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Endogenous mediators that inhibit the leukocyte-endothelium interaction
Abstract
Leukocyte extravasation occurs in many pathophysiological conditions, including inflammation, neoplasia and asthma. In recent years many studies have elucidated the steps that promote the initial interaction between extravasating cells and endothelium of the post-capillary venule; the sequential role of several classes of adhesion molecules (cell-specific chemokines) and activators (multipotent cytokines) is well established. In this review, Mauro Perretti focuses on a less well investigated mechanism by which the host downregulates extravasation at the leukocyte-endothelium interface. The neutrophilic polymorphonuclear leukocyte is used as the prime example of a leukocyte that interacts with the endothelium, and particular emphasis is given to the possibility that novel anti-inflammatory therapies might be developed from a better understanding of the inhibitory mechanisms activated by endogenous mediators such as adenosine, lipocortin 1, NO, prostacyclin and cathepsin G.
... Indeed, a number of recent reports have heightened the awareness that resolution is an active process, one that requires activation of endogenous programs that enable the host tissue to maintain homeostasis (3)(4)(5). In this paper, we shall underscore current topics and definitions of components in resolution as well as outline where gaps in information lie within this new field. ...
... Indeed, several endogenous regulators of the inflammatory response have already been elucidated (for reviews, see refs. [3][4][5], adding support to the idea that this is a widely employed mechanism. Clearly, disturbances in such counter-regulatory circuits could lead to exacerbated inflammatory responses just as effectively (although perhaps less obviously) than excessive activation of the pro-inflammatory cascades (6). ...
... Consistently, agonists at receptor targets utilized by endogenous anti-inflammatory proresolving mediators exert an anti-inflammatory effect when added back into experimental models/specific settings in the laboratory (4,5). We envision that new drugs specifically designed to promote resolving mediators and biochemical pathways will have a much better compliance and be "homeostatic and modulatory" in their actions, mimicking the way inflammation naturally subsides in the body. ...
A recent focus meeting on Controlling Acute Inflammation was held in London, April 27-28, 2006, organized by D.W. Gilroy and S.D. Brain for the British Pharmacology Society. We concluded at the meeting that a consensus report was needed that addresses the rapid progress in this emerging field and details how the specific study of resolution of acute inflammation provides leads for novel anti-inflammatory therapeutics, as well as defines the terms and key components of interest in the resolution process within tissues as appreciated today. The inflammatory response protects the body against infection and injury but can itself become dysregulated with deleterious consequences to the host. It is now evident that endogenous biochemical pathways activated during defense reactions can counter-regulate inflammation and promote resolution. Hence, resolution is an active rather than a passive process, as once believed, which now promises novel approaches for the treatment of inflammation-associated diseases based on endogenous agonists of resolution.--Serhan, C. N., Brain, S. D., Buckley, C. D., Gilroy, D. W., Haslett, C., O'Neill, L. A. J., Perretti, M., Rossi, A. G., Wallace, J. L. Resolution of inflammation: state of the art, definitions and terms.
... The awareness of the complexity of the experimental inflammatory reaction has steadily increased in the past decade, in concomitance with the discovery of endogenous inflammatory and pro-resolving pathways (Nathan, 2002). Together with a few other laboratories worldwide, we have pioneered the concept that the inflammatory response follows-in an ideal fashion-a bell-shaped curve such that an acute strong inflammatory phase is followed by a pro-resolving phase with dampening of the response (Perretti, 1997;Serhan and Savill, 2005;Serhan et al., 2007). This sequence attained by an active involvement of inhibitory pathways and mediators (Gilroy et al., 2004;Serhan et al., 2007). ...
... Here we summarize the actions of Annexin A1 in innate immunity, emphasizing the opportunity that a better appreciation of the mechanisms activated by this protein to inhibit the inflammatory reaction would represent for innovative drug discovery. In fact, as proposed before (Perretti, 1997), we hypothesize that targets activated by endogenous anti-inflammatory mediators could be exploited for novel drug discovery programmes; new molecules obtained and developed in this manner would likely be devoid of major side effects, as their application would be mimicking the way our body naturally disposes of inflammation. Development of adenosine or somatostatin analogues would fit with this approach (Perretti, 1997). ...
... In fact, as proposed before (Perretti, 1997), we hypothesize that targets activated by endogenous anti-inflammatory mediators could be exploited for novel drug discovery programmes; new molecules obtained and developed in this manner would likely be devoid of major side effects, as their application would be mimicking the way our body naturally disposes of inflammation. Development of adenosine or somatostatin analogues would fit with this approach (Perretti, 1997). ...
The appreciation that the inflammatory reaction does not ‘spontaneously’ finish, but rather that inflammatory resolution is an active phenomenon brought about by endogenous anti‐inflammatory agonists opens multiple opportunities for a reassessment of the complexity of inflammation and its main mediators. This review dwells on one of these pathways, the one centred around the glucocorticoid‐regulated protein Annexin A1 and its G protein‐coupled receptor. In recent years, much of the knowledge detailing the processes by which Annexin A1 expresses its anti‐inflammatory role on innate immunity has been produced. Moreover, the generation of the Annexin A1 null mouse colony has provided important proof‐of‐concept experiments demonstrating the inhibitory properties of this mediator in the context of inflammatory and/or tissue‐injury models. Therefore, Annexin A1 acts as a pivotal homeostatic mediator, where if absent, inflammation would overshoot and be prolonged. This new understanding scientific information could guide us onto the exploitation of the biological properties of Annexin A1 and its receptor to instigate novel drug discovery programmes for anti‐inflammatory therapeutics. This line of research relies on the assumption that anti‐inflammatory drugs designed upon endogenous anti‐inflammatory mediators would be burdened by a lower degree of secondary effects as these agonists would be mimicking specific pathways activated in our body for safe disposal of inflammation. We believe that the next few years will produce examples of such new drugs and the validity of this speculation could then be assessed.
This article is part of a themed issue on Mediators and Receptors in the Resolution of Inflammation. To view this issue visit http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009
... In fact, heart damage associated with ischemia-reperfusion is the end result of an intense production of reactive oxygen species, nitric oxide derivatives, proteolytic enzymes, and inflammatory cytokines (13,14). Accordingly, inhibition of PMN accumulation to the myocardium or regulation of PMN activation is an obvious target for the development of novel therapies for myocardial injury following ischemia-reperfusion. Lipocortin 1 (LC1; also referred to as annexin I) is a member of the annexin super-family of proteins that is endowed with a potent leukocyte antimigratory activity (15). Treatment of experimental animals with human recombinant LC1, or with peptide analogues drawn from the LC1 N-terminal region, reduces PMN extravasation in simple models of acute inflammation (16,17). ...
... The profile of effect was similar to that attained with LC1 (i.e., inhibition of IS and IS/LV, reduction in MPO values, TNF-α and MIP-1α levels, and no effect on the parameters of systemic cell activation). Treatment with DEX is likely to increase LC1 levels in circulating leukocytes (18,38,54), thereby augmenting the inhibitory action of endogenous LC1 on neutrophils adherent to the endothelium (15,55,56). Further investigation is required to study the potential role of endogenous LC1 in a systematic manner. ...
... These mediators, termed lipoxins, are downstream products of arachidonate metabolism, generated through a then novel transcellular mechanism, requiring a two-step biosynthetic pathway with enzymes brought into close vicinity by two distinct cell types [1,2]. Lipoxins were the first factors demonstrated to inhibit leukocyte trafficking and promote apoptosis and phagocytosis of apoptotic cells (or efferocytosis) [3] and were highlighted in a 1997 TiPS review on 'endogenous inhibitors of leukocyte trafficking' [4]. Over the past 15 years this field of research has burgeoned with, on one side, the identification of several mediators synthesised in a strict temporal and spatial manner to actively prevent the overshooting of acute inflammatory mechanisms; on the other side, specific processes have been detailed and found to impact on this counterregulation on the host response. ...
... The past few years have witnessed an increment of interest in this field as testified by the number of cutting edge reviews that have detailed the biology of specific proresolving mediators and/or the processes of resolution (e.g., see [4,[6][7][8][9][10]). Herein, we summarise salient aspects of resolution especially if relevant to further discussion; the reader is redirected to these reviews for deeper analyses on mechanisms, as our scope is to reason on the impact that this area of science is having on informing the development of novel anti-inflammatory agents. ...
Current medicines for the clinical management of inflammatory diseases act by inhibiting specific enzymes or antagonising specific receptors or blocking their ligands. In the past decade, a new paradigm in our understanding of the inflammatory process has emerged with the appreciation of genetic, molecular, and cellular mechanisms that are engaged to actively resolve inflammation. The 'resolution of acute inflammation' is enabled by counter-regulatory checkpoints to terminate the inflammatory reaction, promoting healing and repair. It may be possible to harness this knowledge for innovative approaches to the treatment of inflammatory pathologies. Here we discuss current translational attempts to develop agonists at proresolving targets as a strategy to rectify chronic inflammatory status. We reason this new approach will lead to the identification of better drugs that will establish a new branch of pharmacology, 'resolution pharmacology'.
... IL-1, IL-6, TNF , TGF 1 and MCP-1 secretion by monocytes/macrophages are directly stimulated by hyperglycemia [118,119]. Therefore, the overexpression of some genes in GK islets might be attributed, at least in part, to the presence of islet macrophages: this could be the case for the immediate early serum responsive JE gene that corresponded to the MCP-1 gene, and genes coding for pro-inflammatory factors, such as LPSinduced TNF factor [120], galectin 3 [121] or anti-inflammatory factors, such as apolipoprotein E [122] and lipocortin 1 (annexin 1) [123]. ...
... Lipocalin 2 is considered as a sign of leukocyte activation in various diseases, particularly HTA and acute cerebral ischemic attack [125]. Lipocortin 1 (or annexin 1), as mentioned above for macrophages, is also produced by granulocytes [123]. Finally, granulocytes are associated with capillary closure in spontaneously diabetic monkey retinas [126] and granulocytes might play a role in atherosclerosis [127]. ...
Activation of the innate immune system has been recognized as being associated with T2D patients and those being at increased disease risk. Here we show that inflammation takes place in pancreatic islets both in various T2D animal models and in patients, as reflected by the presence of immune cells and various cytokines and chemokines. Under situations of metabolic stress particularly, islet endocrine cells are a source of cytokine and chemokine production, but other pancreatic islet cells such as immune, endothelial and neuronal cells may also be involved. Notably, in various T2D animal models (and probably in humans too), a process of microangiopathy with subsequent fibrosis takes place that results in islet blood flow alterations and eventually leads to a loss of islet architecture. Taking into account that the microvasculature plays an integral role in islet function and that cytokines are deleterious for β-cells, we suggest that endothelial- islet inflammation might contribute to β-cell impairment in T2D. This hypothesis is supported by the fact that treatments that preserve islet architecture and ameliorate endothelial dysfunction, improve β-cell function. Better understanding of the mechanisms involved in early β-cell failure should lead to the identification of new therapeutic targets for the prevention and treatment of T2D.
... Our understanding of the complexity and need for a balance between pro-and anti-inflammatory pathways to retain a homeostatic environment [60][61][62][63] has given rise to a great number of possible drug targets of the FPR family. In particular, targets activated by endogenous anti-inflammatory mediators could be used for drug discovery for both I/R and other disease states because they are likely to have fewer side effects and their application would be similar to mechanisms that the body uses to reject inflammation [60]. ...
... Our understanding of the complexity and need for a balance between pro-and anti-inflammatory pathways to retain a homeostatic environment [60][61][62][63] has given rise to a great number of possible drug targets of the FPR family. In particular, targets activated by endogenous anti-inflammatory mediators could be used for drug discovery for both I/R and other disease states because they are likely to have fewer side effects and their application would be similar to mechanisms that the body uses to reject inflammation [60]. One particular endogenous ligand of interest is the 37-kDa protein AnxA1, whose involvement with the FPR family has been widely studied and which affords protection in a number of I/R models. ...
Ischaemia-reperfusion (I/R) injury is a common feature of several diseases associated with high morbidity and mortality, such as stroke and myocardial infarction. The damaged tissue displays cardinal signs of inflammation and microvascular injury that, unless resolved, lead to long-term tissue damage with associated dysfunction. Current therapies are limited and are often associated with many side effects. Increasing evidence suggests that members of the formyl peptide receptor (FPR) family, in particular human FPR2/ALX, might have an important role in the pathophysiology of I/R injury. It was recently demonstrated that several peptides and non-peptidyl small-molecule compounds have anti-inflammatory and pro-resolving properties via their action on members of the FPR family. Here I review this evidence and suggest that FPR ligands, particularly in the brain, could be novel and exciting anti-inflammatory therapeutics for the treatment of a variety of clinical conditions, including stroke.
... Although this effect has initially been attributed to the capability of annexin A1 to inhibit PLA 2 and thus the production of eicosanoids, it has become clear more recently that the anti-inflammatory activity of the protein is most likely due to interference with granulocyte recruitment, migration, and/or activation at sites of inflammation (for reviews, see Refs. 105,221,222). ...
... Most likely this is due to an inhibitory effect on transmigration of the released annexin A1, which has been described in vitro and in vivo with exogenously applied protein as well as peptides derived from the unique NH 2 -terminal domain of annexin A1 (for reviews, see Refs. 105,221,222). Such NH 2 -terminal peptides typically covering residues 1-25 of the annexin A1 sequence in their NH 2 -terminally acetylated form (Ac1-25), as well as the entire molecule, are also capable of restricting leukocyte migration and thus tissue damage in animal models of splanchic artery occlusion/reperfusion and myocardial ischemia/reperfusion injury (56,58). ...
Annexins are a family of calcium-dependent phospholipid-binding proteins. They are abundant in the eukaryotic kingdom. Though structurally well investigated in the last twenty years the in vivo function of the annexins is still unclear. The determination of the crystal structure of human annexin V was the first milestone in the structural investigation of this protein family. Succeedingly, a variety of three-dimensional structures of annexin crystals as well as of membrane bound annexins were solved. They should provide tools to understand the in vivo function of the annexin family. Based on the structural knowledge mutagenesis studies and biophysical investigations were started to elucidate possible functions of annexins like membrane binding and ion channel activity.
... Based on the data presented here using exogenously administered LC1 and LC1 mimetics, together with a previous in vitro study on the externalization of intracellular LC1 on the plasma membrane of adherent neutrophils, we propose that LC1 is one of the mediator(s) that prevents adherent neutrophils from entering into diapedesis and eventually detaching from the postcapillary venule. Future studies are required to elucidate the mechanism responsible for this pharmacological effect, although it is likely that LC1 may interfere with the neutrophil activation process that occurs at the leukocyteendothelium interface on the inflamed postcapillary venule (41). ...
... Here, we have identified a different target for pharmacological intervention and shown that the ''adherent neutrophil'' is the target for LC1 and associated mimetics. It is conceivable that other mediators endowed with antiinflammatory activity may act in a similar manner (41,42). ...
In this study we investigated, using intravital microscopy, how neutrophil extravasation across mouse mesenteric postcapillary venules is inhibited by the glucocorticoid-regulated protein lipocortin (LC; also termed annexin) 1. Intraperitoneal injection of 1 mg of zymosan into mice induced neutrophil rolling on the activated mesenteric endothelium followed by adhesion (maximal at 2 hr: 5-6 cells per 100-micrometers of vessel length) and emigration (maximal at 4 hr: 8-10 cells per high-powered field). Treatment of mice with human recombinant LC1 (2 mg/kg s.c.) or its mimetic peptide Ac2-26 (13 mg/kg s.c.) did not modify cell rolling but markedly reduced (>/=50%) the degree of neutrophil adhesion and emigration (P < 0.05). Intravenous treatment with peptide Ac2-26 (13 mg/kg) or recombinant human LC1 (0.7-2 mg/kg) promoted detachment of neutrophils adherent to the endothelium 2 hr after zymosan administration, with adherent cells detaching within 4.12 +/- 0.75 min and 2.36 +/- 0.31 min, respectively (n = 20-25 cells). Recruitment of newly adherent cells to the endothelium was unaffected. The structurally related protein LC5 was inactive in this assay, whereas a chimeric molecule constructed from the N terminus of LC1 (49 aa) attached to the core region of LC5 produced cell detachment with kinetics similar to LC1. Removal of adherent neutrophils from activated postcapillary endothelium is a novel pharmacological action, and it is at this site where LC1 and its mimetics operate to down-regulate this aspect of the host inflammatory response.
... Leukocytes are rich in annexin I content and the protein is mainly localized in the cytoplasm of resting cells, accounting for 2–4% of total cytosolic proteins in human PMNs [4, 5]. We have recently reported that after PMN short-term (15–30 min) adhesion to EC monolayers the protein is externalized on the leukocyte plasma membrane [26], so that post-adherent PMNs are significantly depleted in the amounts of cell-associated annexin I [6]. In addition, after a 2-h transmigration in vitro, the annexin I gene is also switched off in migrated PMNs with approximately 50% reduction in specific mRNA levels. ...
... Most of the annexin I produced at 24 h was of the 33-kDa cleaved variety, whereas, at 48 h, intact annexin I (37-kDa species) was clearly detected. The existence of a specific enzyme that removes the amino-terminal portion of the protein has been recently suggested in human PMN [26, 37]. This process would lead to inactivation of annexin I while the intact molecule would remain biologically active [10, 29]. ...
Based on our previous studies showing endogenous annexin I being depleted from migrated neutrophils (PMN) in vitro, we have tested whether the levels of this glucocorticoid-regulated protein in PMN and mononuclear cells (PBMC) were modified after adhesion to endothelial monolayers in vitro and extravasation into skin blisters in vivo. In vitro, annexin I levels were depleted more significantly (-70%) in post-adherent PMNs than in monocytes (-25%) and lymphocytes (-50%, only in the positive fraction). In vivo, a significant time-dependent increase (approximately threefold, P < 0.05) in cell-associated annexin I was measured in PBMCs recovered from the blisters, whereas no significant changes were detected in extravasated PMNs. This was associated with annexin I release in the blister fluids (approximately 35 ng/mL), whereas no detectable protein was found in matched-paired plasmas. In conclusion, we report for the first time an activation of the annexin I pathway during an ongoing experimental inflammatory response in humans, which is differently regulated between PMNs and PBMCs.
... Currently, anti-inflammatory therapies target chemical mediators that are generated during the resolution phase. These mediators function as an activator in the form of receptor agonists in resolution therapies, which can activate protective mechanisms that result in tissue homeostasis (Perretti 1997;Serhan et al. 2004). So, the new drugs are specifically designed in such a way that can promote both pro-resolving as well as antiinflammatory functions. ...
Inflammation is an essential part of the body's healing process. Yet a long-term or chronic inflammatory response is damaging and is linked with several adult human diseases such as heart ailments, diabetes, cancer, asthma, and arthritis. The current therapeutic approach targets the pro-inflammatory mediators by using several commercially available anti-inflammatory drugs or monoclonal antibodies to combat these chronic inflammatory diseases. Although these current approaches seem to be effective, they often fail to provide a 'total therapeutic solution' and thereby complicating the situation and ultimately increasing the infection risk. In this context, the concepts and mechanisms of the resolution could be helpful for the treatment of these chronic inflammatory diseases. The review is devoted to the mechanism of resolution of inflammation and the working procedure of the specialized pro-resolving lipid mediators such as maresins, lipoxins, resolvins, and protectins. Alongside these standard specialized pro-resolving lipid mediators, plant-based lipid mediators in the form of biflavonoids have also emerged as an alternative option, which can limit the inflammatory response by targeting 5-lipoxygenase and also by triggering the switching of leukotrienes to specialized pro-resolving mediators. Other plant-based mediators will also strengthen the approach to the resolution of chronic inflammatory diseases.
... Moreover, in addition to the inhibition of TNF-α and IL-6 in carrageenaninduced animal models, α-PHE also modulated the other inflammatory mediators, such as leukotriene B4, IL-8 and C5a [98][99][100][101]. The inhibitory effect of α-PHE might be associated with the reduced TNF-α and IL-6 release and regulation of the adhesion molecules in the endothelial cells and polymorphonuclear cells [102,103]. ...
Aromatic essential oils play a significant role in pharmaceuticals, food additives, cosmetics, and perfumery. Essential oils mostly comprise aliphatic hydrocarbons, monoterpenoids, sesquiter-penoids and diterpenes. Plant extracts comprise a complex mixture of terpenes, terpenoids, aliphatic and phenol-derived aromatic components. Terpenes are a significant class of hydrocarbons with numerous health benefits. These biological functions of essential oil components are examined in vitro and in vivo studies. Some studies evaluated the properties and functions of α-phellandrene (α-PHE). Detailed evaluation to determine the functions of α-PHE over a spectrum of health care domains needs to be initiated. Its possible mechanism of action in a biological system could reveal the future opportunities and challenges in using α-PHE as a pharmaceutical candidate. The biological functions of α-PHE are reported, including anti-microbial, insecticidal, anti-inflammatory, anti-cancer, wound healing, analgesic, and neuronal responses. The present narrative review summarizes the synthesis, biotransformation, atmospheric emission, properties, and biological activities of α-PHE. The literature review suggests that extended pre-clinical studies are necessary to develop α-PHE-based adjuvant therapeutic approaches.
... Synthetic glucocorticoids play an extensive and crucial role in the treatment of unwanted and uncontrolled inflammation but come with potentially harmful metabolic side effects with long-term use (57). It is believed that therapeutic application of endogenous anti-inflammatory mediators like AnxA1 will be effective and cause fewer side effects by inducing natural pathways of resolution of inflammation (58)(59)(60). AnxA1 could be used therapeutically to treat the multiple inflammatory diseases of the ocular surface especially dry eye and ocular allergy as well as to prevent these diseases or to maintain ocular surface homeostasis in health. ...
The amount of mucin secreted by conjunctival goblet cells is regulated to ensure the optimal level for protection of the ocular surface. Under physiological conditions lipid specialized pro-resolving mediators (SPM) are essential for maintaining tissue homeostasis including the conjunctiva. The protein Annexin A1 (AnxA1) can act as an SPM. We used cultured rat conjunctival goblet cells to determine if AnxA1 stimulates an increase in intracellular [Ca²⁺] ([Ca²⁺]i) and mucin secretion and to identify the signaling pathways. The increase in [Ca²⁺]i was determined using fura2/AM and mucin secretion was measured using an enzyme-linked lectin assay. AnxA1 stimulated an increase in [Ca²⁺]i and mucin secretion that was blocked by the cell-permeant Ca²⁺ chelator BAPTA/AM and the ALX/FPR2 receptor inhibitor BOC2. AnxA1 increased [Ca²⁺]i to a similar extent as the SPMs lipoxin A4 and Resolvin (Rv) D1 and histamine. The AnxA1 increase in [Ca²⁺]i and mucin secretion were inhibited by blocking the phospholipase C (PLC) pathway including PLC, the IP3 receptor, the Ca²⁺/ATPase that causes the intracellular Ca²⁺ stores to empty, and blockade of Ca²⁺ influx. Inhibition of protein kinase C (PKC) and Ca²⁺/calmodulin-dependent protein kinase also decreased the AnxA1-stimulated increase in [Ca²⁺]i and mucin secretion. In contrast inhibitors of ERK 1/2, phospholipase A2 (PLA2), and phospholipase D (PLD) did not alter AnxA1-stimulated increase in [Ca²⁺]i, but did inhibit mucin secretion. Activation of protein kinase A did not decrease either the AnxA1-stimulated rise in [Ca²⁺]i or secretion. We conclude that in health, AnxA1 contributes to the mucin layer of the tear film and ocular surface homeostasis by activating the PLC signaling pathway to increase [Ca²⁺]i and stimulate mucin secretion and ERK1/2, PLA2, and PLD to stimulate mucin secretion from conjunctival goblet cells.
... The rapid expression elevation of TNAIP6 in the presence of TNF and IL-1 is unswerving with its association in inflammatory processes (Wisniewski et al., 1993). It seems that TNAIP6 acts through the circulation to influence an essential process of extreme increase of neutrophils, and modifying the adhesion of them to the endothelium (Perretti, 1997). The fibrotic changes reported following severe lung infection with influenza virus might be related to the elevation in TSG-6. ...
Human rhinovirus (HRV) is one of the most common viruses, causing mild to severe respiratory tract infections in children and adults. Moreover, it can lead to patients’ hospitalization. Nowadays, evaluation of gene expression alterations in host cells due to viral respiratory infections considered essential to understand the viral effects on cells.
Objective
In this study, we aimed to find important differentially expressed genes (DEGs) related to rhinitis and asthma exacerbation stimulated with Poly (I: C) and then to validate their expression in clinical samples of children how were less than 5 years old, hospitalized with severe acute respiratory infection (SARI) due to HRV infection in comparison with healthy cases.
Methods
Eight candidate genes involved in immunity, viral defense, inflammation, P53 pathway, and viral release processes were selected based on the analysis of a gene expression data set (GSE51392) and gene enrichment analysis. Then quantitative real-time PCR on cDNAs was performed for selected genes. The results were analyzed by Livak method and visualized by GraphPad prism software (8.4.3).
Result
CXCL10, CMPK2, RSAD2, SERPINA3, TNFAIP6, CXCL14, IVNS1AB, and ZMAT3 were selected based on the enrichment and topological analysis of the constructed protein-protein interaction (PPI) network. Laboratory validation by real-time PCR showed CXCL10, CMPK2, RSAD2, SERPINA3, and TNFAIP6 (belonged to immunity, inflammatory responses and viral defense) were up-regulated, whereas CXCL14 (related to immunity) and IVNS1AB, ZMAT3 (associated to Influenza and P53 pathway) were down-regulated.
Conclusion
Our results showed, that in children less than 5 years old affected by HRV and hospitalized with SARI, the inflammatory responses, antiviral defense, and type 1 interferon-signaling pathway have significantly affected by viral infection.
... Among these, the annexin-A1 protein (ANXA1) stands out. as an important antiinflammatory mediator, endowed with a potent anti-migratory leukocyte activity, which ensures the transient nature of the inflammatory response 15,16 . Thus, this study aimed to investigate the histopathological aspects of skin lesions and analyze the presence of M1 and M2 type macrophages at the leishmaniasis infection sites and their ANXA1 expression. ...
Introduction:
Cutaneous leishmaniasis is caused by protozoa of the genus Leishmania and transmission occurs through the bite of sandflies. It is an infectious disease, which affects skin and mucosa. The aim was to quantify the macrophages M1 and M2 and the annexin A1 expression in the skin lesions of patients with cutaneous leishmaniasis.
Methods:
Skin biopsies from patients (n = 50) were analyzed and classified according to the lesion type as: exudative cellular reaction, exudative granulomatous reaction, exudative necrotic reaction, exudative necrotic-granulomatous reaction. Using the immunofluorescence technique, macrophages were identified by CD163 marker, differentiated by anti-MHCII and anti-CD206 antibodies, and annexin A1 expression was determined by arbitrary unit (a.u.) densitometry.
Results:
In M1 macrophages, a greater expression of this protein was observed in the exudative cellular reaction type lesions (136.3 ± 2.6 a.u., assuming mean and standard derivation) when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (108.0 ± 2.3, 121.6 ± 3.2 and 124.7 ± 2.4 a.u., respectively). Regarding M2 macrophages, it was observed that patients with exudative cellular reaction lesion also had a higher expression of this protein (128.8 ± 2.6 a.u.), when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (105.6 ± 2, 113.9 ± 2.8, 114.3 ± 2.1 a.u., respectively).
Conclusions:
These data suggest that annexin A1 is assisting macrophages in the phagocytosis process of patients with exudative cellular reaction lesion type.
... Despite the inflammatory severity in Hb SS genotype, this group presented the highest levels of ANXA1 among the SCD genotypes. ANXA1 is abundant in neutrophils, which may externalize large amounts of this protein (50% to 70%), especially in response to cytokines released during the inflammatory propagation and neutrophil transmigration [20,22,40]. Homozygous patients for Hb S usually exhibit more anemic, hemolytic, vaso-occlusive and clinical severity than the double heterozygous genotypes [3] and this may represent a potential stimulus for outsourcing and releasing of ANXA1. ...
[This corrects the article DOI: 10.1371/journal.pone.0165833.].
... For instance, natural catabolism of proinflammatory mediators such as histamine, complement factors, classic eicosanoids and cytokines explains why a vessel does not remain dilated, why oedema formation ceases and why cells no longer migrate from blood into tissues 5 . However, data emergent over the past 20 years or so indicate that endogenous mediators are released or engaged to inhibit the specific cardinal signs of the acute inflammatory response; thus, adenosine, nitric oxide, lipoxins and annexin A1 (AnxA1; previously referred to as lipocortin) have been singled out as inhibitors of the process of leukocyte recruitment 6 . Parallel work has also defined important non-redundant properties of specific mediators in controlling the second (resolution) phase of inflammation, including the following: the induction of haem oxygenase-1 (REF. ...
The past two decades have witnessed major advancements in the clinical management of inflammatory arthritis, with new treatment strategies in some cases providing a marked improvement in patient outcomes. However, it is widely accepted that current strategies do not provide the 'total therapeutic solution', in view of the proportion of patients who do not respond to therapy, the important incidence of adverse effects and the development of an immune response against antibodies or fusion proteins used therapeutically. Moreover, although some therapeutic approaches can effectively bring about an end to inflammation, mechanisms to promote the recovery and/or repair of damage are required. Harnessing the concepts and mechanisms of the resolution of inflammation is a new approach to the treatment of inflammatory pathologies; this approach could help address the unmet need for new therapeutic approaches that not only control but also revert the course of inflammatory rheumatic diseases.
... Despite the inflammatory severity in Hb SS genotype, this group presented the highest levels of ANXA1 among the SCD genotypes. ANXA1 is abundant in neutrophils, which may externalize large amounts of this protein (50% to 70%), especially in response to cytokines released during the inflammatory propagation and neutrophil transmigration [20,22,40]. Homozygous patients for Hb S usually exhibit more anemic, hemolytic, vaso-occlusive and clinical severity than the double heterozygous genotypes [3] and this may represent a potential stimulus for outsourcing and releasing of ANXA1. ...
Sickle cell disease (SCD) is an inherited hemolytic anemia whose pathophysiology is driven by polymerization of the hemoglobin S (Hb S), leading to hemolysis and vaso-occlusive events. Inflammation is a fundamental component in these processes and a continuous inflammatory stimulus can lead to tissue damages. Thus, pro-resolving pathways emerge in order to restore the homeostasis. For example there is the annexin A1 (ANXA1), an endogenous anti-inflammatory protein involved in reducing neutrophil-endothelial interactions , accelerating neutrophil apoptosis and stimulating macrophage efferocytosis. We investigated the expression of ANXA1 in plasma of SCD patients and its relation with anemic , hemolytic and inflammatory parameters of the disease. Three SCD genotypes were considered: the homozygous inheritance for Hb S (Hb SS) and the association between Hb S and the hemoglobin variants D-Punjab (Hb SD) and C (Hb SC). ANXA1 and proinflamma-tory cytokines were quantified by ELISA in plasma of SCD patients and control individuals without hemoglobinopathies. Hematological and biochemical parameters were analyzed by flow cytometry and spectrophotometer. The plasma levels of ANXA1 were about threefold lesser in SCD patients compared to the control group, and within the SCD genotypes the most elevated levels were found in Hb SS individuals (approximately threefold higher). Proinflammatory cytokines were higher in SCD groups than in the control individuals. Anemic and hemolytic markers were higher in Hb SS and Hb SD genotypes compared to Hb SC patients. White blood cells and platelets count were higher in Hb SS genotype and were positively correlated to ANXA1 levels. We found that ANXA1 is down-regulated and differentially expressed within the SCD genotypes. Its expression seems to depend on the inflammatory, hemolytic and vaso-occlusive characteristics of the diseased. These data may lead to new biological targets for therapeutic intervention in SCD.
... In the model of carrageenan-induced peritonitis, α-phellandrene and dexamethasone attenuated the total leukocyte influx into the peritoneal cavity and decreased the number of rolling neutrophils on or adhered to the endothelium, as detected by intravital microscopy. This observation suggests that α-phellandrene can decrease the leukocyte migration by two possible mechanisms: (1) by inhibiting the release of direct inflammatory mediators, such as TNF-α and IL-6, by resident cells; and (2) by modulating the expression of adhesion molecules in endothelial and polymorphonuclear cells [37,38]. Indeed, various cyclic monoterpenes have been shown to exert anti-inflammatory properties through inhibition of cell migration [38] and decreased production of inflammatory mediators, including TNF-α and IL-6 [40,41], and pro-inflammatory enzymes, such as COX-2 [42]. ...
Aims:
We aimed to investigate the modulating effect of α-phellandrene on neutrophil migration and mast cell degranulation processes.
Main methods:
Male Wistar rats or Swiss mice were treated p.o. with vehicle (3% Tween 80, p.o.), α-phellandrene (50, 100, or 200mg/kg, p.o.), or dexamethasone (0.5mg/kg, p.o.) 1h before carrageenan injection. Then, the neutrophil migration in 6-day-old air pouches or peritoneal cavities. The leukocyte rolling and adhesion were measured in real time and assessed by intravital microscopy. ELISA was used to detect TNF-α and IL-6 in peritoneal lavage. Compound 48/80-induced mast cell degranulation was assessed in mesenteric rat tissues.
Key findings:
In all the tested doses, α-phellandrene prevented carrageenan-induced neutrophil accumulation (P<0.05). As detected by intravital microscopy, α-phellandrene also inhibited leukocyte rolling and adhesion, as well as significantly inhibited the production of the pro-inflammatory cytokines TNF-α and IL-6. Moreover, the degranulation of compound 48/80-induced mast cells was also inhibited by α-phellandrene (P<0.001).
Significance:
These results suggest that α-phellandrene plays an important role as an anti-inflammatory agent through neutrophil migration modulation and mast cell stabilization.
... The has been an extensive body of literature published with regard to the process of inflammation in the past decade, resulting in a discovery of endogenous pro-and anti-inflammatory pathways (Nathan, 2002). Scientists have pioneered a hypothesis regarding the process of inflammatory response where under ideal conditions, a normal distribution curve is observed i.e. an inflammatory stage is followed by a proresolution phase with attenuation of the response (Perretti, 1997, Serhan and Savill, 2005, Serhan et al., 2007. This process is achieved through a balanced and active action of inhibitory molecules and processes (Serhan et al., 2007). ...
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.
... For many years, resolution of inflammation was considered a passive phenomenon, merely associated with the removal of inflammatory stimuli, end of chemoattractant production, dilution of chemokine gradients over time, and prevention of further leukocyte recruitment. Some years later, the existence of endogenous inhibitors of leukocyte trafficking was reported, acting as a counteractive mechanism against promoters of cell recruitment, such as chemoattractants and adhesion molecules [reviewed in Ref. (10)]. Since then, several studies, especially those from Serhan's lab at Harvard, showed that the resolution of inflammation is an active process brought about by the biosynthesis of active mediators, which act on key events of inflammation to promote the return to homeostasis (11)(12)(13)(14). ...
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.”
... With low affinity antigen generally induce a Th2 response, whereas high affinity induces differentiation into a Th1 response [13,14]. Annexin-A1 (ANXA1) is an endogenous protein with anti-inflammatory functions, endowed with potent antimigratory activity of neutrophils, ensuring the transitory nature of the inflammatory response [15,16]. This protein is identified in several types of leukocytes [17,18] and positively modulates TCR signaling, making it an important molecular target in the differentiation and proliferation of lymphocytes. ...
... Inflammatory responses play important roles in body homeostasis, and represent the first line of defense against microbial contamination and tissue injury by restoring tissue integrity. While the inflammatory response is essential and beneficial to the host, an improperly mounted response can be harmful [1,2]. Significant progress has been made in understanding the cellular and molecular mechanisms involved in acute and chronic inflammatory responses, but little is known about their genetic control. ...
Objective and design:
AIRmax and AIRmin mice differ in their local acute inflammatory reactions to polyacrylamide beads (Biogel). These lines were developed to identify genes that affect the intensity of the acute inflammatory response (AIR) and to investigate the cellular and molecular mechanisms of acute inflammation. Although these lines are well established, differences in their responses to chronic inflammatory Biogel exposure have not yet been described. We investigated whether the selective process that modified the acute inflammatory responses in these animals also affected the development of their chronic inflammatory responses.
Results:
Inflammatory exudate cell infiltration was more intense in AIRmax than AIRmin mice at both 48 h and 30 days. Genes involved in signal transduction and immune/inflammatory responses were differentially expressed in the treated skin of AIRmax and AIRmin mice, and divergent expression of some acute inflammatory response genes was detected up to 30 days post-Biogel. However, distinct expression of several pro and anti-inflammatory response genes in both periods was observed.
Conclusion:
These results indicate that the selective process for acute inflammation affected the development of chronic inflammatory responses to Biogel, suggesting common genetic control.
... We have pioneered the concept that therapeutic innovation for inflammatory diseases could derive from appreciation and exploitation of the mechanisms operative during the resolution phase of inflammation (39,40). Such an approach, we propose, would yield therapeutics that would not be resolution toxic by repressing or delaying tissue repair processes and the ensuing restoration of homeostasis (40). ...
There is a need for novel approaches to control pathologies with overexuberant inflammatory reactions. Targeting melanocortin (MC) receptors represents a promising therapy for obesity and chronic inflammation, but lack of selectivity and safety concerns limit development. A new way to increase selectivity of biological effects entails the identification of biased agonists. In this study, we characterize the small molecule AP1189 as a biased agonist at receptors MC1 and MC3. Although not provoking canonical cAMP generation, AP1189 addition to MC1 or MC3, but not empty vector, transfected HEK293 cells caused ERK1/2 phosphorylation, a signaling responsible for the proefferocytic effect evoked in mouse primary macrophages. Added to macrophage cultures, AP1189 reduced cytokine release, an effect reliant on both MC1 and MC3 as evident from the use of Mc1r(-/-) and Mc3r(-/-) macrophages. No melanogenesis was induced by AP1189 in B16-F10 melanocytes. In vivo, oral AP1189 elicited anti-inflammatory actions in peritonitis and, upon administration at the peak of inflammation, accelerated the resolution phase by ∼3-fold. Finally, given the clinical efficacy of adrenocorticotropin in joint diseases, AP1189 was tested in experimental inflammatory arthritis, where this biased agonist afforded significant reduction of macroscopic and histological parameters of joint disruption. These proof-of-concept analyses with AP1189, an active oral anti-inflammatory and resolution-promoting compound, indicate that biased agonism at MC receptors is an innovative, viable approach to yield novel anti-inflammatory molecules endowed with a more favorable safety profile.
Copyright © 2015 by The American Association of Immunologists, Inc.
... As cytosolic Annexin A1 is actively mobilized when the neutrophil adheres to the endothelium, glucocorticoid-dependent increase in cellular Annexin A1 content would increase the amount of the protein externalized on the cell surface once the leukocyte adheres to the inflamed vascular endothelium. A survey of human donors indicated that between 50% and 70% of total Annexin A1 is externalized onto the neutrophil surface upon adhesion to endothelial cell monolayers (Perretti 1997). It is likely that the Annexin A1 system in the neutrophil can be finely regulated by post-translational mechanisms. ...
... With low affinity antigen generally induce a Th2 response, whereas high affinity induces differentiation into a Th1 response [13,14]. Annexin-A1 (ANXA1) is an endogenous protein with anti-inflammatory functions, endowed with potent antimigratory activity of neutrophils, ensuring the transitory nature of the inflammatory response [15,16]. This protein is identified in several types of leukocytes [17,18] and positively modulates TCR signaling, making it an important molecular target in the differentiation and proliferation of lymphocytes. ...
Malaria is the most prevalent parasitic disease in the world. In Brazil, the largest number of malaria cases (98%) is within the Legal Amazon region, where Plasmodium vivax is responsible for over 80% of diagnosed cases. The aim of this study was to investigate the annexin-A1 expression in CD4+, CD8+ T cells, regulatory T cells (Treg) and cytokine IL-10 quantification in plasma from patients with malaria caused by P. vivax.
The quantification of the cytokine IL-10 of patients infected with P. vivax and healthy controls were evaluated by enzyme-linked immunosorbent assay (ELISA). The determination of the expression of annexin-A1 in lymphocytes from patients and healthy controls was determined by immunofluorescence staining. All results were correlated with the parasitaemia and the number of previous episodes of malaria.
The cytokine IL-10 plasma levels showed a significant increase in both patients with low (650.4 +/- 59.3 pg/mL) and high (2870 +/- 185.3 pg/mL) parasitaemia compared to the control (326.1 +/- 40.1 pg/mL). In addition, there was an increase of this cytokine in an episode dependent manner (individuals with no previous episodes of malaria - primoinfected: 363.9 +/- 31.1 pg/mL; individuals with prior exposure: 659.9 +/- 49.4 pg/mL). The quantification of annexin-A1 expression indicated a decrease in CD4+ and CD8+ T cells and an increase in Treg in comparison with the control group. When annexin-A1 expression was compared according to the number of previous episodes of malaria, patients who have been exposed more than once to the parasite was found to have higher levels of CD4+ T cells (96.0 +/- 2.5 A.U) compared to primoinfected (50.3 +/- 1.7). However, this endogenous protein had higher levels in CD8+ (108.5 +/- 3.1) and Treg (87.5 +/- 2.5) from patients primoinfected.
This study demonstrates that in the patients infected with P. vivax the release of immunoregulatory molecules can be influenced by the parasitaemia level and the number of previous episodes of malaria. annexin-A1 is expressed differently in lymphocyte sub-populations and may have a role in cell proliferation. Furthermore, annexin-A1 may be contributing to IL-10 release in plasma of patients with vivax malaria.
... Thus, the development of compounds with innovative mechanisms of action, without the toxicity of conventional immunosuppressive agents, that are effective in preventing acute and chronic rejection is essential (Kędzierska et al., 2011). For this reason, research has been carried out aimed at developing new drugs and mediators responsible for regulating the mechanisms of cell migration (Perretti, 1997;Levy et al., 2001;Gil et al., 2006;Araujo et al., 2012). ...
Immunosuppressive drugs have a critical role in inhibiting tissue damage and allograft rejection. Studies have demonstrated the anti-inflammatory effects of the annexin A1 (AnxA1) in the regulation of transmigration and apoptosis of leucocytes. In the present study, an experimental skin allograft model was used to evaluate a potential protective effect of AnxA1 in transplantation survival. Mice were used for the skin allograft model and pharmacological treatments were carried out using either the AnxA1 mimetic peptide Ac2-26, with or without cyclosporine A (CsA), starting 3 days before surgery until rejection. Graft survival, skin histopathology, leucocyte transmigration and expression of AnxA1 and AnxA5 post-transplantation were analysed. Pharmacological treatment with Ac2-26 increased skin allograft survival related with inhibition of neutrophil transmigration and induction of apoptosis, thereby reducing the tissue damage compared with control animals. Moreover, AnxA1 and AnxA5 expression increased after Ac2-26 treatment in neutrophils. Interestingly, the combination of Ac2-26 and cyclosporine A showed similar survival of transplants when compared with the cyclosporine A group, which could be attributed to a synergistic effect of both drugs. Investigations in vitro revealed that cyclosporine A inhibited extracellular-signal-regulated kinase (ERK) phosphorylation induced by Ac2-26 in neutrophils. Overall, the results suggest that AnxA1 has an essential role in augmenting the survival of skin allograft, mainly owing to inhibition of neutrophil transmigration and enhancement of apoptosis. This effect may lead to the development of new therapeutic approaches relevant to transplant rejection. Copyright © 2013 John Wiley & Sons, Ltd.
... Over recent years, the definition of an endogenous proresolving mediator has evolved. Inhibition of leukocyte egress from the vasculature into the tissue was originally the main criteria for classification (then referred to as endogenous inhibitors of inflammation) [66] . It is now known that proresolving mediators also regulate antimicrobial defense, apoptosis and efferocytosis as well as monocyte recruitment and myeloid cell egress from the tissue into the lymphatic system. ...
Following tissue injury or microbial invasion, neutrophils are robustly recruited to inflammatory loci, which is a hallmark of the host inflammatory response. This event initiates a series of processes required to activate resolution, including recruitment of monocytes, clearance of microbes, cellular debris and apoptotic neutrophils, the egress of phagocytes and, ultimately, regain of tissue homeostasis. Substantial evidence now signifies that resolution of inflammation is a highly coordinated, active process dictated by the spatial-temporal generation of proresolving mediators that act on specific receptors to modulate cell and tissue reactivity. This review will focus on the mediators, targets and pathways initiated to orchestrate resolution. Importantly, disruption of the key processes involved in inflammatory resolution could result in delayed restoration of tissue homeostasis, leading to fibrosis and/or persistent inflammation.
... Adenosine is known to elicit its cellular responses through its interaction with its cell-surface receptors (A 1 R, A 2A R, A 2B R, and A 3 R) (18). All of these receptors have been implicated in the regulation of pulmonary inflammation either in a cytoprotective and/or anti-inflammatory role (31,36,37). Conversely, activation of these receptors, particularly A 1 R and/or A 3 R, has been implicated in a proinflammatory role leading to tissue damage (3,12,21). ...
Mucociliary clearance, vital to lung clearance, is dependent on cilia beat frequency (CBF), coordination of cilia, and the maintenance of periciliary fluid. Adenosine, the metabolic breakdown product of ATP, is an important modulator of ciliary motility. However, the contributions of specific adenosine receptors to key airway ciliary motility processes are unclear. We hypothesized that adenosine modulates ciliary motility via activation of its cell surface receptors (A(1), A(2A), A(2B), or A(3)). To test this hypothesis, mouse tracheal rings (MTRs) excised from wild-type and adenosine receptor knockout mice (A(1), A(2A), A(2B), or A(3), respectively), and bovine ciliated bronchial epithelial cells (BBECs) were stimulated with known cilia activators, isoproterenol (ISO; 10 μM) and/or procaterol (10 μM), in the presence or absence of 5'-(N-ethylcarboxamido) adenosine (NECA), a nonselective adenosine receptor agonist [100 nM (A(1), A(2A), A(3)); 10 μM (A(2B))], and CBF was measured. Cells and MTRs were also stimulated with NECA (100 nM or 10 μM) in the presence and absence of adenosine deaminase inhibitor, erythro-9- (2-hydroxy-3-nonyl) adenine hydrochloride (10 μM). Both ISO and procaterol stimulated CBF in untreated cells and/or MTRs from both wild-type and adenosine knockout mice by ~3 Hz. Likewise, CBF significantly increased ~2-3 Hz in BBECs and wild-type MTRs stimulated with NECA. MTRs from A(1), A(2A), and A(3) knockout mice stimulated with NECA also demonstrated an increase in CBF. However, NECA failed to stimulate CBF in MTRs from A(2B) knockout mice. To confirm the mechanism by which adenosine modulates CBF, protein kinase activity assays were conducted. The data revealed that NECA-stimulated CBF is mediated by the activation of cAMP-dependent PKA. Collectively, these data indicate that purinergic stimulation of CBF requires A(2B) adenosine receptor activation, likely via a PKA-dependent pathway.
... In analogy to the variety of pro-inflammatory mediators, there are short-lived lipids such as lipoxins, proteins and peptides, autacoids such as adenosine and even, as recently reported, adhesion molecules 12 within the umbrella of endogenous anti-inflammation. [13][14][15] It is important to establish how each of these mediators, part of specific homeostatic circuits, would operate. Appreciation of specific patho-physiological pathways is important to characterise the cellular and tissue-restricted events that each of these checkpoints mediators bring about; similarly, in terms of pharmacology, the investigation of the specific receptor target(s) that mediate the actions at the molecular level of a given anti-inflammatory mediator is of crucial importance. ...
Over 20 years of research based upon application of experimental models of inflammation and tissue injury have revealed exquisite controlling functions for melanocortin hormones and, subsequently, their synthetic derivatives. More recent discoveries have shed light on the receptor targets responsible for these effects, leading to what could be the next step-change for this line of research, the development of novel therapeutics for the control of human inflammatory pathologies. Here we review some of this work with particular emphasis on more recent studies that have substantiated the activities of melanocortin peptides to reveal important regulatory functions for their receptors in vascular inflammation and disease models. Moreover, we summarise the drug discovery activities (for what is published knowledge) attempting to capitalise on this wealth of research on melanocortins, though we should not forget the successful employment of ACTH to treat human gouty arthritis. Altogether, this chapter would corroborate and flare the enthusiasm for this line of research, as we are confident that the right times might have arrived to develop novel anti-arthritic and tissue-protective compounds that will be acting by mimicking the way our endogenous melanocortins would act to exert their homeostatic and check-point functions.
... The concept of endogenous anti-inflammation has gained ground recently with various mediators being identified: they are actively involved in controlling and attenuating the potential over-reaction of the body's immune system, thereby affording a degree of protection for the host, assuring a strict timedependence of the acute inflammatory response and promoting rapid regain of homeostasis [1,2,3,4] . Among the group of proresolving endogenous anti-inflammatory mediators, glucocorticoids represent one of the main pathways. ...
Our understanding of pro-resolution factors in determining the outcome of inflammation has recently gained ground, yet not many studies have investigated whether specific genes or patterns of genes, are modified by these mediators. Here, we have focussed on the glucocorticoid modulated pro-resolution factor annexin A1 (AnxA1), studying if its interaction with the ALX receptor would affect downstream genomic targets.
Using microarray technology in ALX transfected HEK293 cells, we discovered an over-lapping, yet distinct gene activation profile for AnxA1 compared to its N-terminal mimetic peptide Ac2-26, which may be suggestive of unique downstream inflammatory outcomes for each substance. When the up-regulated genes were explored, consistently induced was the sphingosine phosphate phosphatase-2 gene (SGPP2), involved in regulation of the sphingosine 1 phosphate chemotactic system. Up-regulation of this gene, as well as JAG1 (and down-regulation of JAM3), was confirmed using real time PCR both with transfected HEK293 cells and human peripheral blood leukocytes. Furthermore, lymph nodes taken from AnxA1(null) mice displayed lower SGPP2 gene activity. Finally, connectivity map analysis for AnxA1 and peptide Ac2-26 indicated striking similarities with known anti-inflammatory therapeutics, glucocorticoids and aspirin-like compounds, as well as with histone deacetylase inhibitors.
We believe these new data raise the profile of AnxA1 from being solely a short-term anti-inflammatory factor, to being a 'trigger' of the endogenous pro-resolution arsenal.
... Whilst for TH2 responses in the asthmatic lung activation of integrins on circulating eosinophils followed by tethering and rolling on bronchial endothelial cells [4] with increases in IL-4 and IL-5 leading to further activation and subsequent transmigration [5]. To aid in the resolution of this response a number of endogenous modulators of inflammation are produced including annexin 1 [6], galectin [7], lipoxins [8] and melanocortins [9] [10] and these have been shown to inhibit leukocyte trafficking and help prevent tissue damage. ...
In this study we set out to ascertain whether melanocortin peptides could be potential therapeutic agents in allergic and non-allergic models of lung inflammation by identifying the receptor(s) involved using a molecular, genetic and pharmacological approach. Western blot analyses revealed expression of the melanocortin receptor (MCR) type 1 and 3 on alveolar macrophages from wild-type mice. Alveolar macrophage incubation, with the selective MC3R agonist [D-TRP(8)]-gamma-MSH and pan-agonist alpha-MSH but not the selective MC1R agonist MS05, led to an increase in cAMP in wild-type macrophages. This increase occurred also in macrophages taken from recessive yellow (e/e; bearing a mutant and inactive MC1R) mice but not from MC3R-null mice. In an allergic model of inflammation, the pan-agonist alpha-MSH and selective MC3R agonist [D-TRP(8)]-gamma-MSH displayed significant attenuation of both eosinophil and lymphocyte accumulation but not IL-5 levels in wild-type and recessive yellow e/e mice. However in MC3R-null mice, alpha-MSH failed to cause a significant inhibition in these parameters, highlighting a preferential role for MC3R in mediating the anti-inflammatory effects of melanocortins in this model. Utilising a non-allergic model of LPS-induced lung neutrophilia, the pan-agonist alpha-MSH and selective MC3R agonist [D-TRP(8)]-gamma-MSH displayed significant attenuation of neutrophil accumulation and inhibition of TNF-alpha release. Thus, this study highlights that melanocortin peptides inhibit leukocyte accumulation in a model of allergic and non-allergic inflammation and this protective effect is associated with activation of the MC3R. The inhibition of leukocyte accumulation is via inhibition of TNF-alpha in the non-allergic model of inflammation but not IL-5 in the allergic model. These data have highlighted the potential for selective MC3R agonists as novel anti-inflammatory therapeutics in lung inflammation.
... Whereas the first group comprises the widely known cytokine interleukin (IL) -1, IL-8, and tumor necrosis factor (TNF) -␣ (to quote just a few) and adhesion molecules as well as proteolytic enzymes, the effectors of the latter group of mediators have been discovered in more recent years (3,4). The portfolio of endogenous inhibitors of inflammation, which often possess proresolving properties, include short-lived lipids such as lipoxins, resolvins, and cyclopentanone prostaglandins; purine bases such as adenosine and peptides; and proteins, such as IL-10, galectin-1, and annexin-1 (5)(6)(7). ...
The existence of anti-inflammatory circuits centered on melanocortin receptors (MCRs) has been supported by the inhibitory properties displayed by melanocortin peptides in models of inflammation and tissue injury. Here we addressed the pathophysiological effect that one MCR, MCR type 3 (MC3R), might have on vascular inflammation. After occlusion (35 min) and reopening of the superior mesenteric artery, MC3R-null mice displayed a higher degree of plasma extravasation (45 min postreperfusion) and cell adhesion and emigration (90 min postreperfusion). These cellular alterations were complemented by higher expression of mesenteric tissue CCL2 and CXCL1 (mRNA and protein) and myeloperoxydase, as compared with wild-type animals. MC1R and MC3R mRNA and protein were both expressed in the inflamed mesenteric tissue; however, no changes in vascular responses were observed in a mouse colony bearing an inactive MC1R. Pharmacological treatment of animals with a selective MC3R agonist ([D-Trp(8)]-gamma-melanocyte-stimulating hormone; 10 microg i.v.) produced marked attenuation of cell adhesion, emigration, and chemokine generation; such effects were absent in MC3R-null mice. These new data reveal the existence of a tonic inhibitory signal provided by MC3R in the mesenteric microcirculation of the mouse, acting to down-regulate cell trafficking and local mediator generation.
Chronic diseases that affect our society are made more complex by comorbidities and are poorly managed by the current pharmacology. While all present inflammatory etiopathogeneses, there is an unmet need for better clinical management of these diseases and their multiple symptoms. We discuss here an innovative approach based on the biology of the resolution of inflammation. Studying endogenous pro-resolving peptide and lipid mediators, how they are formed, and which target they interact with, can offer innovative options through augmenting the expression or function of pro-resolving pathways or mimicking their actions with novel targeted molecules. In all cases, resolution offers innovation for the treatment of the primary cause of a given disease and/or for the management of its comorbidities, ultimately improving patient quality of life. By implementing resolution pharmacology, we harness the whole physiology of inflammation, with the potential to bring a marked change in the management of inflammatory conditions.
Expected final online publication date for the Annual Review of Pharmacology and Toxicology, Volume 63 is January 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Malaria is one of the most widespread and serious parasitic diseases worldwide. Currently available antimalarial drugs have side effects, and many strains of Plasmodia have developed resistance to such drugs. The present review examines the use of annexins (ANXs) and of natural peptides from snake venom as a new class of anti-malarial agents, with the key property of reducing inflammation. Severe cases of malaria manifest elevated serum levels of liver enzymes, inflammation, fibrin deposition, apoptosis, and reduction in peripheral CD8+ T cells. The annexin-A1/5 (ANXA-1/5) proteins trigger inflammation via increased expression of diverse cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin- (IL-10), however, by shielding microbial phospholipids they prevent injury via damage-associated molecular patterns (DAMPs). Here, we also review an in silico -based bioengineering approach that may allow for a better design, synthesis and characterization of novel peptides from snake venom as a more effective approach for treatment due to their improved antimalarial activity.
The vascular endothelium lines the vascular tree and covers several square meters. One of its characteristics is its heterogeneity, which actually makes it difficult to discuss ‘the endothelial cell’ and to draw conclusions on results from different types of experiment. Endothelial cells are no longer considered as passive bystanders, but play a central role in the coordination of the inflammatory response. Endothelial cells are dynamic participants in the communication with circulating blood cells, acting as doorkeepers in blood cell margination and extravasation. Endothelial cells are important sites of synthesis of mediators including those providing an adequate vascular tone. Endothelial cells further regulate coagulation and fibrinolysis, maintain selective permeability and at the same time serve as target cells for many substances released during traumatic and septic shock [1]. Endothelial cell regulated vascular growth is beyond the scope of this chapter. Since only a few endothelial cells are circulating [2], it is difficult to monitor their status, and this will be discussed further below.
Adhesion of inflammatory cells to the vascular endothelium is an essential step in the movement of these cells out of the blood and into tissue sites and is therefore required for adequate host defence. However, the excessive recruitment of leucocytes to the lung, seen in diseases such as asthma and chronic obstructive pulmonary disease, is equally dependent upon this process. Therefore, leucocyte-endothelial adhesion presents a potential drug target for the development of new anti-inflammatory agents that would reduce the tissue damage and inflammation associated with the presence of these cells in the lung. This chapter discusses the adhesion molecules and pathways involved in adhesion of different leucocyte types to the vascular endothelium and some approaches that have been made in limiting these mechanisms.
The glucocorticoid-regulated protein annexin I (lipocortin I) has been shown to mediate antiinflammatory activities of glucocorticoids, but the molecular basis of its action has remained elusive. Here we show that annexin I acts through the formyl peptide receptor (FPR) on human neutrophils. Peptides derived from the unique N-terminal domain of annexin I serve as FPR ligands and trigger different signaling pathways in a dose-dependent manner. Lower peptide concentrations possibly found in inflammatory situations elicit Ca2+ transients without fully activating the MAP kinase pathway. This causes a specific inhibition of the transendothelial migration of neutrophils and a desensitization of neutrophils toward a chemoattractant challenge. These findings identify annexin I peptides as novel, endogenous FPR ligands and establish a mechanistic basis of annexin I–mediated antiinflammatory effects.
Inflammation is a primordial response that organisms set up to counteract the invasion of pathogens and attack by any other xenobiotic; the symptoms characteristic of inflammation are well known, the classic rubor, dolor, calor, tumor and functio laesa being described since ancient times. This response of the host is complex and multifactorial, and crucial for survival: abnormal inflammation, as in the case of patients affected by leukocyte adhesion deficiency, a genetic disease whereby specific adhesion molecules are absent, is associated with poor life quality and precocious death. Fighting inflammation is a common problem that physicians have to face when dealing with a wide variety of diseases. Hence, understanding the molecular and cellular mechanism responsible for it is important for the design of a better therapy. Modern theories in this field of research have suggested that one way to achieve a better and more efficient anti-inflammatory therapy is to exploit the body's own arsenal of endogenous antinflammatory mediators. One such protein is Annexin-1 (AnxA1): an endogenous anti-inflammatory protein whose activity correlates to the pharmacological effects of glucocorticoids. In this review we will summarize the most recent studies on the biological effects of AnxA1 and in particular we will be focusing on the differential and yet complementary role of this protein in the innate and adaptive immune systems.
Atherosclerosis has been recognized as an inflammatory disease. The innate and adaptive immunity mechanisms are involved in atherogenesis. Low-grade inflammation is associated with innate immunity activation. The CRP has been proposed as the best indicator of low-grade inflammation and a predictor of cardiovascular events. A triumvirate of targets in the prevention of CVD are: LDL-C aggressive lowering, HDL-C increasing and CRP lowering. The statins' role in lipids and CRP lowering is well established. Moreover, statins may be beneficial in many other inflammatory immune conditions. Among PPAR-α agonists, anti-inflammatory properties of fenofibrate are the best demonstrated. PPAR-γ agonists (thiazolidinediones) pleiotropic effects are also promising. PPAR α/γ agonists have been proved to cause severe side effects. Ezetimibe exerts beneficial anti-inflammatory actions when administered with statins. Colesevelam, a novel bileacid sequestrant has beneficial lipid-lowering and anti-inflammatory actions. CETP and ACAT inhibitors actions call for further studies. Inhibition of CRP, CRP-targeted antisenses, HDL-particle modification and immunomodulation of atherosclerosis are possible novel approaches in atherosclerosis treatment and prevention.
It is now evident that several endogenous anti-inflammatory pathways are activated in parallel with the host inflammatory
response to maintain a homeostatic control. From this idea has arisen the concept of anti-inflammation, a term used to describe
the balance that exists between pro-inflammatory and anti-inflammatory mediators/pathways that operate in concert to initiate,
maintain and finally resolve the inflammatory reaction.
Ischaemia–reperfusion injury underlies many of the most important cardiovascular diseases such as myocardial infarction, thrombotic stroke, embolic vascular occlusions and peripheral vascular insufficiency. Neutrophils feature prominently in this inflammatory component of post-ischaemic injury. Experimental therapies, shown to reduce neutrophil-mediated ischaemia–reperfusion injury include neutrophil depletion, direct inhibitors of neutrophil activators, antibodies against neutrophil adhesion molecules and the endothelial adhesion molecules. However, aside from these approaches, it is increasingly recognised that glucocorticoids are potent inhibitors of neutrophil-mediated injury. The anti-inflammatory actions of glucocorticoid include the activation of classical cytoplasmic receptors leading to changes in gene transcription as well as the induction of regulatory proteins, such as annexin 1. Annexin 1 is a potent inhibitor of neutrophil extravasation in vivo. Administration of the annexin 1 or peptides derived from its N-terminal domain, reduce neutrophil extravasation in models of acute inflammation. In addition, as reviewed by this article, annexin 1 protects against ischaemia–reperfusion in the heart and mesenteric microcirculation, as well as in multiple organ failure associated with splanchnic ischaemia–reperfusion. Such findings would suggest annexin 1 is a novel anti-inflammatory agent with a potential for the treatment of cardiovascular pathologies associated with neutrophil activation and recruitment.
Certains micro-organismes utilisent d'autres organismes comme hôtes. Résultent de ces interactions hétérotypiques des perturbations plus ou moins durables de l'homéostasie cellulaire/tissulaire, voire systémique des hôtes. Parmi de multiples effecteurs cellulaires et moléculaires qui sous-tendent ces processus infectieux/ parasitaires, opèrent des cytokines chimiokines et leurs récepteurs. Deux exemples illustrent le caractère plus ou moins durable et très dynamique de leurs fonctions dans les sites colonisés soit par Listeria monocytogenes, soit par Leishmania major, chez les souris de laboratoire.
Annexin-1 (ANX1) is an anti-inflammatory protein as well as an important modulator in inflammation. However, the precise action of ANX1 remains unclear. To elucidate the protective effects of ANX1 on lipopolysaccharide (LPS)-induced murine macrophage Raw 264.7 cells, we constructed a cell-permeable Tat-ANX1 protein. The transduced Tat-ANX1 protein markedly inhibited the expression of cyclooxygenase-2, production of prostaglandin E(2), and generation of pro-inflammatory cytokines in the cells. Furthermore, transduced Tat-ANX1 protein caused a significant reduction in the activation of nuclear factor- kappa B (NF-kB) and mitogen-activated protein kinase (MAPK). The results indicate that Tat-ANX1 inhibits the production of inflammatory response cytokines and enzymes by blocking NF-kB and MAPK. Therefore, Tat-ANX1 protein may be useful as a therapeutic agent against various inflammatory diseases.
Neutrophil extravasation, a critical component of innate immunity must be tightly regulated to prevent inadvertent or prolonged inflammation and subsequent tissue damage. We have shown previously that endothelial ERK1/2 signaling essential for neutrophil transendothelial migration is induced by a soluble factor produced by activated neutrophils. In this study, we demonstrate that the soluble neutrophil factor is a truncated form of annexin A1 (AnxA1) that can be generated by calpain 1 cleavage of the N terminus, thus identifying a novel proinflammatory function to AnxA1. In contrast, neither the full-length protein nor the N-terminal 26 aa peptide, previously shown to be antiinflammatory, were able to activate Erk. Our data suggest that two different fragments of AnxA1 have opposing functions in inflammation. We also provide evidence that C-terminal AnxA1 functions by increasing ICAM1 clustering around adherent neutrophils to anchor them to the endothelium and promote transmigration through the transcellular route.
The need for novel anti-inflammatory drugs justifies the search for innovative targets that could satisfy this goal. For quite some time now, we have proposed the study of endogenous anti-inflammation as a distinctive approach to the discovery of new drugs. This approach requires development of new compounds that activate specific receptor targets to downregulate the cellular and tissue pathways operative in the host during inflammation. Here we dwell on a family of G-protein coupled receptors (GPCR) termed FPRs, acronym for formyl-peptide receptors. With three and seven members in man and mouse, respectively, these receptors harness many biological functions, spanning odour perception and hair growth, to the control of multiple facets (pain; cell migration; oxidative burst; xenobiotic engulfment) of the inflammatory reaction. We focus on FPR biology with particular attention to molecules able to produce pharmacological effects by interacting with these GPCRs, describing endogenous agonists of FPRs and, more relevantly, the current development of synthetic agonists. Besides being potential leads for the development of the anti-inflammatory therapeutics of the future, these compounds could also help clarify the properties and roles that each FPR might play in the complex network of pathways that is inflammation. We conclude that FPR2 agonists could be valid warhorses for defining a novel philosophy for anti-inflammatory drug discovery programmes: mimicking - with new compounds - the way our body disposes of inflammation could be a viable approach to regulate aberrant inflammatory responses as in the case of several chronic rheumatic and cardiovascular pathologies.
The human formyl-peptide receptor (FPR)-2 is a G protein-coupled receptor that transduces signals from lipoxin A(4), annexin A1, and serum amyloid A (SAA) to regulate inflammation. In this study, we report the creation of a novel mouse colony in which the murine FprL1 FPR2 homologue, Fpr2, has been deleted and describe its use to explore the biology of this receptor. Deletion of murine fpr2 was verified by Southern blot analysis and PCR, and the functional absence of the G protein-coupled receptor was confirmed by radioligand binding assays. In vitro, Fpr2(-/-) macrophages had a diminished response to formyl-Met-Leu-Phe itself and did not respond to SAA-induced chemotaxis. ERK phosphorylation triggered by SAA was unchanged, but that induced by the annexin A1-derived peptide Ac2-26 or other Fpr2 ligands, such as W-peptide and compound 43, was attenuated markedly. In vivo, the antimigratory properties of compound 43, lipoxin A(4), annexin A1, and dexamethasone were reduced notably in Fpr2(-/-) mice compared with those in wild-type littermates. In contrast, SAA stimulated neutrophil recruitment, but the promigratory effect was lost following Fpr2 deletion. Inflammation was more marked in Fpr2(-/-) mice, with a pronounced increase in cell adherence and emigration in the mesenteric microcirculation after an ischemia-reperfusion insult and an augmented acute response to carrageenan-induced paw edema, compared with that in wild-type controls. Finally, Fpr2(-/-) mice exhibited higher sensitivity to arthrogenic serum and were completely unable to resolve this chronic pathology. We conclude that Fpr2 is an anti-inflammatory receptor that serves varied regulatory functions during the host defense response. These data support the development of Fpr2 agonists as novel anti-inflammatory therapeutics.
Inclui apêndice Orientadora : Helena Cristina da Silva de Assis. Tese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduaçao em Processos Biotecnológicos. Defesa: Curitiba, 2006 Inclui bibliografia Área de concentração: saúde animal e humana
The purpose of this study was twofold: to reveal cellular events associated with the protective role of endogenous annexin A1 (AnxA1) in inflammation and to highlight the potential involvement of members of the formyl peptide receptor (Fpr) family in this process. We found that wild-type, AnxA1-null, and Fpr1-null mice all displayed an intense neutrophil recruitment into the peritoneal cavity as assessed 4 hours after carrageenin injection, and that this recruitment was most pronounced in AnxA1-null mice. In addition, this cell influx could be inhibited by the AnxA1 pharmacophore peptide, Ac2-26, in wild-type, AnxA1-null, and Fpr1-null mice, but was restored when co-treated with the pan-receptor antagonist Boc2. Using the LacZ gene reporter assay, an enhancement of AnxA1 gene promoter activity in extravasated neutrophils was evident in AnxA1-null mice; again this response was reduced after peptide treatment. The lack of functional involvement of Fpr1 prompted us to monitor the structurally related receptor Fpr2. We report, for the first time, the ultrastructural immunocytochemical co-localization of Fpr2 with AnxA1 in neutrophils that migrate into the mesenteric microcirculation and extravasate into the peritoneal fluid. Collectively, these data provide in vivo support to the hypothesis that endogenous AnxA1 is an essential effector of endogenous anti-inflammation and provide an ultrastructural indication that this mediator interacts with Fpr2 in murine neutrophils. We believe that these findings could significantly affect the development of novel therapeutics, which are modeled after the anti-migratory actions of AnxA1.
Migration of blood-derived leukocytes to tissue sites of inflammation is a hallmark of the response that the host organizes to counteract an insult or a trauma or an infection. A cascade of events is then activated to allow interaction between the leukocyte and the endothelium of postcapillary venule, and this cascade is finely regulated such that mechanisms of negative control are operating side by side with pathways that promote and sustain the extravasation process. Examples of both these positive and negative regulatory systems are discussed here.
In the present study we demonstrate that human monocytes activated by lipopolysaccharides (LPS) were able to produce high levels of interleukin 10 (IL-10), previously designated cytokine synthesis inhibitory factor (CSIF), in a dose dependent fashion. IL-10 was detectable 7 h after activation of the monocytes and maximal levels of IL-10 production were observed after 24-48 h. These kinetics indicated that the production of IL-10 by human monocytes was relatively late as compared to the production of IL-1α, IL-1β, IL-6, IL-8, tumor necrosis factor Oi(TNFα), and granulocyte colony-stimulating factor (G-CSF), which were all secreted at high levels 4-8 h after activation. The production of IL-10 by LPS activated monocytes was, similar to that of IL-1α, IL-1β, IL-6, IL-8, TNFα, granulocyte-macrophage colony-stimulating factor (GM-CSF), and G-CSF, inhibited by IL-4. Furthermore we demonstrate here that IL-10, added to monocytes, activated by interferon γ(IFN-γ), LPS, or combinations of LPS and IFN-γ at the onset of the cultures, strongly inhibited the production of IL-1α, IL-1β, IL-6, IL-8, TNFα, GM-CSF, and G-CSF at the transcriptional level. Viral-IL-10, which has similar biological activities on human cells, also inhibited the production of TNFα and GM-CSF by monocytes following LPS activation. Activation of monocytes by LPS in the presence of neutralizing anti-IL-10 monoclonal antibodies resulted in the production of higher amounts of cytokines relative to LPS treatment alone, indicating that endogenously produced IL-10 inhibited the production of IL-1α, IL-1β, IL-6, IL-8, TNFα, GM-CSF, and G-CSF. In addition, IL-10 had autoregulatory effects since it strongly inhibited IL-10 mRNA synthesis in LPS activated monocytes. Furthermore, endogenously produced IL-10 was found to be responsible for the reduction in class II major histocompatibility complex (MHC) expression following activation of monocytes with LPS. Taken together our results indicate that IL-10 has important regulatory effects on immunological and inflammatory responses because of its capacity to downregulate class II MHC expression and to inhibit the production of proinflammatory cytokines by monocytes.
Corticosteroids are the preeminent antiinflammatory agents although the molecular mechanisms that impart their efficacy have not been defined. The endothelium plays a critical role in inflammation by directing circulating leukocytes into extravascular tissues by expressing adhesive molecules for leukocytes [e.g., endothelial-leukocyte adhesion molecule 1 (ELAM-1) and intercellular adhesion molecule 1 (ICAM-1)]. We therefore determined whether corticosteroids suppress inflammation by inhibiting endothelial expression of adhesion molecules for neutrophils (polymorphonuclear leukocytes). Preincubation of endothelial cells with endotoxin [lipopolysaccharide (LPS), 1 microgram/ml] led to a 4-fold increase in subsequent adherence of polymorphonuclear leukocytes (P < 0.0001, n = 10) to endothelial cells, an increase that was markedly attenuated when endothelial cells were treated with dexamethasone (IC50 < 1 nM, P < 0.0001, n = 6 or 7) during preincubation with LPS. Moreover, the steroid receptor agonist cortisol (10 microM), but not its inactive metabolite tetrahydrocortisol (10 microM), diminished LPS-induced endothelial cell adhesiveness. Further evidence that the action of dexamethasone was mediated through ligation of corticosteroid receptors [human glucocorticoid receptors (hGRs)] was provided by experiments utilizing the steroid antagonist RU-486. RU-486 (10 microM), which prevents translocation of ligated hGR to the nucleus by inhibiting dissociation of hGR from heat shock protein 90, completely aborted the effect of dexamethasone on adhesiveness of endothelial cells (P < 0.0005, n = 3). Treatment of endothelial cells with LPS (1 microgram/ml) stimulated transcription of ELAM-1, as shown by Northern blot analysis, and expression of membrane-associated ELAM-1 and ICAM-1, as shown by quantitative immunofluorescence (both P < 0.001, n = 9). Dexamethasone markedly inhibited LPS-stimulated accumulation of mRNA for ELAM-1 and expression of ELAM-1 and ICAM-1 (IC50 < 10 nM, both P < 0.001, n = 4-9); inhibition of expression by dexamethasone was reversed by RU-486 (both P < 0.005, n = 4-6). As in the adhesion studies, cortisol but not tetrahydrocortisol inhibited expression of ELAM-1 and ICAM-1 (both P < 0.005, n = 3 or 4). In contrast, sodium salicylate (1 mM) inhibited neither adhesion nor expression of these adhesion molecules. These studies suggest that antagonism by dexamethasone of endotoxin-induced inflammation is a specific instance of the general biological principle that the glucocorticoid receptor is a hormone-dependent regulator of transcription.
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.
Interleukin 1 receptor antagonist (IL-1ra), a naturally occurring polypeptide with amino acid sequence homology to interleukin 1 alpha (IL-1 alpha) and interleukin 1 beta (IL-1 beta), prevents Escherichia coli-induced shock and death. Both IL-1 and IL-1ra are produced by monocytes stimulated with lipopolysaccharide (LPS). Because interleukin 4 (IL-4) suppresses IL-1 production, we investigated whether IL-4 modulated IL-1ra synthesis in LPS-stimulated human peripheral blood mononuclear cells. IL-1 beta and IL-1ra were measured by specific RIAs. IL-4 alone (0.01-100 ng/ml) did not stimulate IL-1 beta synthesis but rather induced IL-1ra (4.82 +/- 0.94 ng/ml). LPS induced synthesis of both IL-1 beta (6.67 +/- 1.06 ng/ml) and IL-1ra (10.77 +/- 2.79 ng/ml). IL-4 suppressed LPS-induced IL-1 beta mRNA accumulation and synthesis. However, IL-4 acted synergistically with LPS in inducing IL-1ra. IL-4 enhanced LPS-induced IL-1ra mRNA accumulation 4-fold and IL-1ra protein synthesis nearly 2-fold. Moreover, IL-1ra mRNA levels were maximal after 6 hr of exposure to LPS but peaked within the first 3 hr in the presence of IL-4. IL-4 added as late as 12 hr after LPS stimulation still enhanced IL-1ra synthesis. In human peripheral blood mononuclear cells stimulated with IL-1 alpha, IL-4 markedly suppressed IL-1 beta production but enhanced IL-1ra synthesis greater than 2-fold. Because IL-4 favors synthesis of the natural antagonist IL-1ra over synthesis of the agonist IL-1, IL-4 may exert potent antiinflammatory effects on host responses to Gram-negative infections.
The objective of this study was to determine whether endogenous nitric oxide (NO) inhibits leukocyte adhesion to vascular endothelium. This was accomplished by superfusing a cat mesenteric preparation with inhibitors of NO production, NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME), and observing single (30-microns diameter) venules by intravital video microscopy. Thirty minutes into the superfusion period the number of adherent and emigrated leukocytes, the erythrocyte velocity, and the venular diameter were measured; venular blood flow and shear rate were calculated from the measured parameters. The contribution of the leukocyte adhesion glycoprotein CD11/CD18 was determined using the CD18-specific monoclonal antibody IB4. Both inhibitors of NO production increased leukocyte adherence more than 15-fold. Leukocyte emigration was also enhanced, whereas venular shear rate was reduced by nearly half. Antibody IB4 abolished the leukocyte adhesion induced by L-NMMA and L-NAME. Incubation of isolated cat neutrophils with L-NMMA, but not L-NAME, resulted in direct upregulation of CD11/CD18 as assessed by flow cytometry. Decrements in venular shear rate induced by partial occlusion of the superior mesenteric artery in untreated animals revealed that only a minor component of L-NAME-induced leukocyte adhesion was shear rate-dependent. The L-NAME-induced adhesion was inhibited by L-arginine but not D-arginine. These data suggest that endothelium-derived NO may be an important endogenous modulator of leukocyte adherence and that impairment of NO production results in a pattern of leukocyte adhesion and emigration that is characteristic of acute inflammation.
The adhesion of polymorphonuclear leukocytes (PMNs) to vascular endothelial cells (EC) is an early and fundamental event in acute inflammation. This process requires the regulated expression of molecules on both the EC and PMN. EC stimulated with histamine or thrombin coexpress two proadhesive molecules within minutes: granule membrane protein 140 (GMP-140), a member of the selectin family, and platelet-activating factor (PAF), a biologically active phospholipid. Coexpression of GMP-140 and PAF is required for maximal PMN adhesion and the two molecules act in a cooperative fashion. The component of adhesion mediated by EC-associated PAF requires activation of CD11/CD18 integrins on the PMN and binding of these heterodimers to counterreceptors on the EC. GMP-140 also binds to a receptor on the PMN; however, it tethers the PMN to the EC without requiring activation of CD11/CD18 integrins. This component of the adhesive interaction is blocked by antibodies to GMP-140 or by GMP-140 in the fluid phase. Experiments with purified GMP-140 indicate that binding to its receptor on the PMN does not directly induce PMN adhesiveness but that it potentiates the CD11/CD18-dependent adhesive response to PAF by a mechanism that involves events distal to the PAF receptor. Tethering of the PMN to the EC by GMP-140 may also be required for efficient interaction of PAF with its receptor on the PMN. These observations define a complex cell recognition system in which tethering of PMNs by a selectin, GMP-140, facilitates juxtacrine activation of the leukocytes by a signaling molecule, PAF. The latter event recruits the third component of the adhesive interaction, the CD11/CD18 integrins.
Although commonly used to control a variety of inflammatory diseases, the mechanism of action of a low dose of methotrexate remains a mystery. Methotrexate accumulates intracellularly where it may interfere with purine metabolism. Therefore, we determined whether a 48-hr pretreatment with methotrexate affected adenosine release from [14C]adenine-labeled human fibroblasts and umbilical vein endothelial cells. Methotrexate significantly increased adenosine release by fibroblasts from 4 +/- 1% to 31 +/- 6% of total purine released (EC50, 1 nM) and by endothelial cells from 24 +/- 4% to 42 +/- 7%. Methotrexate-enhanced adenosine release from fibroblasts was further increased to 51 +/- 4% (EC50, 6 nM) and from endothelial cells was increased to 58 +/- 5% of total purine released by exposure to stimulated (fMet-Leu-Phe at 0.1 microM) neutrophils. The effect of methotrexate on adenosine release was not due to cytotoxicity since cells treated with maximal concentrations of methotrexate took up [14C]adenine and released 14C-labeled purine (a measure of cell injury) in a manner identical to control cells. Methotrexate treatment of fibroblasts dramatically inhibited adherence to fibroblasts by both unstimulated neutrophils (IC50, 9 nM) and stimulated neutrophils (IC50, 13 nM). Methotrexate treatment inhibited neutrophil adherence by enhancing adenosine release from fibroblasts since digestion of extracellular adenosine by added adenosine deaminase completely abrogated the effect of methotrexate on neutrophil adherence without, itself, affecting adherence. One hypothesis that explains the effect of methotrexate on adenosine release is that, by inhibition of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase, methotrexate induces the accumulation of AICAR, the nucleoside precursor of which (5-aminoimidazole-4-carboxamide ribonucleoside referred to hereafter as acadesine) has previously been shown to cause adenosine release from ischemic cardiac tissue. We found that acadesine also promotes adenosine release from and inhibits neutrophil adherence to connective tissue cells. The observation that the antiinflammatory actions of methotrexate are due to the capacity of methotrexate to induce adenosine release may form the basis for the development of an additional class of antiinflammatory drugs.
Since adenosine and its analogue 2-chloroadenosine prevent neutrophils from generating superoxide anion in response to chemoattractants, we sought to determine whether these agents could inhibit neutrophil-mediated injury of endothelial cells. The chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP, 0.1 microM) enhanced the adherence of neutrophils to endothelial cells twofold (18 +/- 2% vs. 39 +/- 3% adherence, P less than 0.001) and caused substantial neutrophil-mediated injury to endothelial cells (2 +/- 2% vs. 39 +/- 4% cytotoxicity, P less than 0.001). 2-Chloroadenosine (10 microM) not only inhibited the adherence of stimulated neutrophils by 60% (24 +/- 2% adherence, P less than 0.001) but also diminished the cytotoxicity by 51% (20 +/- 4% cytotoxicity, P less than 0.002). Furthermore, depletion of endogenously released adenosine from the medium by adenosine deaminase-enhanced injury to endothelial cells by stimulated neutrophils (from 39 +/- 4% to 69 +/- 3% cytotoxicity, P less than 0.001). Indeed, in the presence of adenosine deaminase, even unstimulated neutrophils injured endothelial cells (19 +/- 4% vs. 2 +/- 2% cytotoxicity, P less than 0.001). These data indicate that engagement of adenosine receptors prevents both the adhesion of neutrophils and the injury they cause to endothelial cells. Adenosine inhibits injury provoked not only by cells that have been stimulated by chemoattractants but also by unstimulated cells. Based on this model of acute vascular damage we suggest that adenosine is not only a potent vasodilator, but plays the additional role of protecting vascular endothelium from damage by neutrophils.
Unlabelled:
Intraarterial bolus treatment with the prostacyclin analogue iloprost appears to have a prolonged beneficial effect on femorodistal bypass graft flow which extends beyond the duration of its vasodilator properties. The effect of iloprost on the microcirculation rendered ischaemic over the time course of a distal bypass operation was investigated in this study without the use of fluorescent light.
Methods:
A rat model was designed to allow prolonged direct observation of leukocyte-venular endothelial adhesion in a femorodistal bypass simulation. The extensor digitorum longus (EDL) muscle of 10 rats was subjected to two 30 minute periods of ischaemia by a non-venous occluding tourniquet and to simulate some of the changes of chronic ischaemia the adverse effect of ischaemia was accentuated by indirect electrical stimulation via the lateral popliteal nerve.
Results:
Intraarterial bolus treatment with iloprost significantly reduced the total numbers of leukocytes observed in EDL venules, and the numbers exhibiting evidence of adhesion by rolling or sticking to venule endothelium compared with saline controls at one hour post ischaemia. Ischaemia induced vasodilatation and reduced shear stress by a similar and significant amount in both groups.
Conclusion:
Two periods of ischaemia and reperfusion similar to those which occur during bypass grafting resulted in changes in the distal microcirculation consistent with reperfusion injury. Intraarterial bolus treatment with iloprost prevented these leucocyte-endothelial changes. It appears iloprost may have a role in reducing leukocyte-induced reperfusion injury in femorodistal bypass surgery.
To test the hypothesis that nitric oxide (NO) limits endothelial activation, we treated cytokine-stimulated human saphenous vein endothelial cells with several NO donors and assessed their effects on the inducible expression of vascular cell adhesion molecule-1 (VCAM-1). In a concentration-dependent manner, NO inhibited interleukin (IL)-1 alpha-stimulated VCAM-1 expression by 35-55% as determined by cell surface enzyme immunoassays and flow cytometry. This inhibition was paralleled by reduced monocyte adhesion to endothelial monolayers in nonstatic assays, was unaffected by cGMP analogues, and was quantitatively similar after stimulation by either IL-1 alpha, IL-1 beta, IL-4, tumor necrosis factor (TNF alpha), or bacterial lipopolysaccharide. NO also decreased the endothelial expression of other leukocyte adhesion molecules (E-selectin and to a lesser extent, intercellular adhesion molecule-1) and secretable cytokines (IL-6 and IL-8). Inhibition of endogenous NO production by L-N-monomethyl-arginine also induced the expression of VCAM-1, but did not augment cytokine-induced VCAM-1 expression. Nuclear run-on assays, transfection studies using various VCAM-1 promoter reporter gene constructs, and electrophoretic mobility shift assays indicated that NO represses VCAM-1 gene transcription, in part, by inhibiting NF-kappa B. We propose that NO's ability to limit endothelial activation and inhibit monocyte adhesion may contribute to some of its antiatherogenic and antiinflammatory properties within the vessel wall.
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.
Serine proteinases of human polymorphonuclear neutrophils play an important role in neutrophil-mediated proteolytic events; however, the non-oxidative mechanisms by which the cells can degrade extracellular matrix in the presence of proteinase inhibitors have not been elucidated. Herein, we provide the first report that human neutrophils express persistently active cell surface-bound human leukocyte elastase and cathepsin G on their cell surface. Unstimulated neutrophils have minimal cell surface expression of these enzymes; however, phorbol ester induces a 30-fold increase. While exposure of neutrophils to chemoattractants (fMLP and C5a) stimulates modest (two- to threefold) increases in cell surface expression of serine proteinases, priming with concentrations of lipopolysaccharide as low as 100 fg/ml leads to striking (up to 10-fold) increase in chemoattractant-induced cell surface expression, even in the presence of serum proteins. LPS-primed and fMLP-stimulated neutrophils have approximately 100 ng of cell surface human leukocyte elastase activity per 10(6) cells. Cell surface-bound human leukocyte elastase is catalytically active, yet is remarkably resistant to inhibition by naturally occurring proteinase inhibitors. These data indicate that binding of serine proteinases to the cell surface focuses and preserves their catalytic activity, even in the presence of proteinase inhibitors. Upregulated expression of persistently active cell surface-bound serine proteinases on activated neutrophils provides a novel mechanism to facilitate their egress from the vasculature, penetration of tissue barriers, and recruitment into sites of inflammation. Dysregulation of the cell surface expression of these enzymes has the potential to cause tissue destruction during inflammation.
Polymorphonuclear leukocytes (PMN) have been identified as cells capable of producing a number of pro- and antiinflammatory cytokines in response to specific agonists. Previously, we showed that tumor necrosis factor (TNF), interleukin-1 beta (IL-1 beta), and IL-8, are produced by PMN after stimulation with agonists, such as lipopolysaccharide (LPS). In this study, we demonstrate that LPS is also a potent stimulus for the mRNA expression and release of the IL-1 receptor antagonist (IL-1ra). In addition, we show that the release of IL-1ra from LPS-stimulated PMN is markedly potentiated in the presence of IL-10 (from two to threefold after 18 h of stimulation). Moreover, we observed that this upregulation of IL-1ra production by IL-10 in LPS-stimulated PMN took place through IL-1ra mRNA stabilization. Indeed, the half-life of IL-1ra mRNA was prolonged in PMN stimulated in the presence of IL-10 and LPS, as compared with cells stimulated with LPS alone. That IL-10 selectively upregulates IL-1ra production in LPS-activated PMN, while it inhibits the production of IL-1 beta, TNF, and IL-8 under the same conditions, suggests that IL-10 may be an important physiologic regulator of cytokine production from PMN, and emphasizes the potential role of IL-10 in inflammatory responses.
A rat model of inflammation was used to investigate the biological effects of thrombin. The thrombin-specific inhibitor Hirulog markedly attentuated the carrageenin-induced edema of the paw of the rat. Injection of thrombin into the paw also produced edema. The effect of thrombin was due to activation of its receptor; a thrombin receptor activating peptide (TRAP) reproduced the effects of thrombin in causing edema. TRAP also increased vascular permeability as demonstrated by extravasation of Evans blue and 125I-labeled serum albumin. The release of bioactive amines played an important role in mediating the TRAP-induced edema; the serotonin/histamine antagonist cryproheptadine and the histamine H2 receptor antagonist cimetidine reduced significantly the edema caused by TRAP. Treatment of rats with the mast cell degranulator 48/80 to deplete these cells of their stores of histamine and serotonin abolished completely the ability of TRAP to produce edema. Histochemical examination confirmed that TRAP treatment led to mast cell degranulation. Thus, it has been possible to demonstrate that thrombin acts as an inflammatory mediator in vivo by activating its receptor, which in turn leads to release of vasoactive amines from mast cells.
The role played by endogenous lipocortin 1 in the anti-migratory action exerted by dexamethasone (Dex) on monocyte recruitment in an in vivo model of acute inflammation was investigated by use of several neutralizing polyclonal antibodies raised against lipocortin 1 or a lipocortin 1-derived N-terminus peptide (peptide Ac2-26). The efficacy of peptide Ac2-26 in inhibiting monocyte and polymorphonuclear leucocyte (PMN) recruitment was also tested.
Intraperitoneal (i.p.) injection of zymosan A (1 mg) produced a time-dependent cell accumulation into mouse peritoneal cavities which followed a typical profile of acute inflammation: PMN influx was maximal at 4 h post-zymosan (between 15 and 20×106 cells per mouse), and this was followed by an accumulation of monocytes which peaked at the 24 h time-point (between 10 and 15×106 cells per mouse).
Dex administration to mice reduced zymosan-induced 4 h PMN infiltration and 24 h monocyte accumulation with similar efficacy: approximately 50% of inhibition of recruitment of both cell types was achieved at the dose of 30 μg per mouse (∼1 mg kg−1, subcutaneously (s.c.)). Maximal inhibitions of 64% and 67% on PMN and monocyte recruitment, respectively, were measured after a dose of 100 μg per mouse (∼3 mg kg−1, s.c.).
Dex (30 μg s.c.) inhibited monocyte (53%) and PMN (69%) accumulation in response to zymosan application in mice which had been treated with a non-immune sheep serum (50 μl s.c.). In contrast, the steroid was no longer active in reducing cell accumulation in mice which had been passively immunized against full length human recombinant lipocortin 1 (serum LCS3), or against lipocortin 1 N-terminus peptide.
Treatment of mice with vinblastine (1 mg kg−1, intravenously (i.v.)) produced a remarkable leucopenia as assessed 24 h after administration. This was accompanied by a 60% reduction in 4 h-PMN influx, and by a 27% reduction in 24 h-monocyte accumulation, measured after zymosan administration. The inhibitory effect of Dex on monocyte recruitment was not significantly modified in vinblastine-treated mice, with 36% and 57% of inhibition calculated at the dose of 30 μg Dex, and 70% and 60% of inhibition at 100 μg Dex, in vehicle- and vinblastine-treated mice, respectively.
Treatment of mice with peptide Ac2-26 dose-dependently attenuated PMN influx at 4 h post-zymosan with a significant effect at 100 μg per mouse (45% of inhibition, n=9, P<0.05) and a maximal effect of 61% inhibition at the highest dose tested of 200 μg s.c. (n=14, P<0.05). No effect of peptide Ac2-26 (200 μg s.c.) was seen on zymosan-induced 24 h monocyte recruitment. In contrast, administration of 200 μg peptide Ac2-26 every 6 h was effective in reducing the number of monocytes harvested from the inflamed peritoneal cavities at 24 h post-zymosan: 9.40±0.58×106 monocytes per mouse (n=13) and 5.74±0.34 monocytes per mouse (n=14) in vehicle- and peptide Ac2-26-treated mice, respectively (P<0.05).
Finally, peptide Ac2-26 produced a concentration-dependent inhibition of the rate of phagocytosis of mouse resident peritoneal macrophages as measured by flow cytometry, with a maximal reduction of 34% at the highest concentration tested of 100 μg ml−1 (n=8 experiments performed in duplicate; P<0.05).
In conclusion, this study suggests that in vivo monocyte recruitment during acute inflammation is, at least in part, under the negative modulatory control of endogenous lipocortin 1 (as seen after administration of Dex by using the specific antisera) and exogenous lipocortin 1 mimetics (as observed with peptide Ac2-26). In addition to the neutrophil, we can now propose that the monocyte also can be a target for the in vivo anti-inflammatory action of lipocortin 1.
British Journal of Pharmacology (1997) 120, 1075–1082; doi:10.1038/sj.bjp.0701029
Annexin 1 is a protein induced by glucocorticoids endowed with extracellular anti-inflammatory properties. In this study, the local expression and secretion of annexins 1–6, in rat proximal colon, were studied at different times after intracolonic administration of 30 mg trinltrobenzenesulfonic acid in 50% ethanol. Secretion was identified by incubating colonic tissues in a culture medium. The expression of annexins was detected by immunoblotting in tissue homogenates and incubation media. Inflammatory stages were evaluated by measuring myeloperoxidase activity. Annexin 1 expression in colons increased after trinitrobenzenesulfonic acid treatment and was maximal between days 1 to 9, during the cellular stage of the inflammation that corresponded to maximal myeloperoxidase activity. From 12 h to 9 days after trinitrobenzenesulfonic acid/ethanol treatment, annexin 1 was specifically secreted. Annexin 3 was also overexpressed during the cellular stage, but the expression of annexins 2, 4, 5, and 6 was unchanged; none of these annexins were secreted. Annexin 1 was shown to be physiologically secreted because its release was specific, abundant, and not correlated with cellular lysis. Annexin 1 may be considered as a putative candidate in the control of the gut inflammatory processes.
Platelet-activating factor (PAF; 100 ng i.v.) transiently modified the number of circulating neutrophils in the mouse, inducing a fast neutropenia (2 min) followed by a late onset neutrophilia (2 h). The potential involvement in PAF-induced neutrophilia of granulocytotic agents such as interleukin-1 and tumor necrosis factor-α could be excluded on the basis of the ineffectiveness of interleukin-1 receptor antagonist and of a specific monoclonal antibody anti-murine tumor necrosis factor-α. PAF granulocytosis was preceded by a significant rise in plasma corticosterone at 20 min. The involvement of endogenous corticosteroids was confirmed by the experiments with adrenalectomized mice and in animals pretreated with the steroid antagonist RU486 (11β-(4-dimethyl amino-phenyl) 17β-hydroxy, 17α(prop-1-ynyl) estra 4,9-dien-3-one), where PAF-induced neutrophilia was greatly reduced (∼50%). Moreover, sustained increase in plasma corticosterone by administration of adrenocorticotropic hormone was paralleled by an intense neutrophilia. We show evidence that endogenous corticosterone acts through the glucocorticoid-inducible protein lipocortin 1.
Propentofylline is an atypical xanthine derivative that blocks adenosine uptake and has been shown to protect against ischemia-induced cerebral damage. We have studied the effect of propentofylline on recruitment of polymorphonuclear leukocytes during acute peritonitis induced by zymosan in mice. Following i.p. injection of zymosan, recruitment of polymorphonuclear leukocytes, reflected by myeloperoxidase activity in the peritoneal cavity, increased from 2 h onwards, peaked at 4 h and then decreased gradually. Propentofylline antagonized the zymosan-induced peritoneal myeloperoxidase accumulation in a concentration-dependent manner. This effect of propentofylline was counteracted by the non-selective adenosine receptor antagonist theophylline (50 mg/kg), and by the selective adenosine A2A receptor antagonists, 4-amino-8-chloro-1-phenyl-[1,2,4]-triazolo[4,3-a]quinoxaline (CP 66713) and 1,3-dipropyl-8-[3,4-dimethoxystyryl]-7-methylxanthine (KF 17387) (both at 2 mg/kg). The results indicate that propentofylline can reduce polymorphonuclear leukocyte recruitment in vivo and that this effect is related to an action on adenosine A2A receptors.
Sialidase, fucoidin and a peptide corresponding to most of lipocortin 1 N-terminus, termed LC1-(Ac2-26)-peptide, induced an intense 2 h neutrophilia whereas a monoclonal antibody to murine CD11b induced an effect by 1 h. The neutropenic response stimulated by platelet-activating factor (PAF) was significantly reduced in the presence of sialidase, fucoidin, LC1-(Ac2-26)-peptide and monoclonal antibody anti-CD11b. Neutrophil migration into a 6-day-old mouse air-pouch induced by interleukin-1 was inhibited by all the pharmacological agents. In vitro, PAF up-regulated CD11b expression on the neutrophil surface but neither human or mouse LC1-(Ac2-26)-peptide inhibited this response. CD11b up-regulation on neutrophils occurred after PAF administration in vivo and was maximal at 2 min. LC1-(Ac2-26)-peptide mimics the action of agents interfering with leucocyte rolling and adhesion in vivo, however, does not inhibit CD11b up-regulation in vitro suggesting other phenomena are important in the activity of this peptide.
We have recently demonstrated that human neutrophils (PMN) possess two different classes of adenosine receptors (A1 and A2) that, when occupied, promote chemotaxis and inhibit the generation of reactive oxygen species (e.g., O2- and H2O2), respectively. We have previously demonstrated that adenosine protects endothelial cells (EC) from injury by stimulated neutrophils (PMN) both by diminishing generation of H2O2 and inhibiting adherence of PMN to EC. We therefore determined whether occupancy of A1 or A2 adenosine receptors regulated adherence of PMN to EC. At concentrations similar to those required to inhibit release of O2- by ligation of A2 receptors, both adenosine (IC50 = 56 nM) and 5'N-ethylcarboxamidoadenosine (NECA, IC50 = 8 nM), the most potent A2 agonist, inhibited adherence to EC by stimulated PMN (FMLP, 0.1 microM). In direct contrast, the specific A1 agonists N6-phenylisopropyladenosine and N6-cyclopentyladenosine (CPA) promoted PMN adherence to EC at concentrations of 1-100 nM. To further investigate the mechanisms by which adenosine receptor agonists affected the adherence of stimulated PMN we examined the effect of NECA (A2) and CPA (A1) on the adherence of PMN to fibrinogen (a ligand for the beta 2 integrin CD11b/CD18) and to gelatin. In a dose-dependent manner (IC50 = 2 nM), NECA inhibited the adherence of FMLP-treated PMN to fibrinogen- but not gelatin-coated plates. In contrast, CPA (A1) promoted adherence of stimulated PMN to gelatin-(EC50 = 13 pM) but not fibrinogen-coated plates. Theophylline (10 microM), an adenosine receptor antagonist, reversed the inhibition by NECA (0.3 microM) of stimulated neutrophil adherence to fibrinogen. These observations not only confirm the presence of A1 and A2 receptors on PMN but also suggest two opposing roles for adenosine in inflammation. Occupancy of A1 receptors promotes neutrophil adherence to endothelium and chemotaxis (a proinflammatory role) whereas occupancy of A2 receptors inhibits adherence and generation of toxic oxygen metabolites (an antiinflammatory role).
To investigate the inhibitory effect of adenosine released by endothelium on neutrophil superoxide (O2-) production, we treated confluent monolayers of cultured human umbilical vein endothelial cells with the enzyme adenosine deaminase, and then added human neutrophils. Superoxide (O2-) production by human neutrophils stimulated with 10(-6) M formyl-methionyl-leucyl-phenylalanine was inhibited by 49% in the presence of a confluent monolayer of human umbilical vein endothelial cells (5.1 +/- 0.1 versus 2.6 +/- 0.3 nmols O2-/10(6) neutrophils). Addition of 0.25 U/ml adenosine deaminase to neutrophils plus endothelial cells restored formyl-methionyl-leucyl-phenylalanine-stimulated neutrophil superoxide production to the level seen with neutrophils alone. Deoxycoformycin (10(-4) M), an inhibitor of adenosine deaminase activity, prevented the increase in superoxide production associated with adenosine deaminase addition. The adenosine analogue 5'-(N-ethylcarboxamido)- adenosine (3 x 10(-4) M) caused increased inhibition of formyl-methionyl-leucylphenylalanine-stimulated superoxide release by neutrophils in the presence of endothelial cells and prevented neutrophil-mediated endothelial cell damage, as measured by release of 3H-2-deoxy-D-glucose. Pairing 2-chloroadenosine (10(-5) M) or 5'-(N-ethylcarboxamido)-adenosine (3 x 10(-4) M) with a cyclic adenosine monophosphate phosphodiesterase inhibitor, 3-isobutyl-l-methyl-xanthine (10-4 M), produced greater inhibition of neutrophil superoxide production than occurred with either compound alone. The results support the hypothesis that vascular endothelial cells protect themselves from neutrophil attack by releasing adenosine to inhibit superoxide production.
This review has attempted to dissect several early events in stimulated neutrophils and relate the significance of these changes to various biologic activities of the cell. It is apparent from studies in this area that signal transduction in the in vivo situation reflects a complex sequence of interrelated events. Depending on the experimental conditons employed, the requirements for a putative second messenger product can be directly demonstrated or completely eliminated. To account for these seemingly disparate observations, it is likely that, in addition to the proposed mechanisms which have been described, auxiliary or regulatory signals are generated in parallel and participate in mediating functional responses. It is also possible that divergent pathways exist. In order to better resolve many discrepancies encountered during in vivo studies, the ultimate goal is to reconstitute the activation process in vitro. In this regard, the development of the cell-free NADPH-dependent oxidase system may be a useful paradigm. Investigations utilizing this system may not only contribute to the discernment of general stimulus-secretion coupling mechanisms in neutrophils, but also have particular relevance to understanding the biochemical basis of chronic granulomatous disease, a disorder which appears to represent a breakdown of the activation pathway at either of two locations.
Glucocorticoids block the localized accumulation of leukocytes as sites of inflammation by preventing their adherence to vascular endothelium. This implies that glucocorticoids are acting either on the leukocytes, endothelium, or cells which produce adherence-promoting factors (such as interleukin 1 (IL-1)). Previous studies have shown that dexamethasone (DEX) treatment of either polymorphonuclear leukocytes (PMN) or human umbilical vein vascular endothelial cells (VEC) or both in vitro does not prevent adherence induced by thrombin or formylmethionyl-leucyl-phenylalanine (f-met peptide). We now show that pretreatment of PMN and/or VEC for 24 hr with 0.1 microM DEX had no effect on adherence of PMN to VEC activated with IL-1 (2 U/ml), lipopolysaccharide (1 microgram/ml), or 12-O-tetradecanoylphorbol-13-acetate (30 ng/ml) suggesting that glucocorticoids may inhibit adherence in vivo by blocking formation of IL-1 and other adherence-inducing stimuli. We have recently established that cultured human lung fragments produce IL-1 in vitro. To investigate whether glucocorticoids could inhibit the production of adherence-inducing factors, we examined the effect of glucocorticoids on IL-1 production from human lung tissue. Treatment of human lung fragments in vitro for 18 hr with glucocorticoids such as DEX and hydrocortisone resulted in dose dependent inhibition of IL-1 production; these and other glucocorticoids, at concentrations ranging between 0.1 and 1 microM, produced greater than 50% inhibition of IL-1 release. Nonglucocorticoid steroids including testosterone and beta-estradiol (1 microM) had no effect. Inhibition of IL-1 production occurred after a lag period 5 of 16 hr, and the relative glucocorticoid potencies agreed with their known anti-inflammatory potencies in vivo (beta-methasone approximately triamcinolone acetonide greater than DEX greater than fludrocortisone greater than prednisolone greater than hydrocortisone). Inhibition of IL-1 production in vivo may, in part, explain the remarkable ability of glucocorticoids to prevent the adherence of leukocytes to endothelium and their accumulation at an inflammatory site.
Catalytically active cathepsin G that is bound to the cell surface of human neutrophils may play a variety of roles in normal neutrophil biology and in pathobiology associated with inflammation. In this study, we describe expression of neutrophil cell surface-bound cathepsin G in response to TNF-alpha and platelet-activating factor (PAF) under conditions in which minimal free release of cathepsin G is detected. TNF-alpha and PAF alone induced modest (two- to threefold) increases in cell surface-bound cathepsin G, but exhibited a marked dose- and time-dependent priming effect for subsequent chemoattractant-induced responses (up to 15- to 25-fold increases in cell surface expression). When optimally primed (TNF-alpha, 100 U/ml, or PAF, 10(-9) M), neutrophils expressed five- to sixfold more cell surface-bound cathepsin G, in comparison with cells exposed to FMLP alone. Priming responses were more rapid with PAF (15 s to 5 min) than with TNF-alpha (1 to 60 min). Optimally primed and FMLP-stimulated neutrophils express approximately 160 ng of catalytically active cathepsin G per 10(6) cells, which represents approximately 11% of the cellular content of unstimulated cells. Cathepsin G binds to the cell surface by a charge-dependent mechanism since: 1) incubation of cells with highly positively charged molecules abrogated agonist-induced up-regulation of the cell surface expression of cathepsin G and 2) cathepsin G was eluted from the cell surface by high concentrations of NaCl. These data indicate that interactions between biologically relevant pro-inflammatory cytokines and chemoattractants serve to markedly up-regulate cell surface-bound cathepsin G. The focused catalytic activity of cell surface-bound cathepsin G may alter endothelial and epithelial barriers, promote thrombogenesis, injure extracellular matrix, and/or facilitate directed migration of neutrophils during inflammation.
The human anticoagulant factor, Protein C, is a plasma glycoprotein that has reported anti-ischaemic and anti-inflammatory
properties. To explore potential mechanisms for these reported activities, we examined the effect of Protein C on the process
of cell adhesion to vascular endothelial cells, which plays a critical role during inflammatory responses. We show that both
human plasma-derived and human cell-produced recombinant Protein C inhibit E-selectin-mediated cell adhesion. This effect
was not mediated through the serine protease activity of Protein C, but through its carbohydrates. Using oligosaccharides
isolated from human cell-produced Protein C, we have defined a polylactosamine structural determinant that inhibits adhesion.
This uncharged detenminant appears to be a more potent ligand for E-selectin than the sialylated Lewis X antigen. Our data
suggest a potential mechanism for the reported anti-inflammatory effects of Protein C and describe a new ligand for selectin-mediated
adhesion.
Adenosine and adenosine analogues exhibit anti-inflammatory effects in vitro and in vivo, but their usefulness is limited by profound cardiovascular side effects. Therefore, we synthesized inhibitors of an enzyme involved in adenosine metabolism, adenosine kinase (AK) (EC 2.7.1.20), to enhance endogenous adenosine concentrations at sites of inflammation. GP-1-515 (4-amino-1-(5-amino-5-deoxy-1-beta-D- ribofuranosyl)-3-bromo-pyrazolo[3,4-d]pyrimidine), a novel AK inhibitor, decreased adhesion of activated human neutrophils to cultured endothelial cell monolayers by increasing local adenosine levels. The mechanism of inhibition in this assay seemed to involve selectin blockade and was independent of the beta 2 integrins. GP-1-515 and 2-chloroadenosine (a nonmetabolizable adenosine analogue) had no effect on the surface expression or shedding of adhesion molecules. An agent that disrupts the cytoskeleton, cytochalasin B, mimicked the effect of adenosine on cell adhesion. Interactions between L-selectin and the neutrophil cytoskeleton might be altered by adenosine and could contribute to adenosine-mediated adhesion inhibition.
The extent of mobilization of four different intracellular compartments was measured during in vivo exudation of neutrophils into skin chambers and compared with resting neutrophils obtained from blood. Exudation of neutrophils induced increased surface expression of alkaline phosphatase, complement receptor 1, and Mac-1, and a complete loss of L-selectin. The increase in the content of surface molecules in the plasma membrane is in accordance with complete mobilization of secretory vesicles. Granule matrix proteins were secreted into the chamber fluid by the exudated neutrophils and the exocytosed proteins were recovered in the skin chamber fluid. Release of gelatinase from gelatinase granules was 38.1%, lactoferrin release from specific granules was 21.9%, and myeloperoxidase release from azurophil granules was 7.0%, clearly illustrating a hierarchy in mobilization among granules. When exudate neutrophils were stimulated with FMLP, additional mobilization of granules was observed and the rank order regarding release was preserved. This is the first report to evaluate the mobilization of secretory vesicles during in vivo exudation of human neutrophils. It is shown that secretory vesicles are regulated exocytotic vesicles that are fully mobilized during in vivo exudation. Once exocytosed, secretory vesicles are not re-formed within a period of 6 h.
We have studied the ability of the glucocorticoid, dexamethasone, to induce annexin 1 secretion by either human blood monocytes or rat peritoneal leukocytes.
The in vivo treatment of rats with dexamethasone (1.25 mg kg ⁻¹ ) selectively induced secretion of annexin 1 by peritoneal leukocytes, as assessed by incubating these cells in culture medium. Annexin 1 secretion was also induced in human cultured monocytes, in vitro , by 10 ⁻⁶ m dexamethasone.
Annexin 1 secretion was inhibited in the presence of 20 mM NH 4 C1 or by conducting the experiments at 18°C. In contrast, it was not inhibited by monensin, nocodazole or brefeldin A.
The time necessary for annexin 1 synthesis and secretion was less than 15 min.
These data indicate that glucocorticoids induce annexin 1 secretion by monocytes or peritoneal leukocytes. Because it is not inhibited by monensin, nocodazole or brefeldin A and it is rapid, annexin 1 secretion seems to occur by the secretory pathway similar to that used by several cytosolic proteins such as interleukin‐iβ.
This study investigates the effect of dexamethasone on leukocyte extravasation in the post-capillary venules of the hamster cheek pouch, using an intravital microscopy technique, and seeks to clarify the potential involvement of the steroid-inducible protein lipocortin 1. Topical application of FMLP (10 nmol), or substance P (10 nmol), to the superfused cheek pouch induced at the level of the post-capillary venules the three characteristic phenomena of leukocyte rolling, adhesion, and transmigration. Pretreatment of hamsters with an anti-inflammatory dose of dexamethasone (1 mg/kg) increased lipocortin 1 levels in circulating leukocytes as assessed by flow cytometry, but did not modify either leukocyte rolling or the number of adherent cells; however approximately 65% of the adherent leukocytes subsequently detached and returned to the blood stream, whereas those that entered into the diapedesis process exhibited a long latency (approximately three- to fourfold longer than in control animals) before transmigration. In hamsters passively immunized with a polyclonal anti-lipocortin 1 serum, leukocyte diapedesis started at similar times in both control and dexamethasone-treated animals, whereas a significant prolongation was observed in those animals treated with a non-immune sheep serum. These observations indicate that 1) lipocortin 1 is elevated in circulating leukocytes following dexamethasone treatment; 2) the step of leukocyte extravasation affected by dexamethasone in the actual transmigration process, and 3) this specific effect upon leukocyte diapedesis is mediated by endogenous lipocortin 1.
Objective:
The acute antiinflammatory effects of methotrexate are mediated, at least in part, by increased extracellular adenosine concentrations at inflamed sites. This observation suggests that other agents that increase extracellular adenosine concentrations might also reduce inflammation. Since adenosine can be rapidly taken up by cells, phosphorylated by adenosine kinase, and maintained intracellularly as adenine nucleotides, we investigated whether a potent inhibitor of adenosine kinase, GP-1-515, could increase exudate adenosine concentration and thereby diminish inflammation in the murine air pouch model of inflammation.
Methods:
We studied the effect of various oral doses of GP-1-515 on carrageenan-induced inflammation in air pouches induced on BALB/c mice. Adenosine concentration in pouch exudates was determined by high performance liquid chromatography, and intensity of inflammation was determined by leukocyte counts in the exudate fluid.
Results:
There was a greater concentration of adenosine in the pouch exudates of animals treated with GP-1-515 than of those treated with saline (P < 0.002). GP-1-515 inhibited, in a dose-dependent manner (P < 0.01), leukocyte accumulation in the murine air pouch in response to carrageenan. Inhibition of inflammation by GP-1-515 in this model depended upon increased adenosine concentration in the inflamed pouch since injection of adenosine deaminase into the air pouch with the carrageenan completely reversed the antiinflammatory effects of GP-1-515 at all doses of GP-1-515 tested. Moreover, as previously demonstrated, the antiinflammatory effects of adenosine were mediated via occupancy of adenosine A2 receptors, since the specific adenosine A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine, but not the A1 receptor antagonist 8-cyclopentyl-dipropylxanthine, completely reversed the antiinflammatory effects of GP-1-515. GP-1-515 also decreased tumor necrosis factor alpha levels in the air pouch exudates by 51%, most likely as a result of the direct action of adenosine on macrophages.
Conclusion:
These results indicate that the antiinflammatory actions of GP-1-515 are mediated by adenosine. The development of agents that promote adenosine release at sites of inflammation is a novel strategy for the treatment of inflammatory diseases such as rheumatoid arthritis.
There is growing evidence that nitric oxide (NO.), a biologically active gas continuously produced by endothelium, is a homeostatic regulator of leukocyte adhesion in the microcirculation. Inhibition of NO. production leads to increased leukocyte rolling and adhesion in various vascular beds and two adhesion molecules, P-selectin and CD11/CD18, have been implicated in these processes. The role of mast cells and mast cell-derived mediators as potential contributors to the increased adhesion are discussed in this review. Moreover, oxidants may initiate the leukocyte recruitment after NO. synthesis inhibition. Recent data demonstrating increased oxidative stress in endothelium deprived of NO. are summarized. The role of NO. as an anti-inflammatory and antiadhesive modulator in postischemic venules of various organs is also discussed. The beneficial effect of NO. donors in this inflammatory condition is summarized. Finally, the potential use of NO. donating drugs in concert with available pharmaceutical compounds to reduce inflammation is reviewed.
We examined the effects of taprostene (2.5 x 10(-8) M to 1 x 10(-7) M), a stable prostacyclin analog, on PMN-endothelial interaction (i.e., adherence) and subsequent vasocontraction and endothelial dysfunction. Taprostene effectively inhibited the adherence of leukotriene B4-stimulated autologous cat polymorphonuclear (PMN) leukocytes to isolated cat coronary artery endothelium. Taprostene also inhibited coronary artery vasocontraction to leukotriene B4-stimulated PMNs (p < 0.01). In isolated coronary artery rings stimulated with either 2 U/ml of thrombin or 100 microM hydrogen peroxide (H2O2), adherence of unstimulated PMNs to coronary endothelium was significantly increased, resulting in vasocontraction and subsequent endothelial dysfunction. However, taprostene (1 x 10(-7) M) significantly attenuated unstimulated PMN adherence to stimulated coronary endothelium. This antiadherence action effectively attenuated PMN-induced coronary artery vasocontraction (p < 0.01) and significantly blunted the subsequent PMN-induced endothelial dysfunction (p < 0.01) characterized by a loss of endothelium-derived nitric oxide (NO). Thus, taprostene exerts a profound inhibitory effect on PMN-endothelium interaction and subsequent PMN-mediated coronary endothelial dysfunction, which may be beneficial in ischemia-reperfusion and other inflammatory states.
A novel Ca(2+)-binding protein, which we have named S100C (Ohta et al. (1991) FEBS Lett. 295, 93-96), was purified to homogeneity from porcine heart by Ca(2+)-dependent dye-affinity chromatography. S100C possesses some properties of S100 proteins, such as self-association and exposure of a hydrophobic site upon binding of Ca2+ but it differs from S100 proteins in forms of its isoelectric point (pI = 6.2), cross-reactivity with antibodies, staining by Stains-all, and its Ca(2+)-dependent interaction with the immobilized dye. S100C bound to cytoskeletal components at physiological concentrations of Ca2+. Moreover, it was found that 125I-labeled S100C interacted with annexin I in a Ca(2+)-dependent manner. S100C also inhibited the phosphorylation of annexin I by protein kinase C. These data suggest that S100C might act to regulate the cytoskeleton in a Ca(2+)-dependent manner via interactions with annexin I.
An early event associated with neutrophil-dependent tissue damage involves the adhesion of neutrophils to the vascular endothelium and the subsequent release of oxygen-derived free radicals and granule constituents. Elevations in intracellular cAMP are known to inhibit free radical release but not lysosomal enzyme release. The role of cAMP in FMLP-induced neutrophil adhesion was examined in this study by using an in vitro model of neutrophil-endothelial cell adhesion. FMLP stimulated a time- and concentration-dependent increase in human neutrophil adhesion to HUVEC. FMLP-mediated adhesion was inhibited by a diverse group of cAMP modulators: forskolin, isoproterenol, phosphodiesterase IV inhibitors (rolipram and Ro 20-1724), but not phosphodiesterase III inhibitors (milrinone and bemoradan). Endogenous adenosine has previously been shown to mediate FMLP-induced increases in cAMP enhanced in the presence of Ro 20-1724. In this study, adenosine deaminase prevented the inhibitory effects observed with rolipram and Ro 20-1724, implicating endogenous adenosine as a co-modulator of inhibition. FMLP stimulated neutrophil shape change and the surface expression of the beta 2 integrins CD11b/CD18 and CD11a/CD18. Both these responses were inhibited by rolipram but not bemoradan. With the use of 4,4'-diisothiocyanostilbene-2,2'disulfonic acid, we showed that mobilization of the intracellular pool of CD11b/CD18 paralleled adhesion. We conclude that neutrophil-endothelial cell adhesion is attenuated by elevating neutrophil intracellular cAMP and that inhibition of neutrophil CD11b/CD18 surface expression by cAMP accounts for this observed inhibition of adhesion.
The annexins are Ca(2+)-regulated, phospholipid-binding proteins which have been suggested to take part in cellular events such as exocytosis. The subcellular localization of annexins in human neutrophils was determined using monoclonal antibodies against annexins I, II, IV and VI and a polyclonal peptide antiserum against an annexin consensus sequence. Several annexins were translocated to the light membrane fraction enriched in plasma membranes and secretory vesicles. Annexins were associated also with the azurophil and specific granules. Whereas annexins I, IV and VI and one unidentified 35 kDa protein translocated to each of the isolated organelles, annexin II, a 66 kDa annexin IV-like protein, and a 38 kDa annexin I-like protein exhibited organelle-related differences in their association with membranes. The 38 kDa annexin associated only with specific granules and the secretory vesicles/plasma membrane but not with azurophil granules. Annexin II and the 66 kDa annexin IV-like protein associated with each of the neutrophil organelles, but the binding to specific granules and secretory vesicles/plasma membrane showed a Ca(2+)-dependency different from that of azurophil granules. This observation suggests that these proteins may contribute to the secretory process in neutrophils.
Lipocortin (annexin) 1 is a putative mediator of the inflammatory effects of glucocorticoids. By flow cytometric analysis (FACS) we have studied the effect of dexamethasone on the cellular localization of lipocortin 1. U-937 cells were incubated with or without 10 nM phorbol 12-myristate 13-acetate (PMA) to induce cell differentiation. Then 1 microM dexamethasone was added and incubation carried out for increasing times (1-24 h). Dexamethasone caused a time-dependent biphasic translocation of lipocortin 1 from the intracellular compartment to the cell membrane with maximal membrane expression at 4 and 24 h. In differentiated U-937 cells the steroid-induced membrane accumulation of lipocortin 1 was significantly higher than that of undifferentiated cells. The accumulation of the protein in the cell membrane may precede its release which is stimulated by dexamethasone in differentiated U-937 cells. Since extracellular lipocortin 1 has anti-inflammatory properties the modulation of the translocation/secretion process of the protein by glucocorticoids may be part of their mechanism of action.
Since polymorphonuclear leukocytes (PMN) rapidly migrate across the endothelial barrier and attach to extracellular matrix components, we tested the hypothesis that adhesion of PMN to matrix proteins can mediate endothelial injury following PMN activation. Studies were made using gelatin- and fibronectin-coated polycarbonate microporous filters (10 microns thick) on which confluent monolayers of bovine pulmonary microvessel endothelial cells were grown. PMN were layered either directly onto endothelial cells (at a ratio of 10:1) ("upright system") or onto gelatin- and fibronectin-coated filters with the endothelial monolayer grown on the underside of the filter without contact between PMN and endothelial cells ("inverted system"). PMN were activated with phorbol 12-myristate 13-acetate (PMA; 5 x 10(-9) M) in both systems. PMN activation increased endothelial permeability to 125I-labeled albumin in upright as well as inverted systems. Pretreatment of PMN with anti-CD18 monoclonal antibodies IB4 or R15.7, which inhibited PMN adherence to matrix constituents as well as to endothelial cells, prevented the permeability increase in both configurations. This effect of anti-CD18 monoclonal antibodies (mAbs) was not ascribed to a reduction in PMN activation, since PMA-induced superoxide generation was unaffected. We conclude that activation of PMN adherent to extracellular matrix proteins increases endothelial permeability to albumin and that this response is dependent on PMN adhesion to the matrix. The results support the concept that PMN-mediated increase in endothelial permeability is the result of "targeted" release of PMN products independent of whether the PMN are adherent to the extracellular matrix or the endothelium.
The effects of a competitive neutrophil elastase (NE) inhibitor, ONO-5046, and a recombinant human superoxide dismutase on leukotriene B4 (LTB4)-induced polymorphonuclear leukocyte (PMN)-mediated increase in microvascular permeability in isolated non-blood-perfused rabbit lungs were studied. Pulmonary microvascular permeability and lung edema were evaluated by use of the fluid filtration coefficient (Kf) and the wet-to-dry lung weight ratio (W/D), respectively. Pulmonary capillary pressure was estimated by the double occlusion technique. NE activity in the perfusate was determined using a spectrophotometric method. The PMNs (2-3 x 10(8) cells) were added into the perfusate in all groups of lungs. Injection of LTB4 (5 micrograms) increased Kf and W/D without affecting pulmonary arterial or capillary pressure. The LTB4-induced lung injury was closely associated with the increase in NE activity in the perfusate. Infusion of ONO-5046 (1 or 10 mg.kg-1 x h-1) inhibited the LTB4-induced increases in Kf, W/D, and perfusate NE activity in a dose-dependent fashion. Infusion of recombinant human superoxide dismutase (80,000 U.kg-1 x h-1) attenuated the LTB4-induced increases in Kf and W/D, although it did not influence the elevation of perfusate NE activity induced by LTB4. These results suggest that both NE and superoxide anion play important roles in the LTB4-induced PMN-mediated increase in pulmonary microvascular permeability.
Adenosine receptors are present on most cells and organs, yet, although the physiological effects of adenosine were first described over 60 years ago, the potential therapeutic uses of adenosine have only been recognized and realized recently. A decade ago the potent anti-inflammatory effects of adenosine were first described; adenosine, acting at specific A2 receptors, inhibits some, but not all, neutrophil functions. Adenosine inhibits phagocytosis, generation of toxic oxygen metabolites, and adhesion (to some surfaces and to endothelial cells) but does not inhibit degranulation or chemotaxis. Occupancy of adenosine A2 receptors modulates leukocyte function by a novel mechanism. Although adenosine A2 receptors are classically linked to heterotrimeric GS signaling proteins and stimulation of adenylate cyclase, adenosine 3',5'-cyclic monophosphate does not act as the second messenger for inhibition of leukocyte function. By a mechanism that still remains obscure, occupancy of adenosine A2 receptors on neutrophils "uncouples" chemoattractant receptors from their stimulus-transduction proteins. The concentrations of adenosine that inhibit inflammatory cell function are similar to those observed in vivo and suggest a role for adenosine in the modulation of inflammation in vivo. Indeed, recent studies indicate that nonmetabolized adenosine receptor agonists are potent anti-inflammatory agents, and other studies indicate that methotrexate, a commonly used anti-inflammatory agent, diminishes inflammation by increasing adenosine release at inflamed sites. The observations reviewed here suggest that adenosine and agents that act through adenosine are excellent candidates for development as anti-inflammatory agents.
The accumulation of leukocytes in inflamed tissue results from adhesive interactions between leukocytes and endothelial cells within the microcirculation. These adhesive interactions and the excessive filtration of fluid and protein that accompanies an inflammatory response are largely confined to one region of the microvasculature: postcapillary venules. The nature and magnitude of the leukocyte-endothelial cell adhesive interactions that take place within postcapillary venules are determined by a variety of factors, including expression of adhesion molecules on leukocytes and/or endothelial cells, products of leukocyte (superoxide) and endothelial cell (nitric oxide) activation, and the physical forces generated by the movement of blood along the vessel wall. The contribution of different adhesion molecules to leukocyte rolling, adherence, and emigration in venules is discussed. Emerging views on potential endogenous antiadhesion molecules produced by endothelial cells as well as the influence of alterations in shear rate on leukocyte adhesion are addressed. Finally, the pathophysiological significance of the microvascular responses to inflammation are discussed in terms of adhesion-directed strategies for the treatment of different cardiovascular diseases and circulatory disorders.
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.
Methotrexate (MTX) reduces neutrophil adhesion to endothelial cell monolayers, possibly via stimulation of adenosine production. However, it remains unclear whether adenosine participates in the anti-inflammatory actions of MTX in postcapillary venules.
Leukocyte-endothelial cell adhesive interactions were measured in rat mesenteric venules (25-35 microns diameter) during superfusion with either bicarbonate-buffered saline (BBS) alone, BBS combined with platelet-activating factor (PAF), or BBS combined with leukotriene B4 (LTB4). In some experiments, either MTX or adenosine was added to a superfusate containing either PAF or LTB4. In other experiments, either adenosine deaminase (ADA), an adenosine A1-receptor antagonist, or an A2-receptor antagonist was added to a superfusate containing PAF and either MTX or adenosine.
Both MTX and adenosine were effective in preventing the leukocyte-endothelial cell adhesive interactions elicited by PAF, but not by LTB4. These actions of adenosine and MTX against PAF-induced leukocyte adhesion were blunted by ADA and the A2-(but not the A1-) receptor antagonist.
These results indicate that both adenosine and methotrexate attenuate PAF-induced leukocyte-endothelial cell adhesion in postcapillary venules via activation of A2-receptors on the leukocyte.
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.
We measured changes in basal release of nitric oxide and its effect on polymorphonuclear leukocyte (PMN) adherence to endothelial cells (ECs) in a feline model of myocardial ischemia (90 minutes) and reperfusion. Basal release of nitric oxide from the left anterior descending coronary artery (LAD) after myocardial ischemia/reperfusion and from the control left circumflex coronary artery (LCX) was assessed by NG-nitro L-arginine methyl ester (L-NAME)-induced vasocontraction. L-NAME induced a significant EC-dependent vasocontraction in control LCX rings (0.28 +/- 0.04 g), which was fully reversed by L-arginine but not D-arginine. L-NAME-induced vasocontraction of LAD rings was not significantly changed after 90 minutes of myocardial ischemia without reperfusion. However, 10 minutes of reperfusion reduced the L-NAME-induced vasocontraction to 0.13 +/- 0.04 g (p < 0.05), and this was restored by addition of 3 mM L-arginine but not D-arginine. Longer periods of reperfusion progressively decreased L-NAME-induced vasocontraction. After 270 minutes of reperfusion, L-NAME-induced vasocontraction was virtually abolished. Myocardial ischemia without reperfusion did not increase PMN adherence to ECs. However, PMN adherence to LAD ECs was significantly increased after 20 minutes of reperfusion (39 +/- 6 to 105 +/- 9 PMNs/mm2, p < 0.01), and incubation of LAD segments with L-arginine significantly attenuated this increase in PMN adherence. After 270 minutes of reperfusion, PMN adherence to LAD ECs was further increased to 224 +/- 10 PMNs/mm2 (p < 0.001). This increase in PMN adherence was almost completely blocked by MAb R15.7, a monoclonal antibody against CD18 of PMNs, and was significantly attenuated by MAb RR1/1, a monoclonal antibody against intercellular adhesion molecule-1 of ECs (p < 0.01). These results indicate that decreased basal release of endothelium-derived relaxing factor after myocardial ischemia/reperfusion precedes enhanced PMN adherence to the coronary endothelium, which may lead to PMN-induced myocardial injury.
IL-1 is a pro-inflammatory cytokine which controls many features of the immune and inflammatory response. When injected into a mouse 6-day-old air-pouch, human rIL-1 (1 to 100 ng) induced in a dose-dependent fashion a migration of PMN that could be reliably assessed 4 h after injection. Both IL-1 alpha and IL-1 beta were active in this model. The effect of the cytokine was inhibited by local administration of actinomycin D (1 to 10 micrograms), alpha-melanocyte-stimulating hormone (200 micrograms), and a mAb recognizing IL-1R type I (10 micrograms). Indomethacin (1 mg/kg), an inhibitor of cyclo-oxygenase, and BW4AC (2 mg/kg), a selective lipoxygenase inhibitor, were without effect but moderate inhibition was seen with the platelet-activating factor antagonist WEB2086 (1 to 10 mg/kg). The glucocorticoid dexamethasone (0.015 to 1.5 mg/kg) potently inhibited the elicitation of neutrophils induced by IL-1 when given systemically 2 h before the cytokine. The steroid-induced anti-inflammatory protein lipocortin 1 (LC1) also produced a dose-dependent inhibition of PMN migration into the pouch with an ED50 of approximately 0.15 to 0.21 mg/kg. The denatured protein was without effect. Passive immunization of mice with a polyclonal sheep antiserum or a mAb raised against LC1 abolished the inhibitory action of dexamethasone whereas preimmune serum or control IgG were without significant effect. These findings provide further evidence that LC1 is involved in the anti-inflammatory action of glucocorticosteroids and suggest that this protein may act as an endogenous regulator of IL-1 action.
We have studied the occurrence, distribution and disposition of lipocortins (annexins) 1, 2 and 5 in mixed peritoneal leucocytes obtained from rats in which glucocorticoid levels were altered by adrenalectomy, administration of the glucocorticoid antagonist, RU486, or by injection of dexamethasone or hydrocortisone, as well as from rats in which the peritoneal cells were elicited by inflammatory stimuli.
In cells obtained from untreated rats with an intact adrenal cortex, lipocortins 1, 2 and 5 were readily detectable: the majority of each of the proteins was apparently located intracellularly with much smaller amounts in the membrane. Lipocortin 1 and to a lesser extent lipocortin 5 were also seen in a Ca ²⁺ ‐dependent association with the external plasma membrane. Following administration of RU486 (2 × 20 mg kg ⁻¹ ) the amounts of lipocortin 1 and 2 in cells were greatly reduced. Conversely, injection of hydrocortisone (1 mg kg ⁻¹ ) or dexamethasone (0.08 mg kg ⁻¹ ) caused an increase in the amount of lipocortin 1 and 2 in peritoneal cells within 30 min. Lipocortin 5 was unchanged by any manipulation of glucocorticoid levels.
Lipocortins 1 and 2 were elevated in both intracellular and membrane‐associated fractions of macrophages elicited by intraperitoneal injection in inflammogens. This phenomenon also occurred in adrenalectomized animals.
Our data indicate that glucocorticoids control the synthesis of some members of the lipocortin family in rat mixed peritoneal cells but also suggest the existence of a separate system for controlling the generation of this protein. The significance of these observations is considered in relation to the mechanism of glucocorticoid hormone action on eicosanoid production.
We previously reported that mast cell degranulation causes histamine and P-selectin-dependent leukocyte rolling and platelet-activating factor (PAF)- and CD18-associated leukocyte adhesion, whereas others have reported serotonin-induced edema formation. The purpose of the present study was to determine whether nitric oxide (NO) could inhibit the mast cell-induced multistep recruitment of leukocytes and the associated microvascular dysfunction in single inflamed venules.
Intravital fluorescence microscopy was used to demonstrate increased leukocyte rolling and adhesion and increased albumin extravasation in single 25- to 40-microns venules that were treated with the mast cell-degranulating agent compound 48/80 (CMP 48/80). The mast cell-induced histamine-dependent rolling and PAF-dependent adhesion were completely inhibited by the addition of the NO donor spermine NO. However, spermine NO did not directly inhibit histamine-induced leukocyte rolling and only partly affected PAF-induced leukocyte adhesion. Compound 48/80-activated mast cells evoked a significant increase in PAF-dependent neutrophil adhesion in vitro. Spermine-NO prevented the mast cell-dependent neutrophil adhesion but failed to affect direct adhesion with PAF. The mast cell-induced albumin leakage was also inhibited by the NO donor.
Taken together, these results suggest that exogenous NO can modulate leukocyte recruitment and microvascular permeability alterations elicited by mast cell activation and raises the possibility that the use of NO donors may be a reasonable therapeutic approach to reducing mast cell-dependent inflammation.
The effect of systemic treatment of mice with murine recombinant interleukin‐4 (IL‐4) or interleukin‐10 (IL‐10) on neutrophil infiltration into a specific tissue site and nitric oxide (NO) production from peritoneal macrophages was investigated.
Intravenously (i.v.) administered IL‐4 (0.01–10 μg per mouse, approximately 0.3–300 μg kg ⁻¹ , i.v.) and IL‐10 (0.01‐1 μg per mouse, approximately 0.3–30 μg kg ⁻¹ , i.v.) dose‐dependently inhibited neutrophil accumulation into a 6‐day‐old murine air‐pouch induced by local application of interleukin‐1β (IL‐1β, 5 ng), with approximate ED 50 s of 0.35 and 0.90 μg, respectively. Neither IL4 (1 μg, 30 μg kg ⁻¹ , i.v.) nor IL‐10 (1 μg, 30 μg kg ⁻¹ , i.v.) prevented leucocyte accumulation in the mouse air‐pouches when interleukin‐8 (IL‐8, 1μg.) was used as chemoattractant. Similarly, neither cytokine had any effect on the in vitro up‐regulation of CD11b antigen on the surface of murine circulating neutrophils.
Treatment of mice with lipopolysaccharide (LPS, 0.3 mg kg ⁻¹ , i.p.) caused an increase in the formation of NO (measured as nitrite accumulation) in the supernatant of peritoneal macrophages ex vivo . Pretreatment of mice with IL4 (0.01‐1 μg i.v., 20 min before LPS), but not with IL‐10 (1 μg i.v., 20 min before LPS), caused a dose‐dependent reduction in this LPS‐stimulated formation of nitrite by peritoneal macrophages ex vivo .
Activation of murine macrophages with LPS (1 μg ml ⁻¹ for 24 h) in vitro caused a significant increase in nitrite release in the supernatant of these cells. Pretreatment of either J774.2 or peritoneal macrophages with IL4 (0.1‐1 μg ml ⁻¹ , 20 min before LPS), but not with IL‐10 (1 μg ml ⁻¹ , 20 min before LPS) caused a concentration‐related attenuation of this LPS‐stimulated nitrite formation.
Thus, both IL‐4 and IL‐10 inhibit the migration of leucocytes (stimulated by IL‐1β) in vivo; IL‐4 (but not IL‐10) inhibits the induction of NO synthase caused by LPS in murine macrophages in vitro and ex vivo .