Lipoxin A4 selectively programs the profile of M2 tumor-associated macrophages which favour control of tumor progression.

In tumor microenvironments, the macrophage population is heterogeneous, but some macrophages can acquire tumor-promoting characteristics. These tumor-associated macrophages (TAM) exhibit an M2-like profile, with deficient production of NO and ROS, characteristics of pro-inflammatory M1 cytotoxic macrophages. Lipoxins (LX) and 15-epi-lipoxins are lipid mediators which can induce certain features of M2 macrophages in mononuclear cells, but their effects on TAM remain to be elucidated. This study tested the hypothesis that ATL-1, a synthetic analogue of 15-epi-lipoxin A4 , could modulate TAM activity profile. We show that human macrophages (MΦ) differentiated into TAM-like cells after incubation with conditioned medium from MV3, a human melanoma lineage cell. Contrasting with the effects observed in other M2 subsets and M1 profile macrophages, ATL-1 selectively decreased M2 surface markers in TAM, suggesting unique behavior of this particular M2 subset. Importantly, these results were replicated by the natural lipoxins LXA4 and the aspirin induced 15-epi-LXA4 (ATL). In parallel, ATL-1 stimulated TAM to produce NO by increasing the iNOS/arginase ratio and activated NADPH oxidase, triggering ROS production. These alterations in TAM profile induced by ATL-1 led to loss of the anti-apoptotic effects of TAM on melanoma cells and increased their cytotoxic properties. Finally, ATL-1 was found to inhibit tumor progression in a murine model in vivo, which was accompanied by alterations in TAM profile and diminished angiogenesis. Together, the results show an unexpected effect of lipoxin, which induces in TAM a change from an M2- to an M1-like profile, thereby triggering tumor cell apoptosis and down-modulating the tumor progression.

ATL-1, a synthetic analog of lipoxin, modulates endothelial permeability and interaction with tumor cells through a VEGF-dependent mechanism.

Lipoxins (LX) and 15-epi-LX are lipids with a potent inhibitory effect on angiogenesis, in different models in vivo and in vitro. ATL-1, a synthetic analog of 15-epi-LXA4, inhibits various actions stimulated by vascular endothelial growth factor (VEGF). However, LX actions on endothelial cells (EC) in tumor-related contexts are still unknown. Here, we investigated the modulation of EC by ATL-1, in a model that mimics tumor extravasation. We observed that the analog inhibited endothelial permeability induced by VEGF, through the stabilization of VE-cadherin/ß-catenin-dependent adherens junctions. We tested the ability of MV3 cells, a highly metastatic melanoma cell line, to transmigrate across unchallenged EC monolayers for 18 h, as compared to NGM normal melanocytes. ATL-1 was able to inhibit only melanoma extravasation. MV3 cells secrete large amounts of VEGF and we observed that ATL-1 per se did not alter this ability. Melanoma cells skills to crossing endothelial monolayers were due to the steady accumulation of tumor-derived VEGF. When endothelial cells were challenged with exogenous VEGF, added at levels comparable to those secreted by MV3 cells over 18 h, and a short-term (4h) transendothelial migration assay was performed, both melanoma and melanocyte cells were able to extravasate, and ATL-1 was able to block the passage of both cells. These results indicate that ATL-1 has a potent inhibitory effect on the permeability induced by VEGF, and that this pharmacological effect could be used to block tumor extravasation across endothelial barriers, with a possible prospect of reducing the haematogenic spread of cancer cells.

A synthetic analog of 15-epi-lipoxin A4 inhibits human monocyte apoptosis: involvement of ERK-2 and PI3-kinase.

Human monocytes play a central function in several steps of the immune response and the process involved in regulating their survival are critical to population control. Lipoxins are lipid mediators and members of the eicosanoid family that exhibit selective stimulatory but nonphlogistic activities in mononuclear cells. In this study, we investigated the effects of 15-epi-16-(para-fluoro)phenoxy-LXA(4) (ATL-1), a synthetic analog of 15-epi-lipoxin A(4), in human monocytes survival and apoptosis. ATL-1 concentration-dependently increased monocyte survival, as a consequence of cell apoptosis reduction by the analog. Treatment of these cells with PD98059 or LY294002 blocked ATL-1 effects, indicating the involvement of ERK-2 and PI3-K, both pathways associated with cell survival. Confirming the activation of these pathways, we demonstrated an increase in ERK-2 nuclear translocation and Akt phosphorylation. Furthermore, we showed that ATL-1 inhibits Bax translocation to the mitochondria. These results confirm a cytoprotective effect of lipoxins in monocytes and might contribute to the elucidation of the mechanisms associated with the resolution phase of the inflammatory process in different pathophysiological events.

A role for the MAPK/ERK pathway in oligodendroglial differentiation in vitro: stage specific effects on cell branching.

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway is important for both long-term survival and timing of the progression of oligodendrocyte differentiation. Oligodendroglial cells treated with MEK inhibitor were distinguished by using stage specific markers: NG2 proteoglycan, A2B5, 2'3'nucleotide-cyclic 3'phosphodiesterase (CNPase) and myelin basic protein (MBP), and classified according to their morphology into different developmental stages. Treatment significantly increased the number of cells with more immature morphologies and decreased the number of mature cells. Furthermore, it increased the number of rounded cells that could not be classified into any of the oligodendroglial developmental stages. The strongest effects were usually observed shortly after treatment. Rounded cells were CNPase/MBP positive and they were not stained by anti-NG2 or A2B5, indicating that they were mature cells unable either to extend and/or to maintain their processes. These data showed an effect of the MAPK/ERK pathway on oligodendroglial branching, with possible consequences for the formation of the myelin sheath.

ATL-1, an analogue of aspirin-triggered lipoxin A4, is a potent inhibitor of several steps in angiogenesis induced by vascular endothelial growth factor.

BACKGROUND AND PURPOSE: Vascular endothelial growth factor (VEGF) is the most important proangiogenic protein. We have demonstrated that ATL-1, a synthetic analogue of aspirin-triggered lipoxin A(4), inhibits VEGF-induced endothelial cell (EC) migration. In the present study, we investigated the effects of ATL-1 in several other actions stimulated by VEGF. METHODS: Human umbilical vein ECs were treated with ATL-1 for 30 min before stimulation with VEGF. Cell proliferation was measured by thymidine incorporation. Adherent cells were determined by fluorescence intensity using a Multilabel counter. Expression and activity of matrix metalloproteinases (MMP) were analysed by western blot and zymography. KEY RESULTS: ATL-1 inhibited EC adhesion to fibronectin via interaction with its specific receptor. Furthermore, VEGF-induced MMP-9 activity and expression were reduced by pretreatment with ATL-1. Because the transcription factor NF-kappaB has been implicated in VEGF-mediated MMP expression and EC proliferation, we postulated that ATL-1 might modulate the NF-kappaB pathway and, indeed, ATL-1 inhibited NF-kappaB nuclear translocation. Pretreatment of EC with ATL-1 strongly decreased VEGF-dependent phosphorylation of phosphainositide 3-kinase (PI3-K) and extracellular signal-regulated kinase-2 (ERK-2), two signalling kinases involved in EC proliferation. Inhibition of VEGF-induced EC proliferation by ATL-1 was antagonized by sodium orthovanadate, suggesting that this inhibitory activity was mediated by a protein tyrosine phosphatase. This was confirmed by showing that ATL-1 inhibition of VEGF receptor-2 (VEGFR-2) phosphorylation correlates with SHP-1 association with VEGFR-2. CONCLUSIONS AND IMPLICATIONS: The synthetic 15-epi-lipoxin analogue, ATL-1, is a highly potent molecule exerting its effects on multiple steps of the VEGF-induced angiogenesis.

Aspirin-triggered Lipoxin A4 inhibition of VEGF-induced endothelial cell migration involves actin polymerization and focal adhesion assembly.

Angiogenesis, the growth of new capillaries from pre-existing ones, occurs through dynamic functions of the endothelial cells (EC), including migration, which is essential to achieve an organized formation of the vessel sprout. We demonstrated previously that an aspirin-triggered lipoxin analog, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A4 (ATL-1), inhibits vascular endothelial growth factor (VEGF)-induced EC migration. In the present study, we investigated the effects of ATL-1 in the actin cytoskeleton reorganization of EC stimulated with VEGF. Pretreatment of EC with ATL-1 caused a reduction in VEGF-induced stress fibers and therefore reduced the intracellular content of filamentous actin. A concomitant impairment in stress-activated protein kinase (SAPK2/p38) phosphorylation suggests that ATL inhibition of VEGF-stimulated actin polymerization involves the SAPK2/p38 pathway. Moreover, ATL-1 treatment inhibited focal adhesion clustering due to inhibition of focal adhesion kinase (FAK) phosphorylation and the subsequent association of FAK with the actin cytoskeleton. This final event, which ultimately allows cell migration, was reverted by an LX receptor antagonist, but not by a cys-LT1R antagonist, indicating an effect via the G-protein-linked LXA4 receptor. Together our results provide evidence that ATL-1 inhibits EC migration via the concerted inhibition of actin polymerization and proper assembly of focal adhesions, supporting a role for these novel lipid mediators as angiogenesis modulators.

Novel lipid mediator aspirin-triggered lipoxin A4 induces heme oxygenase-1 in endothelial cells.

Lipoxins (LX) and aspirin-triggered LX (ATL) are eicosanoids generated during inflammation via transcellular biosynthetic routes that elicit distinct anti-inflammatory and proresolution bioactions, including inhibition of leukocyte-mediated injury, stimulation of macrophage clearance of apoptotic neutrophils, repression of proinflammatory cytokine production, and inhibition of cell proliferation and migration. Recently, it was reported that aspirin induces heme oxygenase-1 (HO-1) expression on endothelial cells (EC) in a COX-independent manner, what confers protection against prooxidant insults. However, the underlying mechanisms remain unclear. In this study, we investigated whether an aspirin-triggered lipoxin A(4) stable analog, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A(4) (ATL-1) was able to induce endothelial HO-1. Western blot analysis showed that ATL-1 increased HO-1 protein expression associated with increased mRNA levels on EC in a time- and concentration-dependent fashion. This phenomenon appears to be mediated by the activation of the G protein-coupled LXA(4) receptor because pertussis toxin and Boc-2, a receptor antagonist, significantly inhibited ATL-1-induced HO-1 expression. We demonstrate that treatment of EC with ATL-1 inhibited VCAM and E-selectin expression induced by TNF-alpha or IL-1beta. This inhibitory effect of the analog is modulated by HO-1 because it was blocked by SnPPIX, a competitive inhibitor that blocks HO-1 activity. Our results establish that ATL-1 induces HO-1 in human EC, revealing an undescribed mechanism for the anti-inflammatory activity of these lipid mediators.

Mechanisms in anti-inflammation and resolution: the role of lipoxins and aspirin-triggered lipoxins

Multicellular host responses to infection, injury or inflammatory stimuli lead to the formation of a broad range of chemical mediators by the host. The integrated response of the host is essential to health and disease; thus it is important to achieve a more complete understanding of the molecular and cellular events governing the formation and actions of endogenous mediators of resolution that appear to control the duration of inflammation. Lipoxins are trihydroxytetraene-containing lipid mediators that can be formed during cell-cell interactions and are predominantly counterregulators of some well-known mediators of inflammation. Since this circuit of lipoxin formation and action appears to be of physiological relevance for the resolution of inflammation, therapeutic modalities targeted at this system are likely to have fewer unwanted side effects than other candidates and current anti-inflammatory therapies. Here, we present an overview of the recent knowledge about the biosynthesis and bioactions of these anti-inflammatory lipid mediators

Mechanisms in anti-inflammation and resolution: the role of lipoxins and aspirin-triggered lipoxins.

Multicellular host responses to infection, injury or inflammatory stimuli lead to the formation of a broad range of chemical mediators by the host. The integrated response of the host is essential to health and disease; thus it is important to achieve a more complete understanding of the molecular and cellular events governing the formation and actions of endogenous mediators of resolution that appear to control the duration of inflammation. Lipoxins are trihydroxytetraene-containing lipid mediators that can be formed during cell-cell interactions and are predominantly counterregulators of some well-known mediators of inflammation. Since this circuit of lipoxin formation and action appears to be of physiological relevance for the resolution of inflammation, therapeutic modalities targeted at this system are likely to have fewer unwanted side effects than other candidates and current anti-inflammatory therapies. Here, we present an overview of the recent knowledge about the biosynthesis and bioactions of these anti-inflammatory lipid mediators.

Cutting edge: nociceptin stimulates neutrophil chemotaxis and recruitment: inhibition by aspirin-triggered-15-epi-lipoxin A4.

The nociceptin receptor (Noci-R) is a G protein-coupled receptor present in neural tissues and its activation by nociceptin is involved in the processing of pain signals. Here, we report that Noci-R is present and functional on peripheral blood polymorphonuclear leukocytes (PMN). Human PMN express mRNA for Noci-R, its nucleotide sequence determined, and specific binding with [(125)I]-labeled nociceptin gave an apparent K(d) approximately 1.5 nM for this PMN opioid receptor. Nociceptin evoked PMN chemotaxis with maximal activity at 100 pM, without intracellular Ca(2+) mobilization. When injected in murine air pouches, nociceptin elicited leukocyte infiltration in a concentration-dependent fashion. Nociceptin-stimulated PMN infiltration was inhibited by treating mice with a synthetic analog of the aspirin-triggered lipid mediator 15-epi-lipoxin A(4). The present results identify nociceptin as a potent chemoattractant and provide a novel link between the neural and immune systems that are blocked by aspirin-triggered lipid mediators and may be relevant in neurogenic inflammation.

Activation of lipoxin A(4) receptors by aspirin-triggered lipoxins and select peptides evokes ligand-specific responses in inflammation.

Lipoxin (LX) A(4) and aspirin-triggered LX (ATL) are endogenous lipids that regulate leukocyte trafficking via specific LXA(4) receptors (ALXRs) and mediate antiinflammation and resolution. ATL analogues dramatically inhibited human neutrophil (polymorphonuclear leukocyte [PMN]) responses evoked by a potent necrotactic peptide derived from mitochondria as well as a rogue synthetic chemotactic peptide. These bioactive lipid analogues and small peptides each selectively competed for specific (3)H-LXA(4) binding with recombinant human ALXR, and its N-glycosylation proved essential for peptide but not LXA(4) recognition. Chimeric receptors constructed from receptors with opposing functions, namely ALXR and leukotriene B(4) receptors (BLTs), revealed that the seventh transmembrane segment and adjacent regions of ALXR are essential for LXA(4) recognition, and additional regions of ALXR are required for high affinity binding of the peptide ligands. Together, these findings are the first to indicate that a single seven-transmembrane receptor can switch recognition as well as function with certain chemotactic peptides to inhibitory with ATL and LX (lipid ligands). Moreover, they suggest that ALXR activation by LX or ATL can protect the host from potentially deleterious PMN responses associated with innate immunity as well as direct effector responses in tissue injury by recognition of peptide fragments.

Studies on the anti-allergic activity of Mikania glomerata.

A fraction (MG1) obtained from the ethanolic extract of Mikania glomerata Sprengel (Compositae), popularly known as 'guaco' and used as 'an' anti-allergic and anti-inflammatory agent, was evaluated for these properties on ovalbumin-induced allergic pleurisy and in models of local inflammation induced by biogenic amines, carrageenan and PAF. Plasma exudation as well as neutrophil and eosinophil infiltration evoked by the intrapleural injection of the antigen were significantly reduced by the fraction. Likewise, PAF-induced pleural neutrophil migration was inhibited by the treatment with MG1. On the other hand, pre-treatment of the animals with MG1 failed to modify the pleurisy induced by histamine, serotonin or carrageenan. These results suggest that MG1 is effective in inhibiting immunologic inflammation but did not affect acute inflammatory response caused by other agents.

Anti-inflammatory profile of N-phenylpyrazole arylhydrazone derivatives in rats.

A series of synthetic N-phenylpyrazole arylhydrazone compounds, rationally designed as mixed-hybrid isosteres of two known inhibitors of prostaglandin synthase and 5-lipoxygenase enzymes, BW-755c and CBS-1108, has been investigated for anti-inflammatory activity in the carrageenan-induced pleurisy model in rats. The compounds have different oxygenated substituent groups in the aryl group of the hydrazone framework to ensure a different range of redox properties. A new arylhydrazone derivative, 2,6-di-tert-butyl-4-(4-nitro-3-methyl-N-phenylpyrazol-5-yl-hydr azonomethyl)phenol, was also synthesized and tested for anti-inflammatory activity. Although all the compounds significantly inhibited (by 30-90%) neutrophil accumulation in the pleural cavity, there was great variability in the anti-oedematogenic effect of the compounds (3-96%). 5-(4'-Hydroxy-3'-methoxybenzylidene)hydrazone-3-methyl-4-nitrop henylpyrazole was the most active compound in this series; it had a remarkable antiinflammatory profile, almost blocking both assays. In contrast, the compound with a 2,6-di-tert-butylated hydroxybenzene ring on the hydrazone group inhibited neutrophil migration only. These results will be useful for further structure-activity relationship studies devoted to improving the dual prostaglandin synthase-5-lipoxygenase activity of these derivatives and determining the minimum structural requirements necessary for this activity.

Induction of NOS in rat blood PMN in vivo and in vitro: modulation by tyrosine kinase and involvement in bactericidal activity.

Intravenous administration of lipopolysaccharide (LPS) to rats increased the production of nitric oxide (NO) metabolites (NOx) by blood polymorphonuclear neutrophils (PMN) in vitro. Both dexamethasone and L-NMMA, added in vitro to neutrophil cultures, inhibited the production of NO. On the other hand, the production of NO was not affected by the treatment, in vivo or in vitro, with different inhibitors of cyclooxygenase or 5-lipoxygenase or with a platelet-activating factor (PAF) antagonist. The incubation of blood PMN from normal rats in vitro with neutrophil activators (PAF, leukotriene B4, and interleukin-8) and different cytokines [interleukin-1, tumor necrosis factor alpha, and interferon-gamma (IFN-gamma)] showed that only IFN-gamma was able to induce the production of high amounts of NO. This induction was directly correlated with the expression of iNOS and an increase in in the enzyme activity in blood PMN. The tyrosine kinase inhibitor genistein inhibited NO production induced by IFN-gamma, suggesting that the signal transduction pathway leading to NOS induction in rat PMN involves phosphorylation by tyrosine kinase. We also showed that NO produced by IFN-gamma activated rat blood PMN involved in the killing of Pseudomonas aeruginosa.

The involvement of nitric oxide in the anti-Candida albicans activity of rat neutrophils.

Rat peritoneal neutrophils (PMN) spontaneously release nitric oxide (NO) when incubated in vitro. Addition of the NO synthase inhibitor L-monomethylarginine (L-NMMA) to the PMN reduces NO production and impairs the killing of the yeast Candida albicans, both effects being reversed by L-arginine. These data strongly suggest that oxidative metabolism of L-arginine by PMN is involved in the candidacidal activity of these cells. Rat blood PMN, which do not produce significant amounts of NO, exhibit a reduced killing capacity compared with peritoneal cells, except when they are obtained from lipopolysaccharide (LPS)-treated rats. In this case they produce measurable amounts of nitrite and express high fungicidal activity in vitro. Confirming the candidacidal activity of NO, the exposure of the C. albicans cultures to different concentrations of NO donors leads to a reduction in their survival. The candidacidal activity related to the NO pathway in rat PMN is phagocytosis dependent, since the activity can be inhibited by cytochalasin B. However, the oxidative products of oxygen released by rat PMN do not seem to be involved in their candidacidal activity, as incubation of the cells with phorbol myristate acetate (PMA) increases release of superoxide anion but does not affect the pattern of killing. Our results suggest that NO could be an important candidacidal pathway in rat neutrophils.

An increase in nitric oxide produced by rat peritoneal neutrophils is not involved in cell apoptosis.

Polymorphonuclear neutrophils (PMN) obtained from carrageenin-stimulated peritoneal cavities of rats, but not blood PMN, spontaneously produced nitric oxide (NO) when incubated in vitro. Incubation of the cells with the NO synthase inhibitors, L-imino-ethyl-L-ornithine (L-NIO) or N(G)-monomethyl-L-arginine (L-NMMA), inhibited NO production. This inhibition could be reversed by L-arginine. Incubation of PMN with lipopolysaccharide (LPS) failed to enhance NO production. Pretreatment of the rats with dexamethasone (DEXA) prior to carrageenin injection or incubation of PMN with the glucocorticoid in vitro partially inhibited the spontaneous release of NO. On the other hand, when PMN obtained from DEXA pretreated rats were incubated in vitro with DEXA, NO synthase activity and hence NO generation were almost abolished. A similar inhibition was also observed following the addition of L-NIO or cycloheximide to cultures of carrageenin-elicited PMN. The NO production by PMN did not appear to be related to cell viability or apoptosis. Indeed, neither the blockade of NO generation by L-NIO nor the incubation of the neutrophils with a NO donor, S-nitroso-acetylpenicillamine (SNAP) modified the pattern of LDH release or DNA fragmentation. In summary, it appears that PMN migration triggers a continuous NO synthesis, and that NO produced by these cells is not related to their apoptosis.