigated the ability of ANXA1 to influence T cell effector function in relapsing/remitting MS (RRMS), an autoimmune disease sustained by proinflammatory Th1/Th17 cells. Circulating expression levels of ANXA1 in naive-to-treatment RRMS subjects inversely correlated with disease score and progression. At the cellular level, there was an impaired ANXA1 production by CD4+CD25- conventional T and CD4+RORγt+ T (Th17) cells from RRMS subjects that associated with an increased migratory capacity in an in vitro model of blood brain barrier. Mechanistically, ANXA1 impaired monocyte maturation secondarily to STAT3 hyperactivation and potently reduced T cell activation, proliferation, and glycolysis. Together, these findings identify impaired disease resolution pathways in RRMS caused by dysregulated ANXA1 expression that could represent new potential therapeutic targets in RRMS.
... Синтезируемый некоторыми генерация-ми Нф противовоспалительный белок аннек-син А1 опосредует эндокринные реакции, реализующие иммуносупрессивные, противо-воспалительные и/или противоаллергические эффекты глюкокортикоидов: угнетает актив-ность фосфолипазы A2 и циклооксигеназы 1-го и 2-го типов, что в конечном счете приводит к снижению синтеза эйкозаноидов (проста-гландинов и лейкотриенов) [15]. Связываясь со специфическим рецептором ALX/FPR2 на мембране лейкоцитов, аннексин A1 угнетает активность лейкоцитов: подавляет способность к адгезии, миграционную способность лейко-цитов, хемотаксис, фагоцитоз, высвобождение провоспалительных медиаторов, лизосомаль-ных ферментов из Нф, макрофагов и тучных клеток [16]. Аннексин А1 регулирует способ-ность макрофагов к эффероцитозу, влияет на продуцирование TNF и IL-6 и снижает спо-собность к дегрануляции тучных клеток [17]. ...
Granulocyte colony-stimulating factor (G-CSF) promotes proliferation, survival, and differentiation of myeloid-lineage cells, as well as normal hematopoietic cells. The immunomodulating effects of G-CSF, which consist in stimulating the Th2 biased type immune response, prevent the development of graft-versus-host disease (GvHD) and, as a special case of GvHD, are responsible for the embryo implantation into the endometrium after the embryo transfer. G-CSF stimulates subpopulations of neutrophils, which display anti-inflammatory properties and are involved in tissue regeneration. The increased secretion of annexin A1 and IL-10 ensures the anti-inflammatory and immunomodulating effects of neutrophils. This review article presents data from four meta-analyzes aimed to explore the efficiency of G-CSF on infertile women undergoing in vitro fertilization. These data demonstrate an increase in the embryo implantation rate and clinical pregnancy rate, which is provided by the change in the endometrial receptivity and/or the invasive potential of the developing embryo.
... Another family of mediators requiring negative regulation during resolution are chemokines ( Table 2). Chemokines are produced in abundance during onset of inflammation and initiate leukocyte migration to the affected tissue [20]. To attain a resolving environment, chemokine cleavage and sequestration are necessary to stop further neutrophil influx. ...
Deciphering the origins of chronic inflammatory and autoimmune diseases remains elusive with reliance on therapies aimed at halting inflammation in its tracks. In recent years, an appreciation of targeting pathways by which inflammation is resolved has begun to rouse interest. Resolution of inflammation is driven by a complex set of mediators that regulate cellular events required to clear inflammatory cells from sites of infection or injury to restore tissue function. However, recent studies suggest that resolution is not the end of innate mediated immune responses to infection/injury. There is further immunological activity occurring after the resolution cascade is complete that alters the immune physiology of tissues, redefining what was once termed restorative homeostasis as adapted homeostasis.
... Briefly, purified PMNs were diluted to 4 × 10 6 cells/ml in RPMI with 0.5% FBS and placed in the upper well in a total volume of 100 μl, and IL-8 (30 ng/ml) or medium (RPMI 1640 with 0.5% FBS) was added to the lower chamber [27]. Plates were then incubated for 2 h in a humidified incubator at 37°C with 5% CO 2 . ...
Inflammatory bowel disease is characterised by intricate immune cell interactions with tissue cells and such cross-talks can become deregulated. The formyl peptide receptor 1 (Fpr1) is expressed by both immune and stromal cells including epithelial cells. We evaluated the development of the physiopathology of the DNBS induced colitis in Fpr1 KO mice on the C57BL/6 genetic background compared to C57BL/6 genetic background animals. We have assessed both macroscopic and histological markers of the diseased, together with the immunohistochemical and molecular changes. DNBS-treated Fpr1 KO mice showed a i) reduction in weight loss, ii) lower extent of colon injury and iii) an increase in MPO activity. Molecular analyses indicated that in absence of Fpr1 there was reduced NF-κB translocation into the nucleus, cytokines levels, FOXP3 and GATA3, CD4, CD8 and CD45 expression as well as a dysregulation of TGF-β signalling. In addition, the colon of DNBS-injected Fpr1 KO mice displayed a lower degree of expression of Bax and higher expression of Bcl-2 compared correspondent WT mice. Finally, intravital microscopy investigation of the microcirculation post-DNBS instillation revealed a lower degree of neutrophil-endothelial cell rolling and adhesion - mediated by P-selectin and ICAM-1 – in Fpr1 KO mice. All the main outcome in the study have a P-value, statistical significance of evidence, less than 0.05. We provide evidence for an important pathogenic role of mouse Fpr1 in experimental colitis, an outcome effected through modulation of immune cell recruitment together with a modulation of local cellular activation and survival.
... Although cytosolic AnxA1 protein bears no canonical signal sequence and therefore is unlikely to be transported via the classical secretory pathway, it is, under inflammatory conditions, however, externalized by yet unknown mechanisms. AnxA1 is released when leukocytes interact with the endothelium, leading to a subsequent detachment [122,123]. On the other hand, AnxA1 externalization might appear independently of leukocyteendothelial interaction [124]. Interestingly, N-terminal peptides, which might be released by proteinase 3 cleavage of the externalized protein [123,124] retain the anti-inflammatory activity. ...
The innate immune system is the first line of defense against pathogenic threats. For the early pathogen recognition and activation of cell protective mechanisms, germline-encoded pattern recognition receptors (PRRs) detect characteristic and evolutionary conserved pathogen-associated molecular patterns (PAMPs). PRRs are therefore key elements in the innate immune response; in addition, they sense danger-associated molecular patterns (DAMPs) that are released by host cell molecules under pathophysiological conditions. Formyl peptide receptors (FPRs) are G-protein-coupled PRRs that respond to a surprisingly broad range of ligands, derived from both pathogens and host cells. Here, we exemplary discuss ligands in order to illustrate the wide pathophysiological relevance of the FPR signaling axis in case of e.g., chronic inflammations and to underscore its potential therapeutic value in the light of “biased agonism”, a modern concept of GPCR (G-protein coupled receptors) activation. These novel insights into the GPCR receptor biochemistry will hopefully (re)stimulate FPR-related research and lead to novel strategies for the urgently needed development of drugs with pharmacologically advantageous characteristics.
... The changes that occur upon a conformational change may reflect a need for interacting with potential receptors and the functions of the proteins. Blood immune cells such as monocytes and granulocytes express high levels of ANXA1 and are the largest source of cellular ANXA1 with lymphocytes possessing moderate expression levels (Perretti et al., 1996b;Oliani et al., 2002). Interestingly, B cells and platelets do not express ANXA1 (Perretti and Dalli, 2009). ...
Influenza A viruses (IAVs) are important human respiratory pathogens which cause seasonal or periodic endemic infections. IAV can result in severe or fatal clinical complications including pneumonia and respiratory distress syndrome. Treatment of IAV infections is complicated because the virus can evade host immunity through antigenic drifts and antigenic shifts, to establish infections making new treatment options desirable. Annexins (ANXs) are a family of calcium and phospholipid binding proteins with immunomodulatory roles in viral infections, lung injury, and inflammation. A current understanding of the role of ANXs in modulating IAV infection and host responses will enable the future development of more effective antiviral therapies. This review presents a comprehensive understanding of the advances made in the field of ANXs, in particular, ANXA1 and IAV research and highlights the importance of ANXs as a suitable target for IAV therapy.
... Even though annexins A1 and A4 are both present in ductal prostate epithelium cells, only annexin A1 is present extracellularly [15]. Several studies have shown that annexin A1 strongly inhibits the transendothelial migration of leukocytes, hence limiting the extent of inflammation [16]. ...
... To date, the leading candidate may be the formyl peptide receptor. Indeed, UPARANT has been shown to bind to the FPR type 1 and 2, expressed in a large variety of cells and organs, including monocytes, macrophages, neutrophils, lymphocytes and microglia [36][37][38]. In addition, the role of these receptors in the inflammatory process has been confirmed by recent study showing an abnormal paw swelling in the FPR2 -/mice in an experimental inflammatory model [17]. ...
Background
Inflammation plays a key role in the pathogenesis of several chronic diseases. The urokinase plasminogen activator receptor (uPAR) exerts a plethora of functions in both physiological and pathological processes, including inflammation. Objective and designIn this study, we evaluated the anti-inflammatory effect of a novel peptide ligand of uPAR, UPARANT, in different animal models of inflammation. Subjects and treatmentRats and mice were divided in different groups (n = 5) for single or repeated administration of vehicle (9% DMSO in 0.9% NaCl), UPARANT (6, 12 and 24 mg/kg) or dexamethasone (2 mg/kg). Animals were subjected to carrageenan-induced paw oedema or zymosan-induced peritonitis. MethodsUPARANT effects were tested on: (1) the carrageenan-induced paw oedema volume, (2) the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and the nitrite/nitrate (NOx) levels in the paw exudates, (3) cells recruitment into the peritoneal cavity after zymosan injection and (4) NOx levels in the peritoneal lavage. ResultsUPARANT (12 and 24 mg/kg) reduced inflammation in both experimental paradigms. Analysis of pro-inflammatory enzymes revealed that administration of UPARANT reduced iNOS, COX2 and NO over-production. Conclusions
Our study provides a solid evidence that UPARANT reduces the severity of inflammation in diverse animal models, thus representing a novel anti-inflammatory drug with potential advantages with respect to the typical steroidal agents.
... Apoptotic cells promote their own clearance by expressing find-me and eat-me signals that attract scavengers and allow the identification of the dying cell, respectively. 34 Among these signals, AnxA1 is a well-known proresolving protein that is able to reduce neutrophil recruitment, 61 induce nonphlogistic recruitment of monocytes, 62 promote neutrophil apoptosis 23,35 and its subsequent phagocytic clearance, 37,63,64 and contribute to tissue repair. 65,66 Notably, Plg KO mice exhibit altered expression of AnxA1 and other phagocytosisrelated proteins in the spleen, which may account for decreased phagocytosis in these mice. ...
Key Points
Plg and Pla induce macrophage reprogramming and promote resolution of acute inflammation. Plg and Pla enhance the efferocytic capacity of macrophages and override the prosurvival effect of LPS on neutrophils via annexin A1.
... In particular, increased Anx-A1 was observed in the intestinal epithelium and infiltrating leukocytes in the mucosa of UC patients compared with normal intestinal mucosa (Leoni et al., 2013). Anx-A1 has also been reported to inhibit neutrophil influx and promote neutrophil apoptosis at the site of resolving inflammation (Perretti et al., 1996). In our experiments, a co-localization between Ly-6G and Anx-A1 positive cells was obtained: FBA reduced Anx-A1 levels as well as Ly-6G + cells. ...
... This molecule increases neutrophil engulfment by macrophages [168,169]. Importantly, AnxA1 also contributes to the resolution of inflammation by negatively regulating neutrophil transmigration, and exogenous administration of AnxA1 causes neutrophil apoptosis in inflamed lungs of humans and mice [170][171][172][173]. With all these properties, it is not surprising that AnxA1-deficient animals exhibit increased susceptibility to DSS-induced colitis, with larger mucosal injury [174]. ...
Inflammatory bowel diseases (IBD), including Crohn’s disease and ulcerative colitis, are characterised by aberrant immunological responses leading to chronic inflammation without tissue regeneration. These two diseases are considered distinct entities, and there is some evidence that neutrophil behaviour, above all other aspects of immunity, clearly separate them. Neutrophils are the first immune cells recruited to the site of inflammation, and their action is crucial to limit invasion by microorganisms. Furthermore, they play an essential role in proper resolution of inflammation. When these processes are not tightly regulated, they can trigger positive feedback amplification loops that promote neutrophil activation, leading to significant tissue damage and evolution toward chronic disease. Defective chemotaxis, as observed in Crohn’s disease, can also contribute to the disease through impaired microbe elimination. In addition, through NET production, neutrophils may be involved in thrombo-embolic events frequently observed in IBD patients. While the role of neutrophils has been studied in different animal models of IBD for many years, their contribution to the pathogenesis of IBD remains poorly understood, and no molecules targeting neutrophils are used and validated for the treatment of these pathologies. Therefore, it is crucial to improve our understanding of their mode of action in these particular conditions in order to provide new therapeutic avenues for IBD.
... In particular, increased Anx-A1 was observed in the intestinal epithelium and infiltrating leukocytes in the mucosa of UC patients compared with normal intestinal mucosa (Leoni et al., 2013). Anx-A1 has also been reported to inhibit neutrophil influx and promote neutrophil apoptosis at the site of resolving inflammation (Perretti et al., 1996). In our experiments, a co-localization between Ly-6G and Anx-A1 positive cells was obtained: FBA reduced Anx-A1 levels as well as Ly-6G + cells. ...
Background and purpose:
Butyrate has shown benefits in inflammatory bowel diseases (IBD). However, its oral administration is infrequent due to rancid smell and unpleasant taste. The efficacy of a more palatable butyrate-releasing derivative, N-(1-carbamoyl-2-phenylethyl) butyramide (FBA), was evaluated in a mouse model of colitis induced by dextran sodium sulphate (DSS).
Experimental approach:
Male 10-week-old BALB/c mice received DSS (2.5%) in drinking water (for 5d) followed by DSS-free water for 7d (DSS group). Oral FBA administration (42.5 mg∙kg(-1) ) started 7d before DSS as preventive (P-FBA), or 2d after DSS as therapeutic (T-FBA), and both treatments lasted at 19d. One DSS-untreated group received only tap water (CON) for totally 4 groups.
Key results:
FBA treatments reduced colitis symptoms and colon damage. P-FBA and T-FBA significantly decreased polymorphonuclear cell infiltration score compared to the DSS group. FBA revert the imbalance between pro- and anti-inflammatory cytokines (reducing inducible NOS protein expression, chemokine (C-C motif) ligand 2 and IL-6 transcripts in colon and increasing TGF-β and IL-10). Morever, P-FBA and T-FBA limit neutrophil recruitment (by expression and localization of the neutrophil granule protease Ly-6G), restore deficiency of butyrate transporter and improve intestinal epithelial integrity, preventing tight-junction impairment (zonulin-1 and occludin). FBA, such as its parental compound sodium butyrate, inhibits histone deacetylase-9 and restores H3 histone acetylation, exerting an anti-inflammatory activity through NF-κB inhibition and PPAR-γ up-regulation.
Conclusions and implications:
FBA reduces inflammatory intestinal damage in mice indicating its potential as post-biotic derivative, to overcome the limits of sodium butyrate's oral administration. This article is protected by copyright. All rights reserved.
... AnxA1 is thought to mediate many anti-inflammatory actions of glucocorticoids [91,92]. In neutrophils, AnxA1 is rapidly mobilized from the cytoplasm to the cell surface following adherence to the endothelium [93]. AnxA1 binds to and activates FPR2/ALX and induces detachment of adhered neutrophils [94]. ...
Resolution of acute inflammation is an active process that requires inhibition of further leukocyte recruitment and removal of leukocytes from inflamed sites. Emigrated neutrophils undergo apoptosis before being removed by scavenger macrophages. Recent studies using a variety of gene knockout, transgenic and pharmacological strategies in diverse models of inflammation established neutrophil apoptosis as a critical control point in resolving inflammation. Analysis of death mechanisms revealed distinct features in executing the death program in neutrophils, which can be exploited as targets for controlling the lifespan of neutrophils. Indeed, anti-inflammatory and pro-resolution lipid mediators derived from essential fatty acids, such as lipoxin A4 and resolvin E1, autacoids and proteins, such as annexin A1 and TRAIL, and cyclin-dependent kinase inhibitors, can enhance the resolution of inflammation through induction of neutrophil apoptosis and promoting their removal by efferocytosis. In this review, we discuss recent advances in understanding the molecular basis of these actions, highlighting the potential of therapeutic induction of neutrophil apoptosis for dampening neutrophil-mediated tissue injury and inflammation underlying a variety of diseases.
... AnxA1 +ve vesicles possess antiinflammatory properties (232) and allow the proper externalization of this pro-resolving mediator to gain access to extracellular surface receptors (i.e., FPR2) and to exert anti-inflammatory effects (39). AnxA1 acts as an exquisite brake for neutrophil adhesion to the microvascular wall, preventing over-exuberant cell transmigration to the inflammatory site (21,(233)(234)(235). We recently identified new properties for AnxA1 +ve vesicles, specifically those abundant in human synovial fluids collected from patients suffering from rheumatoid arthritis: these vesicles ensure the delivery of AnxA1 (and presumably other factors) to the chondrocyte in deep cartilage, enabling the activation of reparative circuits (236). ...
An effective resolution program may be able to prevent the progression from non-resolving acute inflammation to persistent chronic inflammation. It has now become evident that coordinated resolution programs initiate shortly after inflammatory responses begin. In this context, several mechanisms provide the fine-tuning of inflammation and create a favorable environment for the resolution phase to take place and for homeostasis to return. In this review, we focus on the events required for an effective transition from the proinflammatory phase to the onset and establishment of resolution. We suggest that several mediators that promote the inflammatory phase of inflammation can simultaneously initiate a program for active resolution. Indeed, several events enact a decrease in the local chemokine concentration, a reduction which is essential to inhibit further infiltration of neutrophils into the tissue. Interestingly, although neutrophils are cells that characteristically participate in the active phase of inflammation, they also contribute to the onset of resolution. Further understanding of the molecular mechanisms that initiate resolution may be instrumental to develop pro-resolution strategies to treat complex chronic inflammatory diseases, in humans. The efforts to develop strategies based on resolution of inflammation have shaped a new area of pharmacology referred to as “resolution pharmacology.”
... Neutrophils, monocytes and macrophages store large amounts of the glucocorticoid-inducible, pro-resolving protein annexin A1 (AnxA1) [4]. In neutrophils, the majority of AnxA1 is stored in the tertiary (gelatinase) granules which fuse rapidly with the membrane upon attachment to the endothelium [5]. ...
The fundamental role played by neutrophils for an efficient, acute inflammatory response has long been appreciated, with the underlying molecular and cellular mechanisms largely elucidated over the past decades. However, more recent work suggests that the biological functions exerted by this fascinating leucocyte are somewhat more extensive than previously acknowledged. Here we discuss how extravasated neutrophils govern the initiation of the resolution phase of inflammation by enabling activation of pro-resolving circuits to ensure the safe conclusion of the inflammatory response. The neutrophil 'alarm bell' on resolution is effected through release of soluble mediators as well as apoptotic bodies and other vesicles, which, in turn, can inform and modify the microenvironment ultimately leading to termination of the inflammatory response coinciding with re-establishment of tissue homeostasis and functionality.
... 28,31,32 ANXA1 may modulate leukocyte migration by inhibiting adhesion to the endothelium, 33 after being secreted by neutrophils upon adhesion. 34 We have shown that ANXA1 is a virus-regulated host protein, which is induced and cleaved at later time points. This could be a host response, or result of an interplay between the host cell and the proteins of the virus such as NS1 that could modulate ANXA1 expression. ...
The influenza virus infects millions of people each year and can result in severe complications. Understanding virus recognition and host responses to influenza infection will enable future development of more effective anti-viral therapies. Previous research has revealed diverse yet important roles for the annexin family of proteins in modulating the course of influenza A virus (IAV) infection. However, the role of Annexin-A1 (ANXA1) in IAV infection has not been addressed. Here, we show that ANXA1 deficient mice exhibit a survival advantage, and lower viral titers after infection. This was accompanied with enhanced inflammatory cell infiltration during IAV infection. ANXA1 expression is increased during influenza infection clinically, in vivo and in vitro. The presence of ANXA1 enhances viral replication, influences virus binding, and enhances endosomal trafficking of the virus to the nucleus. ANXA1 colocalizes with early and late endosomes near the nucleus, and enhances nuclear accumulation of viral nucleoprotein. In addition, ANXA1 enhances IAV-mediated apoptosis. Overall, our study demonstrates that ANXA1 plays an important role in influenza virus replication and propagation through various mechanisms and that we predict that the regulation of ANXA1 expression during IAV infection may be a viral strategy to enhance its infectivity.Cell Death and Differentiation advance online publication, 4 March 2016; doi:10.1038/cdd.2016.19.
In the context of the electroacupuncture (EA) neurobiological mechanisms, we have previously demonstrated the involvement of formyl peptide receptor 2 (FPR2/ALX) in the antihyperalgesic effect of EA. The present study investigated the involvement of peripheral FPR2/ALX in the antihyperalgesic effect of EA on inflammatory cytokines levels, oxidative stress markers and antioxidant enzymes in an animal model of persistent inflammatory pain. Male Swiss mice underwent intraplantar (i.pl.) injection with complete Freund's adjuvant (CFA). Mechanical hyperalgesia was assessed with von Frey monofilaments. Animals were treated with EA (2/10 Hz, ST36-SP6, 20 minutes) for 4 consecutive days. From the first to the fourth day after CFA injection, animals received i.pl. WRW4 (FPR2/ALX antagonist) or saline before EA. Levels of inflammatory cytokines (TNF, IL-6, IL-4 and IL-10), antioxidant enzymes (catalase and superoxide dismutase), oxidative stress markers (TBARS, protein carbonyl, nitrite/nitrate ratio), and myeloperoxidase activity were measured in paw tissue samples. As previously demonstrated, i.pl. injection of the FPR2/ALX antagonist prevented the antihyperalgesic effect induced by EA. Furthermore, animals treated with EA showed higher levels of IL-10 and catalase activity in the inflamed paw, and these effects were prevented by the antagonist WRW4. EA did not change levels of TNF and IL-6, SOD and MPO activity, and oxidative stress markers. Our work demonstrates that the antihyperalgesic effect of EA on CFA-induced inflammatory pain could be partially associated with higher IL-10 levels and catalase activity, and that these effects may be dependent, at least in part, on the activation of peripheral FPR2/ALX.
Atherosclerosis, a silent chronic vascular pathology, is the cause of the majority of cardiovascular ischaemic events. Atherosclerosis is characterized by a series of deleterious changes in cellularity, including endothelial dysfunction, transmigration of circulating inflammatory cells into the arterial wall, pro-inflammatory cytokines production, lipid accumulation in the intima, vascular local inflammatory response, atherosclerosis-related cells apoptosis and autophagy. Proteins of Annexin A (AnxA) family, the well-known Ca²⁺ phospholipid-binding protein, have many functions in regulating inflammation-related enzymes and cell signaling transduction, thus influencing cell adhesion, migration, differentiation, proliferation and apoptosis. There is now accumulating evidence that some members of the AnxA family, such as AnxA1, AnxA2, AnxA5 and AnxA7, play major roles in the development of atherosclerosis. This article discusses the major roles of AnxA1, AnxA2, AnxA5 and AnxA7, and the multifaceted mechanisms of the main biological process in which they are involved in atherosclerosis. Considering these evidences, it has been proposed that AnxA are drivers- and not merely participator- on the road to atherosclerosis, thus the progression of atherosclerosis may be prevented by targeting the expression or function of the AnxA family proteins.
Chaperonin 60.1 (Cpn60.1) is a protein derived from M. tuberculosis that has been shown, along with its peptide fragment IRL201104, to have beneficial effects in models of allergic inflammation. To further investigate the anti-inflammatory properties of Cpn60.1 and IRL201104, we have investigated these molecules in a model of non-allergic lung inflammation. Mice were treated with Cpn60.1 (0.5-5000ng/kg) or IRL201104 (0.00025-2.5ng/kg), immediately before intranasal instillation of bacterial lipopolysaccharide (LPS). Cytokine levels and cell numbers in mouse bronchoalveolar lavage (BAL) fluid were measured 4h after LPS administration. In some experiments mice were depleted of lung-resident phagocytes. Cells from BAL fluid were analysed for inflammasome function. Human umbilical vein endothelial cells (HUVEC) were analysed for adhesion molecule expression. Human neutrophils were analysed for integrin expression, chemotaxis and cell polarisation. Cpn60.1 and IRL201104 significantly inhibited neutrophil migration into the airways, independently of route of administration. This effect of the peptide was absent in TLR4 and Annexin A1 knock-out mice. Intravital microscopy revealed that IRL201104 reduced leukocyte adhesion and migration into inflamed tissues. However, IRL201104 did not significantly affect adhesion molecule expression in HUVEC or integrin expression, chemotaxis or polarisation of human neutrophils at the studied concentrations. In phagocyte-depleted animals, the anti-inflammatory effect of IRL201104 was not significant. IRL201104 significantly reduced IL-1β and NLRP3 expression and increased A20 expression in BAL cells. This study shows that Cpn60.1 and IRL201104 potently inhibit LPS-induced neutrophil infiltration in mouse lungs by a mechanism dependent on tissue-resident phagocytes and to a much lesser extent the pro-resolving factor Annexin A1.
The annexin superfamily consists of 12 proteins with a highly structural homology that binds to phospholipids depending on the availability of Ca²⁺-dependent. Different studies of overexpression, inhibition, or using recombinant proteins have linked the main function of these proteins to their dynamic and reversible binding to membranes. Annexins are found in multiple cellular compartments, regulating different functions, such as membrane trafficking, anchoring to the cell cytoskeleton, ion channel regulation, as well as pro- or anti-inflammatory and anticoagulant activities. The use of animals deficient in any of these annexins has established their possible functions in vivo, demonstrating that annexins can participate in relevant functions independent of Ca²⁺ signalling. This review will focus mainly on the role of different annexins in the pathological vascular remodelling that underlies the formation of the atherosclerotic lesion, as well as in the control of cholesterol homeostasis.
Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening mucocutaneous adverse drug reactions characterized by massive epidermal detachment. Cytotoxic T cells and associated effector molecules are known to drive SJS/TEN pathophysiology, but the contribution of innate immune responses is not well understood. We describe a mechanism by which neutrophils triggered inflammation during early phases of SJS/TEN. Skin-infiltrating CD8 ⁺ T cells produced lipocalin-2 in a drug-specific manner, which triggered the formation of neutrophil extracellular traps (NETs) in early lesional skin. Neutrophils undergoing NETosis released LL-37, an antimicrobial peptide, which induced formyl peptide receptor 1 (FPR1) expression by keratinocytes. FPR1 expression caused keratinocytes to be vulnerable to necroptosis that caused further release of LL-37 by necroptotic keratinocytes and induced FPR1 expression on surrounding keratinocytes, which likely amplified the necroptotic response. The NETs-necroptosis axis was not observed in less severe cutaneous adverse drug reactions, autoimmune diseases, or neutrophil-associated disorders, suggesting that this was a process specific to SJS/TEN. Initiation and progression of SJS/TEN keratinocyte necroptosis appear to involve a cascade of events mediated by innate and adaptive immune responses, and understanding these responses may contribute to the identification of diagnostic markers or therapeutic targets for these adverse drug reactions.
Neutrophils must navigate accurately towards pathogens in order to destroy invaders and thus defend our bodies against infection. Here we show that hydrogen peroxide, a potent neutrophil chemoattractant, guides chemotaxis by activating calcium-permeable TRPM2 ion channels and generating an intracellular leading-edge calcium “pulse”. The thermal sensitivity of TRPM2 activation means that chemotaxis towards hydrogen peroxide is strongly promoted by small temperature elevations, suggesting that an important function of fever may be to enhance neutrophil chemotaxis by facilitating calcium influx through TRPM2. Chemotaxis towards conventional chemoattractants such as LPS, CXCL2 and C5a does not depend on TRPM2 but is driven in a similar way by leading-edge calcium pulses. Other proposed initiators of neutrophil movement, such as PI3K, Rac and lyn, influence chemotaxis by modulating the amplitude of calcium pulses. We propose that intracellular leading-edge calcium pulses are universal drivers of the motile machinery involved in neutrophil chemotaxis.
Resumen
La superfamilia de anexinas está constituida por 12 proteínas con alta homología estructural que se unen a fosfolípidos de membrana de una manera dependiente de Ca²⁺. Diferentes estudios de sobreexpresión, inhibición o usando proteínas recombinantes han identificado que la función principal de estas proteínas está relacionada con su unión dinámica y reversible a membranas. Estas proteínas se encuentran en múltiples compartimentos celulares participando y regulando diferentes funciones como el tráfico de membranas, el anclaje al citoesqueleto celular, la regulación de canales iónicos, así como actividad proinflamatoria o antiinflamatoria y anticoagulante. El uso de animales deficientes en alguna de estas anexinas ha permitido establecer sus posibles funciones in vivo, demostrando que las anexinas pueden participar en funciones relevantes independientes de la señalización por Ca²⁺. En esta revisión nos centramos principalmente en el papel que juegan las diferentes anexinas en el remodelado vascular patológico que subyace a la formación de la lesión aterosclerótica así como en el control de la homeostasis del colesterol.
Background
Regulatory T (Treg) cells play a negative role in anti-tumor immunity against triple-negative breast cancer, so it is of great significance to find the potential therapeutic target of Treg cells.
Methods
First, Annexin A1 (ANXA1) expression and survival of patients with breast cancer were analyzed using TCGA data. Then plasma ANXA1 levels in patients with malignant and benign breast tumors were detected by ELISA. Next, the effect of ANXA1 on Treg cells was studied through suppressive assays, and how ANXA1 regulates the function of Treg cells was detected by RNA sequencing. Finally, the in vivo experiment in balb/c mice was conducted to test whether the ANXA1 blocker Boc1 could shrink tumors and affect the function of Treg cells.
Results
Our data suggest that ANXA1 expression is associated with lower survival and a higher risk of breast malignancy. Suppressive assays show that ANXA1 can enhance the inhibition function of Treg cells. RNA-Sequencing results indicate that Boc1 could reduce the expression of granzyme A mRNA in Treg cells. Animal experiments have been done to show that Boc1 can reduce tumor size and down regulate Treg cell function.
Conclusions
ANXA1 can enhance the function of Treg cells and reduce the survival rate of patients with breast cancer. Targeting ANXA1 can reduce Treg cell function and shrink breast tumors.
The occurrence, development, infiltration and metastasis of tumors are very complex processes involving the participation of many factors, some of which play a key role. Annexin A1 (ANXA1) is known as an anti-inflammatory protein. However, it has now been recognized to have a broader role beyond the inflammation, including roles in cell proliferation, differentiation, apoptosis, invasion, angiogenesis and metastasis. This review is intended to outline the research surrounding the pathophysiological effects of ANXA1 in tumors and its potential as a therapeutic and diagnostic agent. These studies comprehensively explore the expression changes of ANXA1 in cancer and further explore its mechanism of action in tumors, which is of great clinical significance for the early diagnosis, treatment and prognostic evaluation of tumors.
Annexin-A1 (ANXA1) was first discovered in the early 1980's as a protein, which mediates (some of the) anti-inflammatory effects of glucocorticoids. Subsequently, the role of ANXA1 in inflammation has been extensively studied. The biology of ANXA1 is complex and it has many different roles in both health and disease. Its effects as a potent endogenous anti-inflammatory mediator are well-described in both acute and chronic inflammation and its role in activating the pro-resolution phase receptor, FPR2, has been described and is now being exploited for therapeutic benefit. In the present mini review, we will endeavor to give an overview of ANXA1 biology in relation to inflammation and functions that mediate pro-resolution that are independent of glucocorticoid induction. We will focus on the role of ANXA1 in diseases with a large inflammatory component focusing on diabetes and microvascular disease. Finally, we will explore the possibility of exploiting ANXA1 as a novel therapeutic target in diabetes and the treatment of microvascular disease.
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.
Inflammation is essential to protect the host from exogenous and endogenous dangers that ultimately lead to tissue injury. The consequent tissue repair is intimately associated with the fate of the inflammatory response. Restoration of tissue homeostasis is achieved through a balance between pro-inflammatory and anti-inflammatory/pro-resolving mediators. In chronic inflammatory diseases such balance is compromised resulting in persistent inflammation and impaired healing. During the last two decades the glucocorticoid-regulated protein Annexin A1 (AnxA1) has emerged as a potent pro-resolving mediator acting on several facets of the innate immune system. Here, we review the therapeutic effects of AnxA1 on tissue healing and repairing together with the molecular targets responsible for these complex biological properties.
Introduction: The escalating public health problem represented by obesity has spurred multidisciplinary research into adipose tissue and importantly, the molecular biology of the adipocyte. The concept of adipose tissue as an endocrine organ, in addition to an energy storage compartment, is now pivotal in linking excess adiposity to disease states. Recent studies suggest that obesity related metabolic disorders are characterised by mild chronic inflammation as a result of adipocytokine production from fat tissue leading to dysregulation in the pro/anti?inflammatory systemic balance. Adipokines and pro?inflammatory markers are implicated in insulin insensitivity, blood glucose dysregulation, inflammation and atherosclerosis. There is a considerable amount of research into the characterization of adipokines and pro?inflammatory cytokines, the antiinflammatory adipocytokines warrant further exploration. Research studies and design: This PhD research was set out to investigate potential antiinflammatory molecules that could be used as markers of, and therapeutics for, metabolic syndrome associated maladies. This PhD consists of three studies including Study 1: a characterisation study of pro/anti?inflammatory mediators carried out on 116 men of various BMI and body composition; Study 2: an in vitro study design carried out in human Simpson Golabi Behmel Syndrome (SGBS) adipocyte cell line investigating a glucocorticoid regulated anti?inflammatory protein, annexin A1 (AnxA1) and its role in fat tissue function; and Study 3: a double-blind cross-over randomised trial in 15 borderline metabolic syndrome males investigating the effect of a supplemental antiinflammatory agent, resveratrol (250 mg/day for two weeks, from 500 mg of Polygonum cuspidatum (from root)), on metabolic parameters. Key findings: Study 1: We demonstrated for the first time that AnxA1 is significantly inversely correlated with increasing BMI (R = -0.424**, P < 0.001), increasing body fat % (R = -0.192, P = 0.037) and a larger waist size (R = -0.390**, P < 0.001) in 118 men aged 19 to 61 years, with BMI between 16.8 – 56.4 kg/m2, BF % between 4.3 to 51.8 %. The negative correlation of decreasing plasma AnxA1 was strongest statistically when compared with WHR, rather than total body fat, suggesting that centrally located fat may be more influential at reducing plasma AnxA1 concentrations. Study 2: We have shown that ANXA1 gene is expressed in human SGBS adipocytes and hypoxia reduces the expression of ANXA1 gene showing that AnxA1 may act as a counter regulator of adipose tissue inflammation. We found that CRP expression was significantly down-regulated following 4 (P=0.015), 8 (P=0.035) and 24 (P=0.037) of hypoxia treatment in the cells also treated with Ac2-26 peptide compared to vehicle alone. IL-6 was also found to be significantly down?regulated after 24 hour hypoxia treatment in the Ac2-26 treated cells compared to vehicle (P=0.022). Study 3: The effect of resveratrol on metabolic function had no significant effect on the metabolic markers measured including blood pressure, blood glucose, blood cholesterol and glycated LDL. Conclusion: These data demonstrate that AnxA1 could potentially represent a (fat) depot specific biomarker whose decline with increasing central adiposity may relate to the phenomena of increasing systemic inflammation and associated disease risk. We also demonstrate for the first time that an AnxA1 is expressed in human SGBS preadipocytes and mature adipocytes and AnxA1 mimetic, Ac2-26 peptide, regulates pro?inflammatory markers in human SGBS adipocytes. We showed that it may be difficult to improve the metabolic profile of individuals through supplementation of exogenous anti-inflammatory agent, resveratrol. Whilst anti-inflammatory agents such as AnxA1 may propose novel therapeutics for metabolic syndrome associated diseases, to date regular exercise and weight loss remain the main interventions that significantly.
Adrenal glucocorticoids (GC) and their synthetic analogues are steroid molecules endowed with powerful anti-inflammatory and immunosuppressive properties. The mode of action of GC is very complex and not yet fully elucidated, but a number of mechanisms have been described. Traditionally, selected anti-inflammatory actions of GC have been largely ascribed to the synthesis of a protein termed lipocortin 1 or annexin 1 (AnxAl), which inhibits phospholi-pase A2 activity with subsequent reduction in arachidonic acid release from the cell membrane and pro-inflammatory eicosanoid production [1, 2]. The immunosuppressive effect, on the other hand, is thought to be related to the inhibition of several immune functions, such as cytotoxicity, phagocytosis, and the synthesis of inflammatory cytokines like TNF-α IL-1, IL-2 and IL-8 [3].
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.
Clinical and experimental observations revealed that glucocorticoid-deficient states are associated with an enhanced inflammatory response. The antiinflammatory response of pharmacological doses of glucocorticoids has been tentatively attributed to the induction of lipocortin-I. To determine whether glucocorticoid deficiency causes lipocortin-I down-regulation, the expression of lipocortin-I mRNA and protein was quantified in rats with and without adrenalectomy (ADX). The mRNA of lipocortin-I was quantified by polymerase chain reaction, using a constant amount of modified lipocortin-I cDNA transcript as an internal standard. The lipocortin-I mRNA was decreased by 56 +/- 14% in lung tissue of ADX rats. This down-regulation of lipocortin-I mRNA was not due to a nonspecific effect of ADX, since the mRNA levels of other proteins (c-fos, c-myc, c-erbA beta, and metallothionein-II) remained unchanged. The decrease in lipocortin-I mRNA in ADX rats was reflected by a corresponding decrease in tissue (lung, spleen, liver, and kidney) lipocortin-I protein content, as assessed by quantitative Western blot analysis. Thus, ADX causes a decline in lipocortin-I message and protein, an observation compatible with the increased susceptibility to inflammatory reactions in glucocorticoid deficiency.
In order to examine the role of airway epithelium in the responsiveness of smooth muscle in man, we measured the contractile responses to acetylcholine (ACh), histamine, and prostaglandin F 2α (PGF 2α ) and the relaxation response to isoprenaline (Isop), in 48 bronchi obtained from 10 patients who received surgery. Responses were measured in the presence and absence of the epithelium.
Removal of epithelium (by rubbing the mucosa gently with forceps) significantly increased the contractile responses evoked by ACh, histamine and PGF 2α .
In contrast, removal of epithelium did not alter the relaxation response to Isop.
To clarify the mechanism underlying this epithelial inhibitory effect on smooth muscle contraction, we measured the contractile responses of dog trachea with the epithelium removed to increasing concentrations of ACh. After measuring the control response, we added about 0.1 g of the chopped epithelium in the organ chamber, and measured the response again.
After adding airway epithelium and incubating with tracheal strips, the contractile response of tracheal strips decreased significantly as compared to the control response.
These results show that airway epithelium possesses the ability to decrease the smooth muscle contraction to ACh, histamine and PGF 2α in man and dogs.
The mechanism of this inhibitory effect of the airway epithelium is not explained by a change in mechanical property of the airway nor the change in diffusion of these drugs to the smooth muscle across the epithelium. Thus, these results suggest that airway epithelium may have an important role in modulating smooth muscle tone, possibly by inactivation of these mediators, or by releasing an epithelium‐derived relaxing factor.
Neutrophil chemotaxis plays an important role in the inflammatory response and when excessive or persistent may augment tissue damage. The effects of inhibitors indicated the involvement of one or more serine proteinases in human neutrophil migration and shape change in response to a chemoattractant. Monospecific antibodies, chloromethylketone inhibitors, and reactive-site mutants of alpha 1-antitrypsin and alpha 1-antichymotrypsin were used to probe the specificity of the proteinases involved in chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis. Moreover, rapid inhibitors of cathepsin G and alpha-chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose. The concentrations of antichymotrypsin mutants that reduced chemotaxis by 50% would inactivate free cathepsin G with a half-life of 1.5-3 s, whereas the concentrations of chloromethylketones required to produce a similar inhibition of chemotaxis would inactivate cathepsin G with a half-life of 345 s. These data suggest different modes of action for these two classes of inhibitors. Indeed the chloromethylketone inhibitors of cathepsin G (Z-Gly-Leu-Phe-CMK) and to a lesser extent of chymotrypsin (Cbz-Gly-Gly-Phe-CMK) mediated their effect by preventing a shape change in the purified neutrophils exposed to fMLP. Antichymotrypsin did not affect shape change in response to fMLP even at concentrations that were able to reduce neutrophil chemotaxis by 50%. These results support the involvement of cell surface proteinases in the control of cell migration and show that antichymotrypsin and chloromethylketones have differing modes of action. This opens the possibility for the rational design of anti-inflammatory agents targeted at neutrophil membrane enzymes.
α1-Antitrypsin plasma deficiency variants which form hepatic inclusion bodies within the endoplasmic pathway include the common
Z variant (Glu342→ Lys) and the rarer α1-antitrypsin Siiyama(Ser53→ Phe). It has been proposed that retention of both abnormal proteins is accompanied by a common mechanism of loop-sheet polymerization
with the insertion of the reactive center loop of one molecule into a β-pleated sheet of another. We have compared the biosynthesis,
glycosylation, and secretion of normal, Z and Siiyamavariants of α1-antitrypsin using Xenopus oocytes. Siiyamaand Z α1-antitrypsin both duplicated the secretory defect seen in hepatocytes that results in decreased plasma α1-antitrypsin levels. Digestion with endoglycosidase H localized both variants to a pre-Golgi compartment. The mutation Phe51→ Leu abolished completely the intracellular blockage of Siiyamaα1-antitrypsin and reduced significantly the retention of Z α1-antitrypsin. The secretory properties of M and Z α1-antitrypsin variants containing amino acid substitutions designed to decrease loop mobility and sheet insertion were investigated.
A reduction in intracellular levels of Z α1-antitrypsin was achieved with the replacement of P11/12alanines by valines. Thus a decrease in Z and Siiyamaα1-antitrypsin retention was observed with mutations which either closed the A sheet or decreased loop mobility at the loop
hinge region.
Inflammation is a primary pathological process. The development of an inflammatory reaction involves the movement of white blood cells through the endothelial lining of blood vessels into tissues. This process of transendothelial cell migration of neutrophils has been shown to involve neutrophil beta 2 integrins (CD18) and endothelial cell platelet-endothelium cell adhesion molecules (PECAM-1; CD31). We now show that F(ab')2 fragments of the monoclonal antibody B6H12 against integrin-associated protein (IAP) blocks the transendothelial migration of neutrophils stimulated by an exogenous gradient of the chemokine interleukin 8 (IL-8; 60% inhibition), by the chemotactic peptide N-formyl-methionylleucylphenylalanine (FMLP; 76% inhibition), or by the activation of the endothelium by the cytokine tumor necrosis factor alpha (98% inhibition). The antibody has two mechanisms of action: on neutrophils it prevents the chemotactic response to IL-8 and FMLP, and on endothelium it prevents an unknown but IL-8-independent process. Blocking antibodies to IAP do not alter the expression of adhesion proteins or production of IL-8 by endothelial cells, and thus the inhibition of neutrophil transendothelial migration is selective. These data implicate IAP as the third molecule essential for neutrophil migration through endothelium into sites of inflammation.
Platelet/endothelial cell adhesion molecule 1 (PECAM-1; CD31) is crucial to the process of leukocyte transmigration through intercellular junctions of vascular endothelial cells. A monoclonal antibody to PECAM, or recombinant soluble PECAM, blocks transendothelial migration of monocytes by 70-90%. Pretreating either the monocytes or the endothelial junctions with antibody blocks transmigration. If the endothelium is first activated by cytokines, anti-PECAM antibody or soluble recombinant PECAM again block transmigration of both monocytes and neutrophils. Anti-PECAM does not block chemotaxis of either cell type. Light and electron microscopy reveal that leukocytes blocked in transmigration remain tightly bound to the apical surface of the endothelial cell, precisely over the intercellular junction. Thus, the process of leukocyte emigration can be dissected into three successive stages: rolling, mediated by the selectin class of adhesion molecules; tight adhesion, mediated by the leukocyte integrins and their endothelial cell counter-receptors; and now transmigration, which, based on these studies, requires PECAM-1.
In the 9 years since the last review on leukocyte and endothelial interactions was published in this journal many of the critical structures involved in leukocyte adherence to and migration across endothelium have been elucidated. With the advent of cell and molecular biology approaches, investigations have progressed from the early descriptions by intravital microscopy and histology, to functional and immunologic characterization of adhesion molecules, and now to the development of genetically deficient animals and the first phase I trial of “anti-adhesion” therapy in humans. The molecular cloning and definition of the adhesive functions of the leukocyte integrins, endothelial members of the Ig gene superfamily, and the selectins has already provided sufficient information to construct an operative paradigm of the molecular basis of leukocyte emigration. The regulation of these adhesion molecules by chemoattractants, cytokines, or chemokines, and the interrelationships of adhesion pathways need to be examined in vitro and, particularly, in vivo. Additional studies are required to dissect the contribution of the individual adhesion molecules to leukocyte emigration in various models of inflammation or immune reaction. Certainly, new adhesion structures will be identified, and the current paradigm of leukocyte emigration will be refined. The promise of new insights into the biology and pathology of the inflammatory and immune response, and the potential for new therapies for a wide variety of diseases assures that this will continue to be an exciting area of investigation.
Platelet/endothelial cell adhesion molecule 1 (PECAM-1; CD31) is crucial to the process of leukocyte transmigration through intercellular junctions of vascular endothelial cells. A monoclonal antibody to PECAM, or recombinant soluble PECAM, blocks transendothelial migration of monocytes by 70-90%. Pretreating either the monocytes or the endothelial junctions with antibody blocks transmigration. If the endothelium is first activated by cytokines, anti-PECAM antibody or soluble recombinant PECAM again block transmigration of both monocytes and neutrophils. Anti-PECAM does not block chemotaxis of either cell type. Light and electron microscopy reveal that leukocytes blocked in transmigration remain tightly bound to the apical surface of the endothelial cell, precisely over the intercellular junction. Thus, the process of leukocyte emigration can be dissected into three successive stages: rolling, mediated by the selectin class of adhesion molecules; tight adhesion, mediated by the leukocyte integrins and their endothelial cell counter-receptors; and now transmigration, which, based on these studies, requires PECAM-1.
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.
As a putative mediator of inflammation interleukin-1 has been implicated in the recruitment of leukocytes during the early stages of the inflammatory reaction. In the present report we have investigated the release of endogenous IL-1 in the rat zymosan pleurisy and in the mouse zymosan peritonitis. In both cases the release of the cytokine was maximal 4 hours after zymosan injection and appeared to be time-related to neutrophil migration into the inflammatory site. The effect of in vivo treatment with dexamethasone in rat pleurisy and with polyclonal anti-murine IL-1 beta antibody in mouse peritonitis was also assessed. The steroid reduced both cell migration and the release of IL-1-like activity as well as the formation of exudate and the release of eicosanoids. The anti-IL-1 beta serum inhibited selectively the number of neutrophil that migrated to the inflamed site (approximately 40%) and the IL-1 activity recovered in (approximately 70%) the exudate. In vitro incubation of the inflammatory exudate with polyclonal anti-murine IL-1 alpha or anti-murine IL-1 beta sera allowed the identification of the IL-1 species present. In the rat pleurisy IL-1 biological activity was mainly due to the alpha species, whereas IL-1 beta was the only species apparently present in the mouse peritoneal exudate.
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.
Movement of neutrophils from the bloodstream to inflamed tissue depends on the activation of both the neutrophil and the endothelial cell. Endothelial cells lining the postcapillary venule respond to proinflammatory mediators by expressing adhesion molecules and synthesizing a variety of neutrophil-activating factors. Endothelial cell production of a 77-amino acid variant of interleukin-8 (IL-8) was found to be a requirement for the invasion of neutrophils through a vessel wall model. IL-8 secreted by cytokine- or lipopolysaccharide-stimulated endothelial cells induced the rapid shedding of neutrophil lectin adhesion molecule-1, the up-regulation of leukocyte beta 2 integrins, and the attachment and transmigration of the neutrophils. Thus, endogenous endothelial IL-8 regulates transvenular traffic during acute inflammatory responses.
The effect of dexamethasone on mRNA and protein synthesis of lipocortins (LCT) 1, 2 and 5 has been investigated in U-937 cells. A constitutive expression of both mRNAs and proteins was detected in undifferentiated U-937 cells. This constitutive level was increased time- and dose-dependently by incubation with phorbol myristate acetate (PMA). In U-937 cells differentiated by 24 h incubation with 6 ng/ml PMA, dexamethasone (DEX) (1 microM for 16 h) caused an increased synthesis of the mRNA level of LCT-1 and 2, but not of LCT-5, over the level induced by PMA. DEX had no effect in undifferentiated cells. Moreover, DEX stimulated the extracellular release of LCT-1 and 5, but not of LCT-2, and inhibited the release of PGE2 and TXB2 only in the differentiated U-937 cells. These results suggest that the responsiveness of these cells to glucocorticoids is dependent on the phase of cell differentiation. The selective release of lipocortins by differentiated U-937 cells may explain, at least in part, the inhibition by DEX of the prostanoid release.
The presence and amount of the anti-inflammatory protein lipocortin 1 was determined in plasma and peripheral blood leucocytes by a highly specific, enzyme-linked immunosorbent assay. Within 120 min of a single intravenous dose of 100 mg hydrocortisone, the intracellular concentrations of lipocortin 1 in peripheral monocytes in 7 of 8 healthy men increased by a median of 225% (range 129-507%) compared with pretreatment levels, and mononuclear cell-surface lipocortin increased by a median of 224% (range 76-483%). Placebo injections had no effect. There was no increase at any time in free plasma or polymorph-associated lipocortin. In 3 of 4 subjects, induction of lipocortin was also observed when whole unseparated blood was incubated in vitro after steroid administration, but cells which had first been isolated and purified were refractory to such induction. Thus rapid changes in the concentration of an active anti-inflammatory protein can occur in man after normal therapeutic doses of hydrocortisone.
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.
A permanent human cell line, EA . hy 926, has been established that expresses at least one highly differentiated function of vascular endothelium, factor VIII-related antigen. This line was derived by fusing human umbilical vein endothelial cells with the permanent human cell line A549. Hybrid cells that survived in selective medium had more chromosomes than either progenitor cell type and included a marker chromosome from the A549 line. Factor VIII-related antigen can be identified intracellularly in the hybrids by immunofluorescence and accumulates in the culture fluid. Expression of factor VIII-related antigen by these hybrid cells has been maintained for more than 100 cumulative population doublings, including more than 50 passages and three cloning steps. This is evidence that EA . hy 926 represents a permanent line.
A method allowing quantitative analysis of the expression of foreign antigens at the surface of transformed cells is described. Aminoethyl-Sephadex G-25 beads labelled with different amounts of fluorescein isothiocyanate (FITC) were used to calibrate an Epics V cell sorter. The quantity of FITC molecules bound per bead was found to be a linear function of relative fluorescence intensity expressed by channel number for intermediate and high levels of fluorescence and a non-linear function for low levels. The lowest limit of detectable fluorescence was 3400 FITC molecules bound per bead. Using FITC-conjugated monoclonal antibodies (m.Ab.) the number of HLA class I molecules expressed at the surface of murine LMTK- (H-2Kk) cells transfected by cloned HLA class I genes was estimated and compared with the amount expressed on human B (Raji) and T (MOLT 4) lymphoblastoid reference cells. Murine transformed cells expressed approximately 3 times less HLA class I determinants per surface unit (micrometer 2) than Raji cells and 1.4 times less than MOLT 4 cells. Similar results were obtained by Scatchard analysis of the same populations using radiolabelled m.Ab. A detailed analysis of fluorescent cells showed that 5-20% of transformed cells expressed less than 33,000 HLA class I molecules/cell whereas most MOLT 4 cells exhibited at least 280,000 molecules/cell and the majority of Raji cells more than 700,000 molecules/cell. The expression of foreign antigen did not reduce the amount of H-2Kk molecules at the surface of transformed cells (mean of 350,000 molecules/cell).
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In the 9 years since the last review on leukocyte and endothelial interactions was published in this journal many of the critical structures involved in leukocyte adherence to and migration across endothelium have been elucidated. With the advent of cell and molecular biology approaches, investigations have progressed from the early descriptions by intravital microscopy and histology, to functional and immunologic characterization of adhesion molecules, and now to the development of genetically deficient animals and the first phase I trial of "anti-adhesion" therapy in humans. The molecular cloning and definition of the adhesive functions of the leukocyte integrins, endothelial members of the Ig gene superfamily, and the selectins has already provided sufficient information to construct an operative paradigm of the molecular basis of leukocyte emigration. The regulation of these adhesion molecules by chemoattractants, cytokines, or chemokines, and the interrelationships of adhesion pathways need to be examined in vitro and, particularly, in vivo. Additional studies are required to dissect the contribution of the individual adhesion molecules to leukocyte emigration in various models of inflammation or immune reaction. Certainly, new adhesion structures will be identified, and the current paradigm of leukocyte emigration will be refined. The promise of new insights into the biology and pathology of the inflammatory and immune response, and the potential for new therapies for a wide variety of diseases assures that this will continue to be an exciting area of investigation.
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.
Lipocortin-1, a 37 kDa member of the annexin superfamily of proteins, originally evoked interest as one of the 'second messengers' of the anti-inflammatory actions of the glucocorticoids. Subsequent research has shown that the protein plays a major regulatory role in systems as diverse as cell-growth regulation and differentiation, neutrophil migration, CNS responses to cytokines, neuroendocrine secretion and neurodegeneration. The role of lipocortin-1 in mediating glucocorticoid-induced effects in these systems has been demonstrated using immunoneutralization strategies and by mimicking steroid actions with highly purified or recombinant lipocortin-1 or its biologically active peptide fragments. Originally the mode of action of lipocortin-1 seemed to be largely through inhibition of prostaglandin formation, but it is now clear that it can modify other aspects of cell function, perhaps pointing to a more fundamental mechanism than was originally envisaged. In this article Rod Flower and Nancy Rothwell review the nature, possible mechanisms and clinical relevance of these diverse actions of lipocortin-1.
Phosphorylation of the N-terminal tail by protein kinase C strongly inhibits the ability of bovine or human annexin I to aggregate chromaffin granules by increasing the calcium requirement 4-fold (Wang, W., & Creutz, C. E. (1992) Biochemistry 31, 9934-9936). In the present study three forms of human annexin I truncated in the amino terminus at residue Trp-12, Lys-26, or Lys-29 exhibit dramatic differences in their sensitivities to calcium in a chromaffin granule aggregation assay, while the [Ca2+](1/2)max values for binding of the truncated proteins to granule membranes are similar. Cleavage at Trp-12 causes a 3-fold decrease in calcium sensitivity in the membrane aggregation assay, while cleavage at Lys-26 causes a 4-fold enhancement of calcium sensitivity. In contrast, cleavage at Lys-29 results in virtually no change in calcium sensitivity. Mutagenic substitution with negatively charged amino acids of Ser-27, a site for phosphorylation by protein kinase C, or Tyr-21, a site for phosphorylation by the epidermal growth factor receptor kinase, mimics the inhibition of granule-aggregating activity seen with phosphorylation by protein kinase C. When bovine chromaffin cells are stimulated to secrete by nicotine, annexin I is phosphorylated in the amino terminus. Thr-24 and Ser-28, which are sites for phosphorylation by protein kinase C in vitro, are two of the sites phosphorylated in vivo in stimulated chromaffin cells. These data demonstrate that the ability of annexin I to promote membrane aggregation is highly sensitive to changes in the structure of the N-terminal domain of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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.
Lipocortin 1 (LC1) immunoreactivity in murine peripheral blood leukocytes was quantified by use of a flow cytometric technique associated with a permeabilisation protocol with saponin. Using specific antisera raised against the whole protein or against its N‐terminus peptide, cell‐associated LC1‐like immunoreactivity was easily detected in circulating neutrophils and monocytes, whereas very low levels were found in lymphocytes. Of the total protein measured 17.6% and 36% were associated with the external plasma membrane in neutrophils and monocytes, as assessed in the absence of cell permeabilisation, whereas no signal was detected on lymphocyte plasma membrane.
Treatment of mice with dexamethasone (Dex; 0.5‐5 μg per mouse corresponding to ∼ 0.015‐1.5 mg kg ⁻¹ ) increased LC1 levels in neutrophils and monocytes. The 2–3 fold increase in LC1 levels was time‐dependent with a peak at 2 h. Treatment of mice with the steroid antagonist, RU486 (two doses of 20 mg kg ⁻¹ orally) decreased LC1‐like immunoreactivity in all three types of circulating leukocytes by ≥50%.
Extravasation of blood neutrophils into inflamed tissue sites resulted in a consistent reduction (≥50%) in LC1 levels compared with circulating neutrophils. A high LC1‐like immunoreactivity was also measured in resident macrophages, of which approximately one third was membrane‐associated. Induction of an acute inflammatory response in the murine peritoneal cavity did not modify total LC1 levels measured in macrophages, but reduced membrane‐associated LC1 to a significant extent, i.e. up to 70%.
In conclusion, flow cytometric analysis is a rapid and convenient method for detecting and measuring LC1 in murine leukocytes. We confirmed that LC1 protein expression is controlled by exogenous and endogenous glucocorticoids. Amongst other factor(s) influencing protein concentrations, extravasation was found to be associated with a reduced LC1 expression in the emigrated cells.
1. An immuno-neutralization strategy was employed to investigate the role of endogenous lipocortin 1 (LC1) in acute inflammation in the mouse. 2. Mice were treated subcutaneously with phosphate-buffered solution (PBS), non-immune sheep serum (NSS) or with one of two sheep antisera raised against LC1 (LCS3), or its N-terminal peptide (LCPS1), three times over a period of seven days. Twenty four hours after the last injection several parameters of acute inflammation were measured including zymosan-induced inflammation in 6-day-old air-pouches, zymosan-activated serum (ZAS)-induced oedema in the skin, platelet-activating factor (PAF)-induced neutrophilia and interleukin-1 beta (IL-1 beta)-induced corticosterone (CCS) release. 3. At the 4 h time-point of the zymosan inflamed air-pouch model, treatment with LCS3 did not modify the number of polymorphonuclear leucocytes (PMN) recruited: 7.84 +/- 1.01 and 7.00 +/- 0.77 x 10(6) PMN per mouse for NSS- and LCS3 group, n = 7. However, several other parameters of cell activation including myeloperoxidase (MPO) and elastase activities were increased (2.2 fold, P < 0.05, and 6.5 fold, P < 0.05, respectively) in the lavage fluids of these mice. Similarly, a significant increase in the amount of immunoreactive prostaglandin E2 (PGE2; 1.81 fold, P < 0.05) and IL-1 alpha (2.75 fold, P < 0.05), but not tumour necrosis factor-alpha (TNF-alpha), was also observed in LCS3-treated mice. 4. The recruitment of PMN into the zymosan inflamed air-pouches by 24 h had declined substantially (4.13 +/- 0.61 x 10(6) PMN per mouse, n = 12) in the NSS-treated mice, whereas high values were still measured in those treated with LCS3 (9.35 +/- 1.20 x 10(6) PMN per mouse, n = 12, P < 0.05). A similar effect was also found following sub-chronic treatment of mice with LCPS1: 6.48 +/- 0.10 x 10(6) PMN per mouse, vs. 2.77 +/- 1.20 and 2.64 +/- 0.49 x 10(6) PMN per mouse for PBS- and NSS-treated groups (n = 7, P < 0.05). Most markers of inflammation were also increased in the lavage fluids of LCS3-treated mice: MPO and elastase showed a 2.47 fold and 17 fold increase, respectively (P < 0.05 in both cases); TNF-alpha showed a 11.1 fold increase (P < 0.05) whereas the IL-1 alpha levels were not significantly modified. PGE2 was still detectable in most (5 out of 7) of the mice treated with LCS3 but only in 2 out of 7 of the NSS-treated mice. 5. Intradermal injection of 50% ZAS caused a significant increase in the 2 hoedema formation in the skin of LCS3-treated mice in comparison to PBS- and NSS-treated animals: 16.7 +/- 1.5 microliters vs. 10.8 +/- 1.2 microliters and 10.2 +/- 1.0 microliters, respectively (n = 14 mice per group, P < 0.05). ZAS-induced oedema had subsided by 24 h in control animals but a residual significant amount of extravasation was still detectable in LCS3-treated mice: 4.4 +/- 0.8 microliters (P < 0.05). 6. A recently described model driven by endogenous glucocorticoids is the blood neutrophilia observed following administration of PAF. In our experimental conditions, a single bolus of PAF (100 ng, i.v.) provoked a marked neutrophilia at 2 h (2.43 and 2.01 fold) in NSS- and PBS-treated mice (n = 11), respectively, which was significantly attenuated in the animals treated with LCS3: 1.26 fold increase in circulating PMN (n = 11, P < 0.01 vs. NSS- and PBS-groups). 7. Intraperitoneal injection of IL-1 beta (5 micrograms kg-1) caused a marked increase in circulating plasma CCS by 2 h, to a similar extent in all experimental groups. In contrast, measurement of CCS levels in the plasma of mice bearing air-pouches inflamed with zymosan revealed significant differences between LCS3 and NSS-treated mice at the 4 h time-point: 198 +/- 26 ng ml-1 vs. 110 +/- 31 ng ml-1 (n = 8, P < 0.05). 8. In conclusion, we found a remarkable exacerbation of the inflammatory process with respect to both humoral and cellular components in mice passively immunised agains
Stimulusspecific inhibition of human neutrophil H2O2 production by human recombinant lipocortin 1
- T R J Stevens
- A L Drasdo
- S H Peers
- N D Hall
- R J Flower
- TRJ Stevens