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Antiflammin-2 prevents HL-60 adhesion to endothelial cells and prostanoid production induced by lipopolysaccharides
Abstract
We studied the effect of antiflammin-2 (AF-2) on adhesion molecule expression by HL-60 cells and endothelial (ECV304) cells stimulated by lipopolysaccharides (LPSs), and on leukocyte-endothelial cell interaction in an in vitro coculture system. The action of AF-2 on prostanoid production in these experimental conditions was also tested. LPS increased the adhesion molecule expression, such as lymphocyte function-associated antigen-1 and membrane attack complex-1 on HL-60 cells and E-selectin and intercellular adhesion molecule-1 on ECV304 cells. The LPS-stimulated adhesion molecule expression on HL-60/ECV304 coculture system was higher than on HL-60 or ECV304 cultures. LPS also induced HL-60 adhesion to ECV304 monolayer and thromboxane B(2) and prostaglandin E(2) (PGE(2)) production in HL-60 culture and PGE(2) in ECV304 culture. Prostanoid production by HL-60/ECV304 cocultures was higher than by simple cultures. AF-2 inhibited the enhancement of adhesion molecule expression induced by LPSs, especially E-selectin. Thus, AF-2 significantly reduced the HL-60 adhesion to endothelial cells stimulated by LPSs. AF-2 also inhibited prostanoid synthesis by ECV304 cells or HL-60/ECV304 coculture challenged by LPSs. In conclusion, AF-2 reduced HL-60 adhesion to endothelial cells, suggesting that it reduces inflammation by blocking leukocyte trafficking and the subsequent eicosanoid production.
... Eselectin is usually not expressed under physiological conditions, and its transcription is triggered by cytokines like IL-1, LPS, and TNF-α [50]. As expected, treatment with 1 μg/mL LPS for 24 h augmented the expression of VCAM-1 (Fig. 5A, B), ICAM-1 (Fig. 5C, D), and E-selectin (Fig. 5E, F) on the cell surface of HUVEC cells and their concentration in culture supernatants, in agreement with many reports on the increased expression of adhesion molecules in LPS-treated HUVEC and ECV304 cells [51][52][53][54]. ...
Vascular endothelium plays an important modulatory role due to the production of molecules that mediate vasomotricity, inflammation, and leukocyte adhesion and rolling. Here we addressed whether crotoxin (25–200 μg/mL) – the main component of Crotalus durissus terrificus snake venom – interferes with cell viability, apotosis/necrosis, and cell response to oxidative stress in human umbilical vein endothelial cells (HUVEC) in vitro. We also examined whether crotoxin alters the levels of interleukins, adhesion molecules, and endothelial vasoactive factors in HUVEC cells treated or not with lipopolysaccharide (LPS; 1 μg/mL; 24 h). Crotoxin was not cytotoxic towards HUVEC cells, and downregulated the LPS-induced production of adhesion molecules (VCAM-1, ICAM-1, and E-selectin), vasoactive factors (endothelin-1 and prostaglandin I2), and interleukins (IL-6, IL-8, and IL1β), as well as protected cells against H 2 O 2 –induced oxidative stress. Hence, crotoxin played anti-inflammatory, antioxidant, immunomodulating, and vasoactive actions on HUVEC cells, in vitro. Considering that the initial stages of atherosclerosis is characterized by vasoconstriction, increased levels of adhesion molecules, inflammatory cytokines, and oxidative stress in the vascular endothelium; and crotoxin downmodulated all these events, our findings indicate that the actions of crotoxin here demonstrated suggest that it may have an anti-atherogenic action in vivo, which deserves to be tested in future studies.
... Neutrophil/endothelial interaction (in vitro) ↓ PMN capture, rolling, and adhesion ↑ L-selectin shedding [40,54] Human PMN activated with CCL5 ↓ integrin activation [40] Neutrophil Journal of Immunology Research AnxA1 may also be tightly coupled to the anti-inflammatory properties of other FPR2/ALX agonists such as LXA 4 and antiflammin 2 (AF-2) [50]. The nonapeptide AF-2, which corresponds to region 246-254 of AnxA1 [80], is known to interfere with PMN activation, chemotaxis, and adhesion to endothelial cells [60,61], via FPR2/ALX receptor [81]. Also, LXA 4 is a potent regulator of PMN trafficking in experimental inflammation [9,82]. ...
Neutrophils (also named polymorphonuclear leukocytes or PMN) are essential components of the immune system, rapidly recruited to sites of inflammation, providing the first line of defense against invading pathogens. Since neutrophils can also cause tissue damage, their fine-tuned regulation at the inflammatory site is required for proper resolution of inflammation. Annexin A1 (AnxA1), also known as lipocortin-1, is an endogenous glucocorticoid-regulated protein, which is able to counterregulate the inflammatory events restoring homeostasis. AnxA1 and its mimetic peptides inhibit neutrophil tissue accumulation by reducing leukocyte infiltration and activating neutrophil apoptosis. AnxA1 also promotes monocyte recruitment and clearance of apoptotic leukocytes by macrophages. More recently, some evidence has suggested the ability of AnxA1 to induce macrophage reprogramming toward a resolving phenotype, resulting in reduced production of proinflammatory cytokines and increased release of immunosuppressive and proresolving molecules. The combination of these mechanisms results in an effective resolution of inflammation, pointing to AnxA1 as a promising tool for the development of new therapeutic strategies to treat inflammatory diseases.
... They were obtained from the European Collection of Cell Cultures (Salisbury, UK) and grown in medium 199 with Earl's salts, 25 mM HEPES supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% FBS. Cells were split at confluence 1-3 or at higher passages 1-2 every 5-7 days and plated on gelatin-coated flasks up to passage 20 [14]. ECV304 cells were washed and incubated in normal glucose (5 mM glucose and 25 mM mannitol as an osmotic control of high glucose) or coincubated with high glucose (30 mM) in the presence or absence of 0-120 mM FFAs (oleic or linoleic acid) and 10 mM hydroxytyrosol or 10 mM gallic acid equivalents of PEEVOO for 48 h. ...
Epidemiological and clinical studies have reported that olive oil reduces the incidence of cardiovascular disease. However, the mechanisms involved in this beneficial effect have not been delineated. The endothelium plays an important role in blood pressure regulation through the release of potent vasodilator and vasoconstrictor agents such as nitric oxide (NO) and endothelin-1 (ET-1), respectively, events that are disrupted in type 2 diabetes. Extra virgin olive oil contains polyphenols, compounds that exert a biological action on endothelial function. This study analyzes the effects of olive oil polyphenols on endothelial dysfunction using an in vitro model that simulates the conditions of type 2 diabetes. Our findings show that high glucose and linoleic and oleic acids decrease endothelial NO synthase phosphorylation, and consequently intracellular NO levels, and increase ET-1 synthesis by ECV304 cells. These effects may be related to the stimulation of reactive oxygen species production in these experimental conditions. Hydroxytyrosol and the polyphenol extract from extra virgin olive oil partially reversed the above events. Moreover, we observed that high glucose and free fatty acids reduced NO and increased ET-1 levels induced by acetylcholine through the modulation of intracellular calcium concentrations and endothelial NO synthase phosphorylation, events also reverted by hydroxytyrosol and polyphenol extract. Thus, our results suggest a protective effect of olive oil polyphenols on endothelial dysfunction induced by hyperglycemia and free fatty acids.
© 2014 Published by Elsevier Ltd.
... 2–26, amino acid 2–26, AMVSEFLKQAWIENEEQEYVVQTVK; Ac. 2–12, amino acid 2–12, AMVSEFLKQAWI; and Ac. 13–26, ENEEQEYVVQTVK)12131415 or annexin repeat III-derived nonapeptide antinflammin-2 (amino acid 246–254; HDMNKVLDL) [16,17]. As we will discuss later, these are AnxA1 mimetics whose biological effects might or might not overlap with those of the endogenous protein. ...
... More importantly, a reduction by b-sitosterol in arachidonic acid release and prostanglandin E2 production, induced by oxidized low-density lipoproteins, has been observed [29]. Such data implicate possible mechanism(s) involved in the regulatory effects of b-sitosterol in adhesion molecule expression, cell-cell interaction and inflammation [30]. However, De Jong et al. [31] demonstrated that b-sitosterol did not modify soluble adhesion molecules, neither did it affect markers of antioxidant status or oxidative stress, despite a significant reduction in LDL cholesterol in patients treated with statins. ...
beta-Sitosterol, normally present in vegetable-containing diets, comprises an important component of cholesterol controlling functional foods. It has been associated with cardiovascular protection, exerting its effect mainly through increasing the antioxidant defense system and effectively lowering the serum cholesterol levels in humans. However, its anti-inflammatory effect on endothelium is unknown. Attachment of leukocytes to the vascular endothelium and the subsequent migration of cells into the vessel wall are early events in atherogenesis, this process requiring the expression of endothelial adhesion molecules. We examined the effect of beta-sitosterol (0.1-200 microM) on (i) the expression of vascular adhesion molecule 1 and intracellular adhesion molecule 1 by cell ELISA and (ii) the attachment of monocytes (U937 cells) in tumor necrosis factor-alpha (TNF-alpha)-stimulated human aortic endothelial cells (HAECs) by adhesion assay. The effect on nuclear factor-kB phosphorylation was also examined via a cell-based ELISA kit. Results showed that beta-sitosterol inhibits significantly vascular adhesion molecule 1 and intracellular adhesion molecule 1 expression in TNF-alpha-stimulated HAEC as well as the binding of U937 cells to TNF-alpha-stimulated HAEC and attenuates the phosphorylation of nuclear factor-kB p65. This study extends existing data regarding the cardioprotective effect of beta-sitosterol and provides new insights into understanding the molecular mechanism underlying the beneficial effect of beta-sitosterol on endothelial function.
... There are two possible explanations. MGOinduced COX-2-PGE 2 was related to other ECs inflammatory states such as hyperpermeability (6,19) and/or increased leukocyte-ECs interaction (12,21) rather than direct ECs morphological damage. Alternatively, MGO might directly interact with DNA and damage the cell, since it is suggested that MGO mediates DNA glycation, which may be associated with mutation, DNA strand breaks, and cytotoxicity (32). ...
Methylglyoxal (MGO) is a reactive metabolite of glucose. Since the plasma concentration of MGO is increased in diabetic patients, MGO is implicated in diabetes-associated vascular endothelial cells (ECs) injury, which might be responsible for atherosclerosis. In the present study, we examined effects of treatment of human umbilical vein ECs with MGO on EC morphology and inflammatory responses. MGO (24 h) induced cytotoxic morphological changes in a concentration-dependent manner (0-420 microM). MGO induced mRNA and protein expression of cyclooxygenase (COX)-2 in a concentration (0-420 microM)- and time (6-24 h)-dependent manner. COX-2 induction was associated with increased PGE(2) release. Acute treatment with MGO (20 min) induced concentration-dependent (0-420 microM) activation of JNK and p38 MAP kinase but not ERK or NF-kappaB. Both the JNK inhibitor SP600125 and the p38 inhibitor SB203580 prevented the MGO induction of COX-2. However, inhibiting JNK and p38 or COX-2 was ineffective to the morphological damage by MGO (420 microM, 24 h). EUK134, a synthetic combined superoxide dismutase/catalase mimetic, had no effect on MGO-induced COX-2. Present results indicated that MGO mediates JNK- and p38-dependent EC inflammatory responses, which might be independent of oxidative stress. On the other hand, MGO-induced morphological cell damage seems unlikely to be associated with COX-2-PGE(2).
... Two studies demonstrated the lack of effect of ANXA1 and its peptides (both Ac2-26 and AF-2) on endothelial cell adhesion molecule expression, despite reporting their ability to inhibit neutrophil adhesion molecule expression (hence cell adhesion) in co-culture conditions , Zouki et al. 2000. However, another study has reported that AF-2 inhibits prostanoid generation by an endothelial cell line (ECV340 cells) stimulated with lipopolysaccharide (Moreno 2001). ...
The concept of anti-inflammation is currently evolving with the definition of several endogenous inhibitory circuits that are important in the control of the host inflammatory response. Here we focus on one of these pathways, the annexin 1 (ANXA1) system. Originally identified as a 37 kDa glucocorticoid-inducible protein, ANXA1 has emerged over the last decade as an important endogenous modulator of inflammation. We review the pharmacological effects of ANXA1 on cell types involved in inflammation, from blood-borne leukocytes to resident cells. This review reveals that there is scope for more research, since most of the studies have so far focused on the effects of the protein and its peptido-mimetics on neutrophil recruitment and activation. However, many other cells central to inflammation, e.g. endothelial cells or mast cells, also express ANXA1: it is foreseen that a better definition of the role(s) of the endogenous protein in these cells will open the way to further pharmacological studies. We propose that a more systematic analysis of ANXA1 physio-pharmacology in cells involved in the host inflammatory reaction could aid in the design of novel anti-inflammatory therapeutics based on this endogenous mediator.
... Indeed, structural analysis revealed that, in the presence of calcium ions, the AF-2 sequence would be exposed on the ANXA1 protein surface[16], therefore potentially being able to mediate or sustain protein-protein interactions. Out of three members so far described within this family, and termed FPR, FPRL-1, and FPRL-2, our analysis was restricted to the former two, since expressed by the human neutrophils[17], a cell type susceptible to AF-2 inhibitory effects[6,18]. ...
The anti-inflammatory actions of the nonapeptide antiflammin-2, identified by homology with uteroglobin and annexin-A1 sequences, have been described in some detail, yet its mechanisms of action remain elusive. Since recent data indicate an involvement of the formyl peptide receptor (FPR)-like 1 (or FPRL-1) in the effects of annexin-A1, we have tested here the effect of antiflammin-2 with respect to this receptor family. Using HEK-293 cells expressing either human FPR and FPRL-1, and an annexin-A1 peptide as tracer ([125I-Tyr]-Ac2-26), we found that antiflammin-2 competed for binding only at FPRL-1, and not FPR, with an approximate EC50 of 1 mM. In line with data produced for the full-length protein, genuine receptor activation by antiflammin-2 was confirmed by rapid phosphorylation of extracellular-regulated kinase 1 and 2. Finally, study of the neutrophil interaction with activated endothelium under flow demonstrated an inhibitory effect of antiflammin-2, thus providing functional support to a role for the antiflammin-2/FPRL-1 anti-inflammatory axis.
The objective of the present study is to observe the anti-inflammatory and antioxidant effects of Tripterygium wilfordii extract on LPS-induced acute lung injury (ALI) in rats, and to explore its protective mechanism against ALI. And quantitative determination was performed on the extract. 120 rats were randomly divided into normal control group (saline), model group, dexamethasone group (5 mg/kg, ip), and Tripterygium wilfordii extract low-, mediumand high-dose groups (100, 150, 200 mg/kg, ip), n = 20 in each group. The rats were administered for 3 d, 1 h after the last ip administration, ALI model was induced by ip endotoxin (LPS), 6 h later, the rats were sacrificed, BALF WBC count and protein content, nuclear transcription factor (NF-κB) p65 expression in lung tissue, levels of tumor necrosis factor (TNF-α), interleukin (IL)-6, interleukin (IL)-10, superoxide dismutase (SOD), and malondialdehyde (MDA), as well as neutrophil myeloperoxidase (MPO) activity in each group were observed. Total triterpenoid content in Tripterygium wilfordii extract was determined by UV-V is spectrophotometry with tripterine as the reference. Compared with the control group, in the Tripterygium wilfordii extract medium- and high-dose groups, lung tissue NF-κB p65 expressions were (38.03 ± 4.19)% and (30.69 ± 3.99)%, MDA levels were (1.76 ± 0.21) and (1.36 ± 0.12) nmol/mg, TNF-α levels were (263.66 ± 39.65) and (210.78 ± 30.25) μg/L, IL-6 levels were (289.47 ± 99.99) and (226.58 ± 96.25) ng/L, and MPO activities (2.76 ± 0.54) and (2.43 ± 0.42) U/g, all of which were significantly lower (P<0.05~P<0.01) than those of the model group; IL-10 levels were (17.89 ± 3.89) and (17.29 ± 3.68) μg/L, SOD levels were (69.47 ± 9.23) and (73.65 ± 8.12) U/mg, BALF WBC count and protein content in the model group significantly increased (P<0.05~P<0.01); all of which were significantly higher (P<0.05~P<0.01) than those of the model group. Total triterpenoid content in Tripterygium wilfordii extract was 27.379. Tripterygium wilfordii extract can decrease the pulmonary vascular permeability caused by LPS-induced ALI, reduce inflammation exudation, and lower the degree of oxidative stress injury.
The importance of proresolving mediators in the overall context of the resolution of acute inflammation is well recognized, although little is known about whether these anti-inflammatory and proresolving molecules act in concert. In this article, we focused on lipoxin A(4) (LXA(4)) and annexin A1 (AnxA1) because these two very different mediators converge on a single receptor, formyl peptide receptor type 2 (FPR2/ALX). Addition of LXA(4) to human polymorphonuclear leukocytes (PMNs) provoked a concentration- and time-dependent mobilization of AnxA1 onto the plasma membrane, as determined by Western blotting and flow cytometry analyses. This property was shared by another FPR2/ALX agonist, antiflammin-2, and partly by fMLF or peptide Ac2-26 (an AnxA1 derivative that can activate all three members of the human FPR family). An FPR2/ALX antagonist blocked AnxA1 mobilization activated by LXA(4) and antiflammin-2. Analysis of PMN degranulation patterns and phospho-AnxA1 status suggested a model in which the two FPR2/ALX agonists mobilize the cytosolic (and not the granular) pool of AnxA1 through an intermediate phosphorylation step. Intravital microscopy investigations of the inflamed mesenteric microvasculature of wild-type and AnxA1(-/-) mice revealed that LXA(4) provoked leukocyte detachment from the postcapillary venule endothelium in the former (>50% within 10 min; p < 0.05), but not the latter genotype (∼15%; NS). Furthermore, recruitment of Gr1(+) cells into dorsal air-pouches, inflamed with IL-1β, was significantly attenuated by LXA(4) in wild-type, but not AnxA1(-/-), mice. Collectively, these data prompt us to propose the existence of an endogenous network in anti-inflammation centered on PMN AnxA1 and activated by selective FPR2/ALX agonists.
We have examined the role of platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) during the transendothelial migration of melanoma cells using a novel in vitro system. Comparable studies have suggested the involvement of PECAM-1 in leukocyte transendothelial migration. Such studies have been confirmed using in vivo models of inflammation. These studies prompted us to examine the role of PECAM-1 in tumor cell transendothelial migration. Anti-PECAM-1 monoclonal antibodies, known to block leukocyte transendothelial migration, were tested in co-cultures of human melanoma cells seeded on a monolayer of human lung microvascular endothelial cells. None of these antibodies inhibited the transmigration of melanoma cells. Moreover, confocal microscopy revealed the dissolution of the PECAM-1 adhesion complexes in the endothelial junctions associated with melanoma cells and the lack of PECAM-1 in heterotypic contacts between transmigrating melanoma cells and adjacent endothelial cells. These data, therefore, indicate that PECAM-1 is not required for the transendothelial migration of melanoma cells.
Steroidal anti-inflammatory drugs induce proteins that inhibit phospholipase A(2) (PLA(2)), including uteroglobin and lipocortin-1 (annexin I). Uteroglobin and lipocortin-1 retain several conserved sequences. Based on these sequences, several nonapeptides (antiflammins) were synthesized. These nonapeptides were shown to have anti-inflammatory effects in vitro and in vivo, possibly by inhibiting PLA(2). Subsequent research showed that PLA(2) is activated by transglutaminase 2 (TGase 2). We hypothesize here that TGase 2 inhibitors may increase the anti-inflammatory efficacy of inhibiting PLA(2) activity. To test this theory, we constructed recombinant peptides containing sequences from pro-elafin (for inhibition of TGase 2), and from lipocortin-1, lipocortin-5, and uteroglobin (for inhibition of PLA(2)). The recombinant peptides, which had dual inhibitory effects on purified TGase 2 and PLA(2), reversed the inflammation of allergic conjunctivitis to ragweed in a guinea pig model. The present work suggests that novel recombinant peptides may be safe and effective agents for the treatment of various inflammatory diseases.
Atherosclerosis is the principal contributor to the pathogenesis of myocardial and cerebral infarction, gangrene and loss of function in the extremities. It results from an excessive inflammatory-fibroproliferative response to various forms of insult to the endothelium and smooth muscle of the artery wall. Atherosclerotic lesions develop fundamentally in three stages: dysfunction of the vascular endothelium, fatty streak formation and fibrous cap formation. Each stage is regulated by the action of vasoactive molecules, growth factors and cytokines. This multifactorial etiology can be modulated through the diet. The degree of unsaturation of dietary fatty acids affects lipoprotein composition as well as the expression of adhesion molecules and other pro-inflammatory factors, and the thrombogenicity associated with atherosclerosis development. Thus, the preventive effects of a monounsaturated-fatty acid-rich diet on atherosclerosis may be explained by the enhancement of high-density lipoprotein-cholesterol levels and the impairment of low-density lipoprotein-cholesterol levels, the low-density lipoprotein susceptibility to oxidation, cellular oxidative stress, thrombogenicity and atheroma plaque formation. On the other hand, the increase of high-density lipoprotein cholesterol levels and the reduction of thrombogenicity, atheroma plaque formation and vascular smooth muscle cell proliferation may account for the beneficial effects of polyunsaturated fatty acid on the prevention of atherosclerosis. Thus, the advantages of the Mediterranean diet rich in olive oil and fish on atherosclerosis may be due to the modulation of the cellular oxidative stress/antioxidant status, the modification of lipoproteins and the down-regulation of inflammatory mediators.
To provide some pharmacological evidence for its clinical use in inflammatory diseases, anti-inflammatory effect of the aqueous extract from Radix Ophiopogon japonicus (ROJ-ext), a traditional Chinese herb, was examined in mouse and rat models. ROJ-ext significantly inhibited xylene-induced ear swelling and carrageenan-induced paw edema in mice when given orally at doses of 25 and 50 mg/kg. Moreover, ROJ-ext also remarkably suppressed carrageenan-induced pleural leukocyte migration in rats and zymosan A-evoked peritoneal total leukocyte and neutrophil migration in mice, while had no obvious effect on pleural prostaglandin E2 level. Furthermore, two active compounds were isolated from ROJ-ext and identified as ruscogenin and ophiopogonin D. As the results, ROJ-ext, ruscogenin and ophiopogonin D dose-dependently reduced phorbol-12-myristate-13-acetate (PMA)-induced adhesion of HL-60 cells to ECV304 cells, with IC50 of 42.85 microg/ml, 7.76 nmol/l and 1.38 nmol/l, respectively. However, they showed no inhibitory effect on PMA-induced cyclooxygense-2 (COX-2) mRNA expression in ECV304 cells. Ruscogenin and ophiopogonin D also notably decreased zymosan A-induced peritoneal leukocyte migration, in comparison with ROJ-ext. These results demonstrate that ROJ-ext presents remarkable anti-inflammatory activity and ruscogenin and ophiopogonin D are two of its active components, which supported its traditional use in the treatment of various diseases associated with inflammation.
The in-vivo inhibitory effects of the ethanol extract of Radix Ophiopogon japonicus (ROJ-ext) on venous thrombosis were studied in mouse and rat models and in-vitro endothelial cell-protective and anti-adhesive activities were observed in ECV304 cells injured by sodium dithionite and HL-60 adhesion to ECV304 cells injured by TNF-alpha. The in-vivo results showed that ROJ-ext significantly inhibited venous thrombosis induced by tight ligation of the inferior vena cava for 6 h in mice and for 24 h in rats by once oral administration at doses of 12.5 and 25 mg/kg. Meanwhile, ROJ-ext had no obvious effect on some coagulation parameters, which was different from warfarin, which remarkably prolonged activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) in rats at the same time. Histological analysis under light microscope and scanning electron microscope (SEM) of inferior vena cava indicated that ROJ-ext could protect endothelial cells from anoxic injury and alleviate inflammatory changes in the vein wall. On the other hand, the in-vitro studies approved that ROJ-ext significantly enhanced viability of ECV304 cells injured by sodium dithionite at the concentrations of 0.1, 1.0 and 10 mug/ml when given before and after the anoxic induction. Meanwhile, ROJ-ext remarkably inhibited adhesion of HL-60 cells to ECV304 cells injured by rh TNF-alpha at above concentrations in a dose-dependent manner. The findings of this study showed that ethanol extract of Radix Ophiopogon japonicus (ROJ-ext) inhibited venous thrombosis, which linked with its endothelial cell-protective and anti-adhesive activities. This lends scientific support to the therapeutic use of the plant for thrombotic diseases.
Two anti-inflammatory peptides, named antiflammins (AFs), corresponding to a region with high amino acid similarity between lipocortin-1 and uteroglobin were tested for their ability to inhibit transglutaminase (TG) and low-molecular-mass phospholipase A2 (PLA2). Porcine pancreatic PLA2 activity and guinea pig hepatic TG activity were determined by arachidonyl release from arachidonyl-phosphatidylcholine and by the incorporation of putrescine into succinylated casein, respectively. AFs inhibited TG activity but did not affect PLA2 activity. Moreover, porcine pancreatic PLA2 was activated by TG and AFs decreased porcine pancreatic PLA2 activation induced by TG. Taken together, our results support the hypothesis that the anti-inflammatory effects of AFs are, at least in part, due to the action of AFs on TG activity.
Ischemic stroke is a leading cause of disability. Inflammation of the vessel wall following neutrophil adhesion to vascular endothelium may contribute to ischemic damage. We studied the effect of a platelet inhibitor and an angiotensin II receptor antagonist: alone or in combination, on the adhesion of neutrophils to endothelial cell line in stroke patients. Neutrophils were collected from 12 patients with ischemic stroke within 48 h. Six patients with previous stroke and six healthy volunteers served as control. Neutrophils were incubated with dipyridamole, candesartan or both and allowed to adhere to human endothelial cell line (ECV-304). Adhesion and expression of adhesion molecules (AM) were determined using fluorescence-activated cell-sorting (FACS). Dipyridamole and the combination of dipyridamole and candesartan inhibited significantly the adhesion of neutrophils from ischemic stroke patients as compared to controls with a prominent additive effect. No inhibition was seen in the control groups. These drugs also reduced significantly the expression of the AM Mac-1. Both candesartan and dipyridamole inhibited the adhesion of neutrophils to vascular endothelium in ischemic stroke patients but not in chronic stroke patients or healthy persons. This effect may be related to specific downregulation of Mac-1 by these drugs or other intracellular events.
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 adhesion of granulocytes to endothelial cells requires regulated expression of molecules on both the endothelial cell and the granulocyte. These pro-adhesive molecules have diverse structures and mechanisms of expression, and act either to tether the two cells together or as signals that induce activation-dependent adhesion events. Combinations of tethering and signaling molecules regulate endothelial-cell-granulocyte interactions at the endothelial surface.
Two anti-inflammatory peptides (antiflammins) corresponding to a high amino acid similarity region between lipocortin I and uteroglobin were tested for their ability to inhibit purified human synovial fluid phospholipase A2 (HSF-PLA2). No inhibitory activity was observed, even at such high concentrations of peptides as 50 microM. When antiflammins were preincubated with the enzyme and/or the substrate, no HSF-PLA2 inhibition was detected. In vivo anti-inflammatory activity of these peptides was evaluated in several experimental models of inflammation induced by carrageenan, croton-oil, oxazolone and Naja naja naja venom phospholipase A2 (PLA2). In contrast to the in vitro results, anti-inflammatory activity was observed in all tests, except when inflammation was induced by snake venom PLA2. Taken together, our results do not support the hypothesis that the in vivo anti-inflammatory effect of antiflammins is directly related to inhibition of PLA2 activity.
The anti-inflammatory synthetic polypeptides referred to as antiflammins are thought to be inhibitors of phospholipase A2 (PLA2) (EC 3.1.1.4). These peptides are from the sequence of amino acids of greatest similarity between uteroglobin and lipocortin I. The effect of these peptides was studied on PLA2 activation in rat platelets and on acute inflammatory models after local or parenteral administration of drug. We found that antiflammins decreased collagen-induced platelet activation, but had no effect when arachidonic acid was used as activator. The peptides were able to inhibit acute inflammatory processes induced by carrageenan or phorbol esters when administered locally or parenterally. However, antiflammins had no effect on inflammation induced by exogenous PLA2 administration. These results indicate that the antiflammins may have a direct inhibitory effect on PLA2 activation but not on the enzyme or enzyme-substrate interaction.
LECAM-1 (leukocyte-endothelial cell adhesion molecule 1), the lymphocyte lectin ("selectin") homing receptor for peripheral lymph nodes (PLNs), participates in the earliest interactions of polymorphonuclear neutrophilic leukocytes (PMNs) with inflamed venules. Here, we present evidence that LECAM-1 mediates this function through a novel mechanism--presentation of oligosaccharide ligands to the inducible vascular selectins endothelial-leukocyte adhesion molecule (ELAM-1) and granule membrane protein 140 (GMP-140). PMN, but not lymphocyte, LECAM-1 is modified with the vascular selectin ligand sialyl Lewis x (sLex) and specifically binds ELAM-1-transfected cells. Although only a small fraction of total cell surface sLex, LECAM-1-associated sLex appears to play a prominent role in PMN interactions with cell-associated ELAM-1 and GMP-140, as anti-LECAM-1 monoclonal antibodies or selective removal of cell surface LECAM-1 inhibits PMN binding to vascular selectin transfectants by up to 70%. The enhanced function of LECAM-1-associated sLex may reflect the striking concentration, shown here, of LECAM-1 on PMN surface microvilli, the site of initial cellular contact.
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The phospholipid inflammatory mediator platelet activating factor (PAF) has been shown to stimulate certain functions of polymorphonuclear leukocytes (PMN). However, the effect of PAF on surface complement receptors of PMN has not been described. Using monoclonal antibodies and flow cytometry, we have assessed the effects of PAF on surface expression of membrane receptors for C3bi (CR3) and C3b (CR1) in human PMN. PAF (optimal concentration of 1 x 10(-8) M) increased CR3 190% and CR1 174% compared with unstimulated cells at 37 degrees C, while the PAF analogue lyso-PAF had no stimulatory effect. Both CR3 and CR1 responses to PAF reached maximum levels at 15-30 min. PAF effects were comparable to peak effects induced by LTB4 but less than induced by FMLP. A PAF receptor antagonist, SRI 63-441, blocked the increased complement receptor expression in a dose-dependent manner with maximal inhibition of 80-95% at 5 x 10(-6) M. Extracellular calcium had no effect on CR1 expression but slightly enhanced and EGTA partially inhibited the PAF-induced increase in CR3 expression. Simultaneous incubation with PAF and LTB4 enhanced CR3 and CR1 expression more than either agent alone. These findings indicate that PAF, alone and in combination with LTB4, can induce altered expression of complement receptors on the surface of PMN. This effect may enhance adhesion and phagocytosis by PMN at inflammatory reaction sites.
Blood-borne lymphocytes initiate entry into secondary lymphoid organs, such as peripheral lymph nodes (PN) and gut-associated Peyer's patches (PP), by a highly specific adhesive interaction between the lymphocytes and the endothelium of specialized blood vessels known as a high endothelial venules (HEV). The selectivity with which functional subpopulations of lymphocytes migrate into particular lymphoid organs is believed to be regulated by the expression of cell adhesion receptors and complementary ligands on lymphocytes and HEV, respectively. The entry of lymphocytes into PN and PP has clearly been shown to involve distinct receptor-ligand pairs. Employing the Stamper-Woodruff in vitro adhesion assay, which measures lymphocyte attachment to HEV in cryostat-cut sections of lymphoid organs, we have previously shown that treatment of PN sections with two different sialidases inactivates HEV-adhesive ligands, whereas treatment of PP tissue sections has no effect on HEV-adhesive function. We now report that in vivo exposure of HEV to sialidase (after i.v. injection of the enzyme) also selectively prevents subsequent in vitro attachment of lymphocytes to PN HEV but not to PP HEV. Consistent with this organ-selective impairment of HEV-adhesive function by sialidase, i.v. injection of the enzyme is shown to prevent short term lymphocyte accumulation within peripheral lymph nodes while having no significant effect on accumulation in PP, blood, or nonlymphoid organs. Histologic examination with the sialic acid-specific lectin from Limax flavus verified that i.v. injected sialidase effectively removes stainable sialic acid moieties from HEV in both PN and PP. This study confirms that sialic acid is required for the adhesive function of PN HEV-ligands. A role for sialic acid as either a recognition determinant or as a regulatory molecule can be envisioned. In view of the fact that many pathogens release sialidase and cause substantially elevated serum levels of this enzyme, the present observations may have pathophysiologic significance. One mechanism by which such pathogens may avoid destruction is to inactivate susceptible HEV-ligands and disrupt the entry of lymphocytes into lymphoid organs where immune responses against the pathogens would normally be initiated.
Significant future developments in the effective treatment of inflammatory diseases may arise from non-toxic dual inhibitors of both cyclooxygenase and lipoxygenase pathways in the arachidonate cascade. Inhibition of phospholipase A2(PLA2)(EC3.1.1.4), may provide such a dual action and recent research has concentrated on the role of PLA2-inhibitory proteins as possible anti-inflammatory agents. Blastokinin or uteroglobin is a steroid-induced rabbit secretory protein with PLA2-inhibitory activity. Its biochemical and biological properties have been extensively studied and its crystallographic structure has been resolved at 1.34 A (refs 15, 16). Lipocortins are a family of related proteins, which, it has been suggested, mediate the anti-inflammatory effects of glucocorticoids (for a review, see ref. 23). Some proteins of this group have been purified and the complementary DNA sequences of two human lipocortins are known. Lipocortins inhibit PLA2 in vitro, although their mechanism of action is still unclear. Recombinant lipocortin I inhibits eicosanoid synthesis in isolated perfused lungs from the guinea pig. Here, we report that synthetic oligopeptides corresponding to a region of high amino-acid sequence similarity between uteroglobin and lipocortin I have potent PLA2 inhibitory activity in vitro and striking anti-inflammatory effects in vivo.
Macrophages (M phi s) exhibit variations in their ability to release and metabolize arachidonate (AA) depending on their state of activation, differentiation, and tissue origin. In order to understand these variations on a molecular level, we determined whether differences in AA release and metabolism by murine peritoneal M phi s could be explained in terms of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX) expression. Resident M phi s exhibited greater COX capacity (conversion of exogenous AA to PGE2) but lower phospholipase (PLase) activity (release of endogenous AA) than elicited M phi s. Activation of resident M phi s in vivo with endotoxin increased both their PLase activity and COX capacity. Despite the observed differences in PLase activity, peritoneal M phi s under all conditions expressed similar amounts of cPLA2 mRNA and protein. All M phi s exhibited COX-1 mRNA and protein (i.e., the constitutive isoform of COX), although elicited M phi s exhibited increased mRNA for COX-1 but decreased levels of protein, relative to resident M phi s. Elicited (but not resident) cells also exhibited COX-2 mRNA but not COX-2 protein (i.e., the inducible form of COX). Despite the increased COX capacity of resident cells with in vivo activation, their expression of COX-2 mRNA and protein was equivalent to that of unactivated cells, becoming apparent only after cell adherence in vitro. In sum, there is no simple relationship between the ability of M phi s to release and metabolize AA, and the expression of cPLA2 or COX isoforms. Moreover, adherence appears to be important for the expression of COX-2 by M phi s.
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
Antiflammins are synthetic peptides with sequence homology to proteins inhibitory for phospholipase A2 (EC 3.1.1.4). The effect of antiflammin 2 on murine arachidonate or 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ear oedema has been studied. Topical application of arachidonic acid (AA) produced a short-lived oedema response with rapid onset associated with marked increases in prostaglandin E2 levels. TPA produced a longer-lasting oedema associated with marked influx of neutrophils and mononuclear cells as well as predominant formation of leukotriene B4 (LTB4). Topical pretreatment with indomethacin or dexamethasone reduced plasma leakage, oedema and prostaglandin E2 biosynthesis in AA-induced oedema, whereas antiflammin 2 had no effect. However, topical pretreatment with antiflammin 2 dose-dependently reduced plasma leakage, cell influx, oedema and LTB4 levels in response to TPA. These results indicate that the anti-inflammatory effect of antiflammins can be attributed to AA mobilization and/or 5 lipoxygenase inhibition but can be dissociated from an effect on arachidonic acid metabolism by the cyclooxygenase pathway.
Adhesive interactions play critical roles in directing the migration, proliferation, and differentiation of cells; aberrations in such interactions can lead to pathological disorders. These adhesive interactions, mediated by cell surface receptors that bind to ligands on adjacent cells or in the extracellular matrix, also regulate intracellular signal transduction pathways that control adhesion-induced changes in cell physiology. Though the extracellular molecular interactions involving many adhesion receptors have been well characterized, the adhesion-dependent intracellular signaling events that regulate these physiological alterations have only begun to be elucidated. This article will focus on recent advances in our understanding of intracellular signal transduction pathways regulated by the integrin family of adhesion receptors.
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.
To interpret results of studies on arachidonic acid (AA) mobilization and metabolism in vitro, it is essential that the influence of culture and conditions should be well defined. Thus, we investigated the effects of murine resident peritoneal macrophage adherence and the presence of foetal calf serum in culture medium on arachidonic acid mobilization. The present data demonstrate that [3H] AA mobilization was triggered simply by contact between cell and substrate. The presence of serum can modulate cell-substrate interactions but not AA mobilization. Protein kinase C, and calmodulin inhibitors failed to inhibit [3H] AA release induced by cell adherence. Finally, low molecular weight PLA2 inhibitors were not able to inhibit [3H] AA mobilization stimulated by cell adherence.
We have studied the effects of antiflammin-2, a fragment corresponding to amino acids 246-254 of lipocortin-1 (HDMNKVLDL), on arachidonate mobilization and metabolism and we also determined the effect of antiflammin-2 on the chemotaxis of phagocytes. Our results demonstrated that the antiflammin-2 was not able to diminish significantly [3H]arachidonic acid mobilization stimulated by 4 beta-phorbol-12-myristate 13-acetate or calcium ionophore A23187 in murine 3T6 fibroblasts or resident peritoneal macrophages. Further, antiflammin-2 had no effect on arachidonate metabolism. In contrast, a glucocorticoid such as dexamethasone reduced significantly [3H]arachidonic acid release and arachidonate metabolism induced in both cells. This study confirms the inhibitory effect of antiflammin on leukocyte migration and suggests that it acts partly through the inhibition of leukocyte binding to endothelial cells.
1. Antiflammins are a new family of peptides that share a common sequence with uteroglobin and lipocortin-1, which retain the anti-inflammatory action of these proteins. 2. However, it is controversial whether or not the antiflammins have any effect on enzymes involved in arachidonic acid mobilization and/or arachidonic acid metabolism. 3. This review summarizes our current knowledge of the properties and activity of antiflammins.
Coculture of a monocytic cell line (HL-60) and iliacal endothelial cells as an in vitro model of vascular inflammation was investigated for cooperative regulatory mechanisms of prostanoid synthesis under conditions of selective prestimulation. In coculture of endothelial cells and 12-O-tetradecanoylphorbol 13-acetate (TPA)-prestimulated monocytic HL-60 cells the capacity of prostanoid synthesis from arachidonic acid was strongly increased compared with monocultures. Concomitant with up-regulation of specific adhesion molecules, cyclooxygenase (COX) 2 mRNA was induced in endothelial cells in coculture independent of cell contact. HL-60 cells exhibited no alterations in mRNA expression of cyclooxygenases or thromboxane synthase. Coculture of TPA-prestimulated endothelial cells with unstimulated HL-60 cells led to a selectively increased capacity of thromboxane production. Under this condition HL-60 cells up-regulated COX1 and COX2 mRNA, whereas endothelial mRNA levels did not change. Our data demonstrate that the increase in prostanoid synthesis in coculture essentially depends on rapid induction of COX2 mRNA within 2 h.
The accumulation of neutrophils and mononuclear cells is a characteristic feature of 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ear edema. This cell influx was accompanied by the enhancement of eicosanoid tissue levels and prostaglandin H synthase-2 (PGHS-2) overexpression. Sialidase treatment, which affects the structure of selectins and inhibits leukocyte influx, significantly reduced eicosanoid and PGHS-2 levels and edema. In contrast, skin PGHS-2 overexpression induced by arachidonic acid (AA) application was not affected by sialidase treatment. These results suggest that PGHS-2 overexpression induced by TPA could be induced by AA and/or AA metabolite release by leukocyte infiltrated during the inflammatory process.