Re: omega-3 and inflammation

November 4, 2011

Wayne, this is really interesting, though most of it over my head. Could you give your ideas from this article on whether supplementing with EPA would make a meaningful difference to Samter's patients?Thanks, JoanOn Nov 4, 2011, at 8:02 PM, Wayne wrote:

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References P. Tull1, Clara M. Yates1, H. Maskrey2, B. O'Donnell2, Jackie Madden3, F. Grimble3, Philip C. Calder3, Gerard B. Nash1, G. Ed. Rainger1*1 Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, The Medical School, The University of Birmingham, Birmingham, United Kingdom, 2 Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff, United Kingdom, 3 Institute of Human Nutrition, School of Medicine, University of Southampton, Southampton, United Kingdom

Abstract TopInflammation is a physiological response to tissue trauma or infection, but leukocytes, which are the effector cells of the inflammatory process, have powerful tissue remodelling capabilities. Thus, to ensure their precise localisation, passage of leukocytes from the blood into inflamed tissue is tightly regulated. Recruitment of blood borne neutrophils to the tissue stroma occurs during early inflammation. In this process, peptide agonists of the chemokine family are assumed to provide a chemotactic stimulus capable of supporting the migration of neutrophils across vascular endothelial cells, through the basement membrane of the vessel wall, and out into the tissue stroma. Here, we show that, although an initial chemokine stimulus is essential for the recruitment of flowing neutrophils by endothelial cells stimulated with the inflammatory cytokine tumour necrosis factor-Î±, transit of the endothelial monolayer is regulated by an additional and downstream stimulus. This signal is supplied by the metabolism of the omega-6-polyunsaturated fatty acid (n-6-PUFA), arachidonic acid, into the eicosanoid prostaglandin-D2 (PGD2) by cyclooxygenase (COX) enzymes. This new step in the neutrophil recruitment process was revealed when the dietary n-3-PUFA, eicosapentaenoic acid (EPA), was utilised as an alternative substrate for COX enzymes, leading to the generation of PGD3. This alternative series eicosanoid inhibited the migration of neutrophils across endothelial cells by antagonising the PGD2 receptor. Here, we describe a new step in the neutrophil recruitment process that relies upon a lipid-mediated signal to regulate the migration of neutrophils across endothelial cells. PGD2 signalling is subordinate to the chemokine-mediated activation of neutrophils, but without the sequential delivery of this signal, neutrophils fail to penetrate the endothelial cell monolayer. Importantly, the ability of the dietary n-3-PUFA, EPA, to inhibit this process not only revealed an unsuspected level of regulation in the migration of inflammatory leukocytes, it also contributes to our understanding of the interactions of this bioactive lipid with the inflammatory system. Moreover, it indicates the potential for novel therapeutics that target the inflammatory system with greater affinity and/or specificity than supplementing the diet with n-3-PUFAs.

Author Summary TopInflammation is a physiological response to tissue trauma or infection. Neutrophils, which circulate in the blood stream, are the first inflammatory cells to be recruited to a site of tissue inflammation. In response to recruitment signals provided by chemotactic peptides called chemokines, neutrophils traverse the endothelial cell lining of blood vessels. This process involves a multistep cascade of neutrophil adhesion and activation events on the endothelial barrier. While investigating the anti-inflammatory functions of the omega-3 fatty acid , EPA, which is found, for instance, in dietary fish oil, we identified an additional unexpected lipid-derived signal that is essential for neutrophil migration across the endothelium. Our experiments show that a chemokine delivered the first signal needed to bind neutrophils firmly to the endothelial surface. However, in order to traverse the endothelium, a subsequent signal delivered by prostaglandin-D2 (PGD2), a lipid derived from the omega-6 fatty acid arachidonic acid, was essential. When EPA, was introduced into the experiment, it was used to form PGD3. This alternative lipid blocked interactions between PGD2 and its receptor on neutrophils, preventing the process of migration across the endothelial barrier. Thus, we reveal a new step in the recruitment of neutrophils during inflammation, and a novel anti-inflammatory mechanism of action of dietary EPA.

Citation: Tull SP, Yates CM, Maskrey BH, O'Donnell VB, Madden J, et al. (2009) Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a New Step in Neutrophil Recruitment. PLoS Biol 7(8): e1000177. doi:10.1371/journal.pbio.1000177Academic Editor: Philip M. , NIH/NIAID, United States of AmericaReceived: February 6, 2009; Accepted: July 14, 2009; Published: August 25, 2009Copyright: © 2009 Tull et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Funding: This work was supported by the Biotechnology and Biological Sciences Research Council, United Kingdom (grant BBS/B/0105) and by the British Heart Foundation (grant BS/97001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing interests: The authors have declared that no competing interests exist.Abbreviations: DC, dendritic cell* E-mail: g.e.rainger@...

Introduction TopIn vertebrates, tissue trauma or infection causes the rapid initiation of an inflammatory reaction. The early phase of this phylactic response results in the localised recruitment of cells of the innate immune system from the blood, a tissue infiltrate that is dominated by neutrophils. The molecular processes that support the initial interactions between blood-borne neutrophils and the endothelial cells lining postcapillary venules (the site in the blood vasculature where leukocytes are recruited during inflammation) are well described (see Figure 1 for a schematic representation of the steps in the neutrophil recruitment process). In response to the localised production of inflammatory mediators, such as the cytokine tumour necrosis factor-Î± (TNF), activated endothelial cells decorate themselves with specialised adhesion receptors of the selectin family [1]–[3]. Due to their ability to rapidly form strong but short-lived bonds with carbohydrate counter-ligands on the neutrophil surface, E- and P-selectin are capable of tethering neutrophils from rapidly flowing blood [4]. The sequential formation and dissolution of selectin bonds also support a characteristic and dynamic form of adhesion, referred to as rolling [5]. Rolling adhesion does not require neutrophil activation. However, neutrophil migration through the vessel wall and into the inflamed tissue is dependent upon the receipt of an activating stimulus [5],[6]. We have previously shown that endothelial cells can present peptide agonists of the CXC-chemokine family to rolling neutrophils and thus stabilise adhesion [7],[8]. Ligation of the neutrophil CXC-Receptor-2 (CXCR2) by these agents is essential for activation of the Î²2-intergrin adhesion receptors and the reorganisation of the actin cytoskeleton that support transendothelial cell migration [8]. Here, we show that in the presence of an antibody that blocks chemokine interactions with CXCR2, neutrophils roll on the endothelial cell surface indefinitely (Figure 2), demonstrating that a chemokine signal is essential for neutrophil activation on TNF-stimulated endothelial cells. However, the removal of this primary activating stimulus provides no information on the requirement for additional, downstream signals, which might coordinate transit of the vessel wall and onward migration into stromal tissues.

Figure 1. Steps in the process of neutrophil recruitment.Step 1: Neutrophils are captured from flow and tether and roll on tumour necrosis factor-Î± (TNF)-stimulated endothelial cells. Step 2: Neutrophils are then activated by the action of CXC-chemokines on the chemokine receptor, CXCR2, a process resulting in the activation of neutrophil Î²2-integrins. Step 3: Î²2-integrins engage counter receptors on the endothelial cell surface, and the neutrophil becomes stationary. Step 4: Prostaglandin-D2 (PGD2), generated by the action of cyclooxygenase enzymes on the n-6-PUFA arachidonic acid (AA), binds the PGD2 receptor, DP-1. DP-1 generates signals that stabilise neutrophil adhesion, induce neutrophil shape change, and support the process of transmigration across the endothelial cell monolayer. Step 5: If the endothelial cells have been supplemented with the n-3-PUFA, eicosapentaenoic acid (EPA), the alternative series prostanoid, PGD3, is generated, and this antagonises PGD2-mediated neutrophil responses.doi:10.1371/journal.pbio.1000177.g001

Figure 2. Chemokine signalling through CXCR2 is essential for neutrophil activation on TNF-stimulated endothelial cells.Using a flow-based adhesion assay, neutrophils isolated from whole blood were perfused across endothelial cells. Neutrophils were recruited to endothelial cells treated with TNF, but did not adhere to unstimulated endothelium. Detailed analysis of neutrophil behaviour showed that on TNF-stimulated endothelial cells, the majority of recruited cells transmigrated across the monolayer. When neutrophils were treated with anti-CXCR2, but not anti-CXCR1, neutrophil activation was inhibited so that nearly all of the recruited neutrophils rolled indefinitely on the monolayer and could not migrate; data are mean±SEM of four experiments **p<0.01 for comparison by paired t-test of neutrophil behaviour on TNF stimulated endothelial cells in the presence or absence of anti-CXCR2.doi:10.1371/journal.pbio.1000177.g002

Results/Discussion Top

EPA Released from Endothelial Cell Membrane Phospholipids Inhibits the Recruitment of NeutrophilsDietary omega-3 polyunsaturated fatty acids (n-3-PUFAs) have anti-inflammatory properties. For example, their inclusion in the diet in the form of n-3-PUFA–rich fish oil reduces the symptoms of disease as well as the use of nonsteroidal anti-inflammatory drugs in arthritis patients with severe inflammatory joint disease [9]. In addition, dietary n-3-PUFAs may offer protection against vascular pathology associated with atherosclerosis, having been reported to be efficacious in epidemiological studies [10]. By modulating inflammation within the artery wall, n-3-PUFAS also alter the cellular and the structural composition of advanced atherosclerotic plaques in a manner that could reduce the incidence of plaque rupture or ulceration, a process that precedes tissue infarction (e.g., heart attack or stroke) [11]. Despite these documented anti-inflammatory benefits, the mode(s) of interaction of these lipids with the immune and inflammatory systems are not well understood. When we supplemented endothelial cell culture medium with the major n-3 -PUFA found in dietary fish oil supplements, eicosapentaenoic acid (EPA; 20:5n-3), it was incorporated into cellular phospholipids so that upon withdrawal of the free fatty acid from the culture medium, a pool of esterified EPA remained localised within endothelial cell membranes (Figure 3A and 3B). After withdrawal of free fatty acid and stimulation with TNF, EPA-treated cells were able to support similar levels of neutrophil adhesion to those that had received no lipid supplement (Figure 4A). However, detailed analysis showed that the behaviour of neutrophils on the two populations of endothelial cells was very different. In response to TNF, but in the absence of EPA, a small population of adherent neutrophils (~20%) rolled throughout the duration of a flow adhesion assay (Figure 4B). The remaining cells became activated on the endothelial cell surface, and this population of neutrophils became progressively smaller as cells migrated across the endothelial cell monolayer (Figure 4B). In contrast, when endothelial cells had been treated with EPA prior to TNF stimulation, there was a marked reduction in the number of cells undergoing transendothelial cell migration (Figure 4B). This was reduced at the earliest time point and did not increase over the duration of the experiment. Interestingly however, many of the cells that did become activated on the surface of endothelial cells reverted to a rolling form of adhesion (Figure 4B), implying that after the receipt of an initial chemokine stimulus, a second signal was required to allow prolonged adhesion and migration across the endothelial cell monolayer. Experiments using different concentrations of EPA showed that these inhibitory effects were evident at concentrations as low as 50 nM (Figure 4C).

Figure 3. The effects of EPA supplementation on the fatty acid content of endothelial cell phospholoipds.(A and The concentration of EPA in membrane phospholipids was increased after supplementation of culture medium with EPA for 24 h. Error bars indicate SEM. AA, arachidonic acid; FA, fatty acid; PA, palmitic acid; SA, stearic acid.doi:10.1371/journal.pbio.1000177.g003

Figure 4. The effects of EPA supplementation on the adhesive behaviour of neutrophils.(A) EPA supplementation did not affect the number of neutrophils initially adhering to endothelial cells from flow. ( However, the time course of neutrophil behaviour on TNF-stimulated endothelial cells showed that EPA supplementation drastically altered neutrophil behaviour. Thus, the number of activated and surface adherent neutrophils decreased with time in the presence or absence of EPA. However, on endothelium that had not been supplemented with EPA, this was because neutrophils transmigrated across the monolayer, whereas on EPA-supplemented endothelium this was because activated cells reverted back to a rolling form of adhesion. ANOVA showed that there was a significant effect of treatment (i.e., ±EPA) on the percentage of cells that were rolling, surface adherent, or transmigrated (p<0.01). In addition, there was a significant effect of time on each form of behaviour for the EPA-treated cells (p<0.05âˆ'0.01). Bonferroni tests showed significant differences at specific time points as marked; **p<0.01. © Inhibition of neutrophil transmigration was evident at levels of EPA supplementation as low as 50 nM. Data are mean±SEM of five experiments. ANOVA showed significant effects of treatment (p<0.05); *p<0.05 compared to untreated control by Dunnett's test. Error bars indicate SEM.doi:10.1371/journal.pbio.1000177.g004

EPA Does Not Inhibit the Expression of Inflammatory Genes in Endothelial CellsTo define the molecular mechanisms underlying these observations, we tested two prevalent hypotheses. The first, predicated on the observation that n-3-PUFAs regulate the transcription of endothelial cell inflammatory genes by down-regulating the activity of the nuclear factor-ÎºB, predicts changes in the levels of adhesion receptor and chemokine expression after supplementation with EPA [12]–[14]. However, previous studies have investigated the regulation of gene expression in the presence of up to 100 µM of n-3-PUFAs, whereas physiological blood plasma levels of free fatty acid are of the order of 1 µM [15], a level that can be increased several fold upon supplementation. Using 5 µM EPA as an approximation of the highest concentration achievable in vivo, we did not see changes in the expression of endothelial cell adhesion molecules when assessed for RNA transcripts or protein expression (Figure 5 and Table 1). Neither was there any alteration in the levels of cytokines and chemokines secreted by endothelial cells (Table 2). Thus, in this model, we were able to discount the gene regulation hypothesis.

Figure 5. The effect of EPA on TNF-induced surface adhesion molecule expression.The expression of (A) E-selectin and ( VCAM-1 was measured by ELISA in TNF-stimulated endothelial cells that were either unsupplemented or supplemented with EPA. Data are mean±SEM of seven experiments. ANOVA showed that there was a significant effect of the dose of TNF (p<0.01), but not of EPA, on the expression of both receptors.doi:10.1371/journal.pbio.1000177.g005

Table 1. Effect of EPA treatment on mRNA expression of endothelial cell adhesion molecules and chemokines induced by TNF.doi:10.1371/journal.pbio.1000177.t001

Table 2. Effect of EPA treatment of endothelial cells on the TNF-Î±-induced secretion of cytokines and chemokines.doi:10.1371/journal.pbio.1000177.t002

The Anti-Inflammatory Effects of EPA Supplementation Require the Metabolic Activity of Cyclooxygenase EnzymesThe second hypothesis proposes that upon endothelial cell activation, EPA may compete with the n-6-PUFA arachidonic acid (AA; 20:4n-6) for cyclooxygenase enzymes (COX1 and COX2) after both fatty acids are liberated from membrane phospholipids by endogenous phospholipases [16]. Ordinarily, AA is metabolised by COX into 2-series endoperoxides, with downstream synthases converting these into the biologically active 2-series prostanoids [17]. However, when utilised as a COX substrate, the metabolism of EPA generates alternative 3-series prostanoids [16]. The 2-series prostanoids generated by COX activity are known to regulate aspects of the inflammatory response. For example, in the COX2 knockout mouse, neutrophil recruitment is dramatically reduced upon ischaemia and reperfusion-induced injury of the liver when compared to wild-type control animals [18]. In a murine model of lipopolysaccharide-induced lung inflammation, inhibitors of COX function (indomethacin and aspirin) modestly increased neutrophil recruitment [19]. Conversely in vitro, inhibition of COX function by aspirin can inhibit neutrophil migration [20] while having no effect on the levels of neutrophil adhesion to the endothelial cell monolayer [21]. Moreover, in murine models of acute and chronic inflammation, a reduction in PGE2 production by genetic deletion of its membrane-bound synthase, moderates the formation of inflammation-associated granulation tissue and angiogenesis, as well as decreasing the nociception of pain, indicating that PGE2 is proinflammatory in these models [22]. PGD2 also appears to have proinflammatory functions, as overexpression of the synthase generating this prostanoid increases production of inflammatory cytokines and chemokines, leading to exaggerated levels of eosinophil and lymphocyte recruitment [23], a mechanism that operates through the PGD2 receptor DP-2. In contrast, in acute peritoneal inflammation, knockout of PGD2 synthase increased inflammatory cytokine production and retarded the rate of inflammatory resolution by a mechanism that operated through the DP-1 receptor [24]. Thus, PGD2 appears to have pro- or anti-inflammatory capabilities depending on the nature of the inflammatory insult. The inflammatory activity of the equivalent 3-series prostanoids is not known. Here, by introducing a panel of COX inhibitors into endothelial cell cultures at the same time as they were activated with TNF, the effects of EPA supplementation could be replicated, with neutrophil transmigration being dramatically inhibited (Figure 6). Moreover, adding a molecular excess of AA to EPA-supplemented endothelial cells at the point of TNF activation could reverse the blockade of migration (Figure 6). Taken together, these data imply that a COX-derived product of AA is required for the transmigration of neutrophils across TNF-stimulated endothelial cells, and that in the presence of EPA, this pathway is efficiently antagonised.

Figure 6. A cyclooxygenase (COX)-derived eicosanoid is required for neutrophil transmigration across TNF-stimulated endothelial cells.(A) Inhibition of COX-1 (SC-560), COX-2 (NS-398), or both (indomethacin) caused inhibition of neutrophil transmigration, *p<0.05 by t-test. ( Addition of AA to EPA-supplemented endothelial cells during the period of TNF stimulation restored neutrophil transmigration. All data are mean±SEM of four experiments. ANOVA showed significant effects of treatments in both (A and (p<0.01). In (A), Dunnett test showed significant effects of individual treatments compared to untreated control, *p<0.05. In (, Dunnett's test showed significant difference between EPA-treated and untreated control (**p<0.01), but not between AA+EPA and control.doi:10.1371/journal.pbio.1000177.g006

Prostaglandin D2 Is Required for Neutrophil Migration across Endothelial Cells Stimulated with TNF-Î±Endothelial cells generate COX products constitutively. For example, prostacyclin (PGI2) and prostaglandin D2 (PGD2) are endothelial cell-derived vasoactive prostanoids that are also involved in the regulation of haemostasis, being antagonists of platelet activation [17],[25]. These prostanoids are difficult to measure in the systemic blood, as they have reported half-lives in plasma that are measured in minutes [26],[27]. However, the constitutive nature of their production is demonstrated by the presence in urine and body fluids of their downstream metabolic products, PGF1Î± and delta-PGJ2, respectively [28],[29].Prostanoids have a documented ability to regulate the migration of a number of leukocyte subsets. For example, PGD2 induces chemotaxis of eosinophils and T-lymphocytes [30] in vitro. Interestingly, the migration of monocyte-derived migratory dendritic cells (DCs) may be tightly regulated by interplay between different prostanoids. Thus, the ability of these cells to traffic out of tissue into lymph nodes via the lymphatic circulation is dependent upon the presence of PGE2 as a differentiation signal [31]–[34]. In this context PGE2 promotes the function (but not the expression) of the chemokine receptor CCR7, so that DCs efficiently respond to the chemokines CCL19 and CCL21. Importantly, a number of studies have shown that the presence of PGD2 can in turn inhibit the ability of DCs to migrate out of the lungs or the skin during an inflammatory response, although the molecular mechanism by which this inhibition is achieved remains undescribed [35],[36].As prostanoids are well documented to regulate leukocyte migration [37], we tested the hypothesis that PGD2 was the endothelial cell-derived agent providing the stimulus for neutrophil migration across the monolayer. We established a population of surface adherent neutrophils on TNF-treated endothelial cells supplemented with EPA and perfused synthetic PGD2 across the endothelial cells and neutrophils to see whether this would reintroduce transmigration. The provision of exogenous PGD2 but not PGD3 (which could be derived from EPA), fully restored the ability of neutrophils to cross the endothelial cell monolayer (Figure 7A). Prostaglandin D2 has two receptors. Chemoattractant-receptor homologous molecule expressed on Th2 cells receptor (CRTH2 or DP-2) is not expressed in neutrophils [35], whereas the DP-1 receptor has been reported in these cells [38]. Here, neutrophils perfused across TNF-stimulated endothelial cells in the presence of a DP-1 receptor antagonist (BW868C) showed a greatly diminished efficiency of endothelial cell transmigration (Figure 7B). Conversely, the transmigration of neutrophils was re-established on EPA-supplemented endothelial cells when a synthetic DP-1 receptor agonist (BW245C) was perfused across cells adherent to the surface of the monolayer (Figure 7C). Importantly, we could demonstrate that PGD2 was operating directly on the neutrophils, because when these cells were harvested after migrating across a TNF-stimulated endothelial cells they had up-regulated CD11b (the alpha subunit of the Î²2 integrin CD11b/CD18 that is required for efficient neutrophil transmigration) and proteolytically shed L-selectin (Figure 8). However, neutrophils harvested from the surface of EPA-treated endothelial cells did not shed L-selectin or up-regulate CD11b. Thus, in this assay system, the delivery of a PGD2-mediated signal to the neutrophils was necessary for full cellular activation and efficient transendothelial migration.

Figure 7. The role of AA- and EPA-derived eicosanoids in neutrophil transmigration.(A) When PDG2 was perfused across a population of neutrophils adherent to EPA-treated endothelial cells, neutrophil transmigration was restored. However, PGD3 had no significant effect on neutrophil behaviour. All data are mean±SEM of four experiments. ANOVA showed significant effect of treatment on transmigration. *p<0.05 compared to EPA-supplemented endothelial cells in the absence of PGD2 by Dunnett test. ( The DP1 receptor antagonist BW868c dose-dependently inhibited neutrophil transmigration on TNF-stimulated endothelial cells. All data are mean±SEM of five experiments; ANOVA showed a significant effect of BW868c concentration (p<0.01). © Perfusion of the DP-1 receptor agonist, BW245C, across neutrophils adherent to EPA-supplemented endothelial cells restored neutrophil migration to control levels. Data are mean±SEM of five experiments. ANOVA showed a significant effect of treatment on transmigration. **p<0.01 compared to EPA-supplemented endothelial cells in the absence of BW868C by Dunnett test. (D) Neutrophils perfused across TNF-stimulated endothelial cells in the presence of PGD3 showed a significantly reduced ability to migrate across the monolayer. Data are mean±SEM of three experiments; ANOVA showed a significant effect of PGD3 concentration on transmigration (p<0.01). (E) The levels of PGD3 released from endothelial cells are increased after EPA supplementation. Data are mean±SEM of three experiments. ANOVA showed a significant effect of treatment (p<0.05). **p<0.01 for PGD3 production compared to endothelial cells activated with TNF without EPA supplementation by Dunnett's test.doi:10.1371/journal.pbio.1000177.g007

Figure 8. The effect of adhesion on the expression of L-selectin and CD11b on neutrophils.Neutrophils that were freshly isolated, migrated across endothelial cells stimulated with TNF or adherent to the surface of EPA-supplemented endothelial cells (EC) activated with TNF were harvested and the expression of (A) CD11b or ( L-selectin (CD62L) assessed by flow cytometry. Data are mean±SEM of four experiments; ANOVA showed significant effect of treatments in (A) (p<0.01) and ( (p<0.05). In (A and , Dunnett's test showed significant difference between âˆ'EPA and freshly isolated cells (*p<0.01 and *p<0.05, respectively), but not between +EPA and freshly isolated cells.doi:10.1371/journal.pbio.1000177.g008

PGD3 Generated from EPA Antagonises the PGD2-Mediated Migration of Neutrophils across Endothelial CellsTaken together, these data show that PGD2 operates through the neutrophil DP-1 receptor to provide a signal that is essential for transendothelial cell migration. Moreover, the ability of neutrophils to respond to this signal is abolished when endothelial cells are preloaded with EPA from which PGD3 is generated. This implies that PGD3 may be an effective antagonist of the DP-1 receptor. To test this hypothesis, we perfused neutrophils across TNF-stimulated endothelial cells in the presence of exogenous PGD3. Neutrophil transmigration was inhibited in a dose-dependent manner by PGD3, showing that in the presence of endogenously generated PGD2 it could effectively antagonise this process (Figure 7D). Importantly, we were able to show that cells supplemented with EPA-generated an increased amount of PGD3 (Figure 7E).The absolute levels of PGD2 and PGD3 that are generated in our assay system, and thus the ratio of their abundance, are not easily assessed. The levels of PGD2 measured as an endothelial cell releasate were at the limits of detection (Table 3), presumably due to the short half-life of this prostanoid under physiological conditions; however, significant levels of PGD3 were assayed upon EPA supplementation, and concentrations in the order of 5–10 nM had accumulated over the 24 h of the EPA supplementation period. The half-life of PGD3 in serum or albumin-containing medium has not been reported to our knowledge; thus it is not clear whether the measurements made here report the true levels of PGD3 production, or if metabolic degradation of this prostanoid renders these measurements underestimates. Irrespective of this, our experiments utilising synthetic PGD2 demonstrate that concentrations on the order of 1 nM were sufficient to fully recapitulate the process of migration on EPA-supplemented endothelial cells, and when using PGD3 as an antagonist of TNF-induced transmigration marked effects were evident at 10–100 pM. Thus, the measurable levels of PGD3 that we report would certainly be sufficient to effectively antagonise the transmigration of neutrophils in our experimental model.

Table 3. Analysis of eicosanoids by liquid chromatography tandem mass spectrometry.doi:10.1371/journal.pbio.1000177.t003

ConclusionsOur study demonstrates a hitherto unknown regulatory step in the recruitment of neutrophils by cytokine-stimulated endothelial cells (refer to Figure 1 to see how this new step fits with the known regulatory mechanisms of neutrophil recruitment). After initial tethering by selectin molecules, a chemokine signal induces arrest of the cell on the endothelial cell surface. However, the chemokine signal is not sufficient to support transmigration across the endothelial cell monolayer, and the arachidonic acid-derived prostanoid, PGD2, is an essential downstream regulator of this process. PGD2 operates through the DP-1 receptor and this signal can be effectively antagonised by PGD3 which is generated from EPA released by the action of phospholipase enzymes on the phospholipids of EPA supplemented cells. Not only does this study reveal a new step in the recruitment of neutrophil recruitment during inflammation, it also reveals a novel anti-inflammatory mechanism of action of the dietary n-3-PUFA, EPA.

Materials and Methods Top

Cell Culture and Neutrophil IsolationEndothelial cells (EC) were isolated and cultured, as previously described [39], with or without 0–5 µM EPA (Sigma) for 24 h. TNF-Î± (100 U/ml; R & D systems) was added to the EC for the final 4 h of culture. In some experiments, EC were treated with 100 U/ml TNF-Î± in the presence of 10 µM indomethacin or 0.1 µM SC-560 or 1 µM NS-398 (Sigma). In the AA reconstitution experiments, 5 µM AA (Sigma) was added to the culture medium simultaneously with TNF-Î±.Blood from healthy adult volunteers was collected into tubes coated with EDTA (1.6 mg/ml), and neutrophils were separated using 2-step density gradients of Histopaque 1119 and 1077 (Sigma), as previously described [40]. After washing in 0.15% BSA in PBS, cells were counted using a Coulter Multisizer (Coulter Electronics), and neutrophils, which were >95% pure, were resuspended at a concentration of 1×106/ml in PBS/Alb with calcium and magnesium.

RNA Extraction and Real-Time PCRmRNA was isolated from EC using the Qiagen RNEasy Mini Kit 50 (Qiagen) following the manufacturer's instructions. Real-time PCR (RT-PCR) was performed using QuantiTect probe RT-PCR kit according to the manufacturer's instructions (Qiagen). Primers were purchased from Applied Biosystems. The expression of each target gene was normalised to Î²-actin expression, and the data presented represent fold change compared to untreated EC.

Luminex for Secreted Cytokines and ChemokinesSupernatants were collected from EC that had been incubated with 0–5 µM EPA for 24 h and had 100 U/ml TNF added to the culture for the final 4 h of culture. The Luminex kit was purchased from Upstate/Chemicon, and the experiment performed to the manufacturer's instructions. Data were collected and analysed from the samples using a Luminex100 machine (Luminex).

Flow-Based Adhesion AssayGlass capillaries (microslides) containing treated EC monolayers were incorporated into a flow-based adhesion assay as described [39]. Briefly, microslides were attached to cell and fluid reservoirs by silicon tubing at one end and to a withdrawal syringe pump at the other end. After mounting on the stage of a phase contrast microscope, EC were washed for 2 min with PBS containing 0.15% BSA. Neutrophils were then perfused (1×106/ml) at 0.1 Pa for 4 min, followed by 15 min of wash. Video images were recorded throughout the experiment and neutrophil behaviour analysed offline using Image Pro software (Image-Pro Plus). In some experiments, neutrophils were perfused across EPA- and TNF-treated EC, allowed to adhere, and subsequently perfused with 1 nM PGD2 or 1 nM PGD3 (both from Cayman Chemicals). Alternatively, neutrophils were treated with BW868c (Cayman Chemicals), a DP1 receptor antagonist, for 10 min prior to perfusion over TNF-Î±-stimulated EC. Neutrophils were also treated with the DP1 receptor agonist BW245c (Cayman Chemicals) prior to perfusion over EPA- and TNF-Î±-treated EC. Neutrophils were also treated with 0–1 nM PGD3, immediately prior to perfusion across TNF-stimulated EC. In all experiments, lipid reagents, agonists, and antagonists were stored in 100% ethanol under nitrogen gas. Once diluted, the medium contained <0.01% ethanol. All controls contained equivalent concentrations of ethanol.

Analysis of EPA Incorporation into Cellular PhospholipidsFirst-passage confluent EC were treated with 5 µM of EPA for 24 h. EC were removed from the plastic culture dish by scraping and stored in 0.88% KCl solution at âˆ'20°C until analysis. Total lipid was extracted with chloroform:methanol (2:1, v/v) containing butylated hydroxytoluene (50 mg/l) as antioxidant. Fatty acids were subsequently hydrolysed from the lipid and simultaneously methylated by incubation with methylation reagent (methanol containing 2% v/v H2SO4) at 50°C for 2 h. Fatty acid methyl esters were separated and identified using a Hewlett Packard 6890 gas chromatograph (Hewlett Packard) fitted with a 30 mm×32 mm BPX 70 capillary column, film thickness 0.25 µm. Helium, at the initial flow rate of 1.0 ml/min, was used as the carrier gas. Injector and detector temperatures were 275°C, and the column oven temperature was maintained at 170°C for 12 min after sample injection. The oven temperature was programmed to increase from 170 to 210°C at 5°C/min. Fatty acid methyl esters were identified by comparison with authentic standards. Peak areas were quantified using ChemStation software (Hewlett Packard). Each fatty acid was expressed as weight percent of total fatty acids present.

Eicosanoid Extraction and Reverse-Phase High-Performance Liquid Chromatography AnalysisEicosanoids were extracted from control EC using C18 Sep-Pak cartridges (Waters). Briefly, cartridges were conditioned with 5 ml of high-performance liquid chromatography (HPLC)-grade MeOH and rinsed twice with water. Reactions were terminated with addition of 100% ice-cold MeOH, containing 2 ng of internal standard PGE2-d4. Sample volume was adjusted to 10% MeOH with water and applied to the column. The column was rinsed with water and the sample eluted with 2 ml of MeOH. Nitrogen gas was used to dry the sample, which was resuspended in 100 µl of MeOH and stored at âˆ'80°C until analysis.

Prostaglandin Quantitation using Liquid Chromatography Tandem Mass SpectrometryProstaglandins were separated on a C18 ODS2, 5 µm, 150×4.6-mm column (Waters) using a gradient of 50%–90% B over 20 min (A, water:acetonitrile:acetic acid, 75:25:0.1; B, methanol:acetonitrile:acetic acid, 60:40:0.1) at 1 ml/min. Products were quantitated by directing the HPLC output directly into the electrospray source of a Q-Trap mass spectrometer (Applied Biosystems 4000 Q-Trap) operating in the negative mode (âˆ'4,500 V). Individual prostaglandins were monitored in the Multiple Reaction Monitoring (MRM) mode using specific parent to daughter transitions of m/z 349–269 for PGD3 with collision energies of âˆ'26 V. Products were identified and quantified with reference to the appropriate standards run in parallel under the same conditions, with 2 ng of PGE2-d4 (m/z 355–275) added as an internal standard.

Analysis of Neutrophil Adhesion Molecule Expression by Flow CytometryNeutrophils for analysis were isolated from whole blood as described above. Control cells were stained for flow cytometry directly after isolation. Cells rolling on EPA-treated endothelial cells in microslides were harvested by elution with 0.02% EDTA after 4 min of neutrophil perfusion and 11 min of wash buffer perfusion (totalling 15 min of contact with endothelial cells). In order to harvest neutrophils that had migrated across endothelial cells, endothelium was cultured on 1.5 mg/ml type I collagen gels (Becton Dickinson). After endothelial cell stimulation with TNF for 4 h, neutrophils were added for 15 min. Surface-adherent cells were washed off with 0.02% EDTA, and the gel was dissolved using type VII collagenase (Sigma). Neutrophils were harvested from the dissolved gel by centrifugation. All neutrophil populations were stained for surface expression of CD11b (PE-conjugated, clone 2LPM19c, 2 µg/ml; DAKO) or CD62L (FITC-conjugated, clone Dreg56,10 µg/ml, Beckton Dickinson). Expression was analysed by flow cytometry using a DAKO CyAn, and data analysed using Summit software (Becton Dickinson). Data are expressed as mean fluorescent intensity.

Statistical AnalysisData were analysed using Prism software (GraphPad software). Results are presented as means±standard error of the mean (SEM). Comparisons between individual treatments were by paired t-test where appropriate. ANOVA was performed to assess the effect of EPA concentration on PMN transmigration. Significant findings were investigated further using Bonferroni multiple comparison test or Dunnett's test.

Author Contributions TopThe author(s) have made the following declarations about their contributions: Conceived and designed the experiments: VBO RFG PCC GBN GER. Performed the experiments: SPT CMY BHM JM. Analyzed the data: SPT CMY BHM VBO JM GER. Contributed reagents/materials/analysis tools: VBO RFG PCC GBN. Wrote the paper: GER.

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November 5, 2011

Essentially, the article shows in a clearer way how Omega-6s (here arachidonic

acid) are pro-inflammatory, confirms that EPA plays an opposite role (ie

anti-inflammatory), and gives a new idea for future research.

>

November 5, 2011

Hi Joan,my belief is that supplementing with EPA would benefit many

inflammatory conditions (heart disease,arthritis,and I suspect A.E.R.D.as

well).Also we consume too much omega-6 fatty acids in our diet(ie 20-30:1

omega-6 to omega-3) so dietary change to get this ratio closer to 1:1 should be

a goal.

Tnf-alpha is one of the chemokines associated with TH-2 or Samter's like

immune response.Tnf-alpha is also responsible for neutrophil migration.

The same COX enzyme can use arachidonic acid or EPA as substrate producing

prostaglandin-D2 (PG-D2)or prostaglandin-D3 (PG-D3)respectively.PG-D2

facilitates neutrophils being transported thru the endothelium of the blood

vessel into tissues whereas PG-3 inhibits it.Like aspirin omega-3's have several

different ways of influencing inflammation.

As both Omega-3 and aspirin can thin the blood it would be wise to consult

your M.D. or E.N.T.regarding this ie checking clotting time.Other things such as

Vitamin E ,alcohol e.t.c. also thin the blood.Any medical conditions such as

peptic ulcer,low Iron or Hemoglobin etc might contraindicate this.

Also the dose of aspirin you take would influence how much EPA you could

take.Myself I take a baby aspirin and 2.0 grams of EPA and DHA combined daily.I

also take l-arginine to help with endothelial dysfunction.These are my ideas and

you need to consult with a medical professional for guidance.

The other point I would make is that aspirin probably doesn't help with

cardiovascular disease process itself as much as omega-3 does.

>

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> > Research Article

> > Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a New Step

in Neutrophil Recruitment

> >

> > While investigating new mechanisms by which the dietary omega-3 fatty acids

regulate inflammation, the authors have identified a new step in the regulation

of neutrophil migration across vascular endothelial cells.

> >

> > Article

> > Metrics

> > Related Content

> > Comments: 0

> > To add a note, highlight some text. Hide notes

> > Make a general comment

> > Jump to

> >

> > Abstract

> > Author Summary

> > Introduction

> > Results/Discussion

> > Materials and Methods

> > Author Contributions

> > References

> > P. Tull1, Clara M. Yates1, H. Maskrey2, B.

O'Donnell2, Jackie Madden3, F. Grimble3, Philip C. Calder3, Gerard B.

Nash1, G. Ed. Rainger1*

> >

> > 1 Centre for Cardiovascular Sciences, School of Clinical and Experimental

Medicine, The Medical School, The University of Birmingham, Birmingham, United

Kingdom, 2 Department of Medical Biochemistry and Immunology, School of

Medicine, Cardiff University, Cardiff, United Kingdom, 3 Institute of Human

Nutrition, School of Medicine, University of Southampton, Southampton, United

Kingdom

> >

> > Abstract Top

> >

> > Inflammation is a physiological response to tissue trauma or infection, but

leukocytes, which are the effector cells of the inflammatory process, have

powerful tissue remodelling capabilities. Thus, to ensure their precise

localisation, passage of leukocytes from the blood into inflamed tissue is

tightly regulated. Recruitment of blood borne neutrophils to the tissue stroma

occurs during early inflammation. In this process, peptide agonists of the

chemokine family are assumed to provide a chemotactic stimulus capable of

supporting the migration of neutrophils across vascular endothelial cells,

through the basement membrane of the vessel wall, and out into the tissue

stroma. Here, we show that, although an initial chemokine stimulus is essential

for the recruitment of flowing neutrophils by endothelial cells stimulated with

the inflammatory cytokine tumour necrosis factor-Î±, transit of the endothelial

monolayer is regulated by an additional and downstream stimulus. This signal is

supplied by the metabolism of the omega-6-polyunsaturated fatty acid (n-6-PUFA),

arachidonic acid, into the eicosanoid prostaglandin-D2 (PGD2) by cyclooxygenase

(COX) enzymes. This new step in the neutrophil recruitment process was revealed

when the dietary n-3-PUFA, eicosapentaenoic acid (EPA), was utilised as an

alternative substrate for COX enzymes, leading to the generation of PGD3. This

alternative series eicosanoid inhibited the migration of neutrophils across

endothelial cells by antagonising the PGD2 receptor. Here, we describe a new

step in the neutrophil recruitment process that relies upon a lipid-mediated

signal to regulate the migration of neutrophils across endothelial cells. PGD2

signalling is subordinate to the chemokine-mediated activation of neutrophils,

but without the sequential delivery of this signal, neutrophils fail to

penetrate the endothelial cell monolayer. Importantly, the ability of the

dietary n-3-PUFA, EPA, to inhibit this process not only revealed an unsuspected

level of regulation in the migration of inflammatory leukocytes, it also

contributes to our understanding of the interactions of this bioactive lipid

with the inflammatory system. Moreover, it indicates the potential for novel

therapeutics that target the inflammatory system with greater affinity and/or

specificity than supplementing the diet with n-3-PUFAs.

> >

> > Author Summary Top

> >

> > Inflammation is a physiological response to tissue trauma or infection.

Neutrophils, which circulate in the blood stream, are the first inflammatory

cells to be recruited to a site of tissue inflammation. In response to

recruitment signals provided by chemotactic peptides called chemokines,

neutrophils traverse the endothelial cell lining of blood vessels. This process

involves a multistep cascade of neutrophil adhesion and activation events on the

endothelial barrier. While investigating the anti-inflammatory functions of the

omega-3 fatty acid , EPA, which is found, for instance, in dietary fish oil, we

identified an additional unexpected lipid-derived signal that is essential for

neutrophil migration across the endothelium. Our experiments show that a

chemokine delivered the first signal needed to bind neutrophils firmly to the

endothelial surface. However, in order to traverse the endothelium, a subsequent

signal delivered by prostaglandin-D2 (PGD2), a lipid derived from the omega-6

fatty acid arachidonic acid, was essential. When EPA, was introduced into the

experiment, it was used to form PGD3. This alternative lipid blocked

interactions between PGD2 and its receptor on neutrophils, preventing the

process of migration across the endothelial barrier. Thus, we reveal a new step

in the recruitment of neutrophils during inflammation, and a novel

anti-inflammatory mechanism of action of dietary EPA.

> >

> > Citation: Tull SP, Yates CM, Maskrey BH, O'Donnell VB, Madden J, et al.

(2009) Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a New

Step in Neutrophil Recruitment. PLoS Biol 7(8): e1000177.

doi:10.1371/journal.pbio.1000177

> >

> > Academic Editor: Philip M. , NIH/NIAID, United States of America

> >

> > Received: February 6, 2009; Accepted: July 14, 2009; Published: August 25,

2009

> >

under the terms of the Creative Commons Attribution License, which permits

unrestricted use, distribution, and reproduction in any medium, provided the

original author and source are credited.

> >

> > Funding: This work was supported by the Biotechnology and Biological

Sciences Research Council, United Kingdom (grant BBS/B/0105) and by the British

Heart Foundation (grant BS/97001). The funders had no role in study design, data

collection and analysis, decision to publish, or preparation of the manuscript.

> >

> > Competing interests: The authors have declared that no competing interests

exist.

> >

> > Abbreviations: DC, dendritic cell

> >

> > * E-mail: g.e.rainger@...

> >

> > Introduction Top

> >

> > In vertebrates, tissue trauma or infection causes the rapid initiation of an

inflammatory reaction. The early phase of this phylactic response results in the

localised recruitment of cells of the innate immune system from the blood, a

tissue infiltrate that is dominated by neutrophils. The molecular processes that

support the initial interactions between blood-borne neutrophils and the

endothelial cells lining postcapillary venules (the site in the blood

vasculature where leukocytes are recruited during inflammation) are well

described (see Figure 1 for a schematic representation of the steps in the

neutrophil recruitment process). In response to the localised production of

inflammatory mediators, such as the cytokine tumour necrosis factor-Î± (TNF),

activated endothelial cells decorate themselves with specialised adhesion

receptors of the selectin family [1]–[3]. Due to their ability to rapidly form

strong but short-lived bonds with carbohydrate counter-ligands on the neutrophil

surface, E- and P-selectin are capable of tethering neutrophils from rapidly

flowing blood [4]. The sequential formation and dissolution of selectin bonds

also support a characteristic and dynamic form of adhesion, referred to as

rolling [5]. Rolling adhesion does not require neutrophil activation. However,

neutrophil migration through the vessel wall and into the inflamed tissue is

dependent upon the receipt of an activating stimulus [5],[6]. We have previously

shown that endothelial cells can present peptide agonists of the CXC-chemokine

family to rolling neutrophils and thus stabilise adhesion [7],[8]. Ligation of

the neutrophil CXC-Receptor-2 (CXCR2) by these agents is essential for

activation of the Î²2-intergrin adhesion receptors and the reorganisation of the

actin cytoskeleton that support transendothelial cell migration [8]. Here, we

show that in the presence of an antibody that blocks chemokine interactions with

CXCR2, neutrophils roll on the endothelial cell surface indefinitely (Figure 2),

demonstrating that a chemokine signal is essential for neutrophil activation on

TNF-stimulated endothelial cells. However, the removal of this primary

activating stimulus provides no information on the requirement for additional,

downstream signals, which might coordinate transit of the vessel wall and onward

migration into stromal tissues.

> >

> > Figure 1. Steps in the process of neutrophil recruitment.

> >

> > Step 1: Neutrophils are captured from flow and tether and roll on tumour

necrosis factor-Î± (TNF)-stimulated endothelial cells. Step 2: Neutrophils are

then activated by the action of CXC-chemokines on the chemokine receptor, CXCR2,

a process resulting in the activation of neutrophil Î²2-integrins. Step 3:

Î²2-integrins engage counter receptors on the endothelial cell surface, and the

neutrophil becomes stationary. Step 4: Prostaglandin-D2 (PGD2), generated by the

action of cyclooxygenase enzymes on the n-6-PUFA arachidonic acid (AA), binds

the PGD2 receptor, DP-1. DP-1 generates signals that stabilise neutrophil

adhesion, induce neutrophil shape change, and support the process of

transmigration across the endothelial cell monolayer. Step 5: If the endothelial

cells have been supplemented with the n-3-PUFA, eicosapentaenoic acid (EPA), the

alternative series prostanoid, PGD3, is generated, and this antagonises

PGD2-mediated neutrophil responses.

> >

> > doi:10.1371/journal.pbio.1000177.g001

> >

> > Figure 2. Chemokine signalling through CXCR2 is essential for neutrophil

activation on TNF-stimulated endothelial cells.

> >

> > Using a flow-based adhesion assay, neutrophils isolated from whole blood

were perfused across endothelial cells. Neutrophils were recruited to

endothelial cells treated with TNF, but did not adhere to unstimulated

endothelium. Detailed analysis of neutrophil behaviour showed that on

TNF-stimulated endothelial cells, the majority of recruited cells transmigrated

across the monolayer. When neutrophils were treated with anti-CXCR2, but not

anti-CXCR1, neutrophil activation was inhibited so that nearly all of the

recruited neutrophils rolled indefinitely on the monolayer and could not

migrate; data are mean±SEM of four experiments **p<0.01 for comparison by paired

t-test of neutrophil behaviour on TNF stimulated endothelial cells in the

presence or absence of anti-CXCR2.

> >

> > doi:10.1371/journal.pbio.1000177.g002

> > Results/Discussion Top

> >

> > EPA Released from Endothelial Cell Membrane Phospholipids Inhibits the

Recruitment of Neutrophils

> >

> > Dietary omega-3 polyunsaturated fatty acids (n-3-PUFAs) have

anti-inflammatory properties. For example, their inclusion in the diet in the

form of n-3-PUFA–rich fish oil reduces the symptoms of disease as well as the

use of nonsteroidal anti-inflammatory drugs in arthritis patients with severe

inflammatory joint disease [9]. In addition, dietary n-3-PUFAs may offer

protection against vascular pathology associated with atherosclerosis, having

been reported to be efficacious in epidemiological studies [10]. By modulating

inflammation within the artery wall, n-3-PUFAS also alter the cellular and the

structural composition of advanced atherosclerotic plaques in a manner that

could reduce the incidence of plaque rupture or ulceration, a process that

precedes tissue infarction (e.g., heart attack or stroke) [11]. Despite these

documented anti-inflammatory benefits, the mode(s) of interaction of these

lipids with the immune and inflammatory systems are not well understood. When we

supplemented endothelial cell culture medium with the major n-3 -PUFA found in

dietary fish oil supplements, eicosapentaenoic acid (EPA; 20:5n-3), it was

incorporated into cellular phospholipids so that upon withdrawal of the free

fatty acid from the culture medium, a pool of esterified EPA remained localised

within endothelial cell membranes (Figure 3A and 3B). After withdrawal of free

fatty acid and stimulation with TNF, EPA-treated cells were able to support

similar levels of neutrophil adhesion to those that had received no lipid

supplement (Figure 4A). However, detailed analysis showed that the behaviour of

neutrophils on the two populations of endothelial cells was very different. In

response to TNF, but in the absence of EPA, a small population of adherent

neutrophils (~20%) rolled throughout the duration of a flow adhesion assay

(Figure 4B). The remaining cells became activated on the endothelial cell

surface, and this population of neutrophils became progressively smaller as

cells migrated across the endothelial cell monolayer (Figure 4B). In contrast,

when endothelial cells had been treated with EPA prior to TNF stimulation, there

was a marked reduction in the number of cells undergoing transendothelial cell

migration (Figure 4B). This was reduced at the earliest time point and did not

increase over the duration of the experiment. Interestingly however, many of the

cells that did become activated on the surface of endothelial cells reverted to

a rolling form of adhesion (Figure 4B), implying that after the receipt of an

initial chemokine stimulus, a second signal was required to allow prolonged

adhesion and migration across the endothelial cell monolayer. Experiments using

different concentrations of EPA showed that these inhibitory effects were

evident at concentrations as low as 50 nM (Figure 4C).

> >

> > Figure 3. The effects of EPA supplementation on the fatty acid content of

endothelial cell phospholoipds.

> >

> > (A and The concentration of EPA in membrane phospholipids was increased

after supplementation of culture medium with EPA for 24 h. Error bars indicate

SEM. AA, arachidonic acid; FA, fatty acid; PA, palmitic acid; SA, stearic acid.

> >

> > doi:10.1371/journal.pbio.1000177.g003

> >

> > Figure 4. The effects of EPA supplementation on the adhesive behaviour of

neutrophils.

> >

> > (A) EPA supplementation did not affect the number of neutrophils initially

adhering to endothelial cells from flow. ( However, the time course of

neutrophil behaviour on TNF-stimulated endothelial cells showed that EPA

supplementation drastically altered neutrophil behaviour. Thus, the number of

activated and surface adherent neutrophils decreased with time in the presence

or absence of EPA. However, on endothelium that had not been supplemented with

EPA, this was because neutrophils transmigrated across the monolayer, whereas on

EPA-supplemented endothelium this was because activated cells reverted back to a

rolling form of adhesion. ANOVA showed that there was a significant effect of

treatment (i.e., ±EPA) on the percentage of cells that were rolling, surface

adherent, or transmigrated (p<0.01). In addition, there was a significant effect

of time on each form of behaviour for the EPA-treated cells (p<0.05âˆ'0.01).

Bonferroni tests showed significant differences at specific time points as

marked; **p<0.01. © Inhibition of neutrophil transmigration was evident at

levels of EPA supplementation as low as 50 nM. Data are mean±SEM of five

experiments. ANOVA showed significant effects of treatment (p<0.05); *p<0.05

compared to untreated control by Dunnett's test. Error bars indicate SEM.

> >

> > doi:10.1371/journal.pbio.1000177.g004

> > EPA Does Not Inhibit the Expression of Inflammatory Genes in Endothelial

Cells

> >

> > To define the molecular mechanisms underlying these observations, we tested

two prevalent hypotheses. The first, predicated on the observation that

n-3-PUFAs regulate the transcription of endothelial cell inflammatory genes by

down-regulating the activity of the nuclear factor-ÎºB, predicts changes in the

levels of adhesion receptor and chemokine expression after supplementation with

EPA [12]–[14]. However, previous studies have investigated the regulation of

gene expression in the presence of up to 100 µM of n-3-PUFAs, whereas

physiological blood plasma levels of free fatty acid are of the order of 1 µM

[15], a level that can be increased several fold upon supplementation. Using 5

µM EPA as an approximation of the highest concentration achievable in vivo, we

did not see changes in the expression of endothelial cell adhesion molecules

when assessed for RNA transcripts or protein expression (Figure 5 and Table 1).

Neither was there any alteration in the levels of cytokines and chemokines

secreted by endothelial cells (Table 2). Thus, in this model, we were able to

discount the gene regulation hypothesis.

> >

> > Figure 5. The effect of EPA on TNF-induced surface adhesion molecule

expression.

> >

> > The expression of (A) E-selectin and ( VCAM-1 was measured by ELISA in

TNF-stimulated endothelial cells that were either unsupplemented or supplemented

with EPA. Data are mean±SEM of seven experiments. ANOVA showed that there was a

significant effect of the dose of TNF (p<0.01), but not of EPA, on the

expression of both receptors.

> >

> > doi:10.1371/journal.pbio.1000177.g005

> >

> > Table 1. Effect of EPA treatment on mRNA expression of endothelial cell

adhesion molecules and chemokines induced by TNF.

> >

> > doi:10.1371/journal.pbio.1000177.t001

> >

> > Table 2. Effect of EPA treatment of endothelial cells on the TNF-Î±-induced

secretion of cytokines and chemokines.

> >

> > doi:10.1371/journal.pbio.1000177.t002

> > The Anti-Inflammatory Effects of EPA Supplementation Require the Metabolic

Activity of Cyclooxygenase Enzymes

> >

> > The second hypothesis proposes that upon endothelial cell activation, EPA

may compete with the n-6-PUFA arachidonic acid (AA; 20:4n-6) for cyclooxygenase

enzymes (COX1 and COX2) after both fatty acids are liberated from membrane

phospholipids by endogenous phospholipases [16]. Ordinarily, AA is metabolised

by COX into 2-series endoperoxides, with downstream synthases converting these

into the biologically active 2-series prostanoids [17]. However, when utilised

as a COX substrate, the metabolism of EPA generates alternative 3-series

prostanoids [16]. The 2-series prostanoids generated by COX activity are known

to regulate aspects of the inflammatory response. For example, in the COX2

knockout mouse, neutrophil recruitment is dramatically reduced upon ischaemia

and reperfusion-induced injury of the liver when compared to wild-type control

animals [18]. In a murine model of lipopolysaccharide-induced lung inflammation,

inhibitors of COX function (indomethacin and aspirin) modestly increased

neutrophil recruitment [19]. Conversely in vitro, inhibition of COX function by

aspirin can inhibit neutrophil migration [20] while having no effect on the

levels of neutrophil adhesion to the endothelial cell monolayer [21]. Moreover,

in murine models of acute and chronic inflammation, a reduction in PGE2

production by genetic deletion of its membrane-bound synthase, moderates the

formation of inflammation-associated granulation tissue and angiogenesis, as

well as decreasing the nociception of pain, indicating that PGE2 is

proinflammatory in these models [22]. PGD2 also appears to have proinflammatory

functions, as overexpression of the synthase generating this prostanoid

increases production of inflammatory cytokines and chemokines, leading to

exaggerated levels of eosinophil and lymphocyte recruitment [23], a mechanism

that operates through the PGD2 receptor DP-2. In contrast, in acute peritoneal

inflammation, knockout of PGD2 synthase increased inflammatory cytokine

production and retarded the rate of inflammatory resolution by a mechanism that

operated through the DP-1 receptor [24]. Thus, PGD2 appears to have pro- or

anti-inflammatory capabilities depending on the nature of the inflammatory

insult. The inflammatory activity of the equivalent 3-series prostanoids is not

known. Here, by introducing a panel of COX inhibitors into endothelial cell

cultures at the same time as they were activated with TNF, the effects of EPA

supplementation could be replicated, with neutrophil transmigration being

dramatically inhibited (Figure 6). Moreover, adding a molecular excess of AA to

EPA-supplemented endothelial cells at the point of TNF activation could reverse

the blockade of migration (Figure 6). Taken together, these data imply that a

COX-derived product of AA is required for the transmigration of neutrophils

across TNF-stimulated endothelial cells, and that in the presence of EPA, this

pathway is efficiently antagonised.

> >

> > Figure 6. A cyclooxygenase (COX)-derived eicosanoid is required for

neutrophil transmigration across TNF-stimulated endothelial cells.

> >

> > (A) Inhibition of COX-1 (SC-560), COX-2 (NS-398), or both (indomethacin)

caused inhibition of neutrophil transmigration, *p<0.05 by t-test. ( Addition

of AA to EPA-supplemented endothelial cells during the period of TNF stimulation

restored neutrophil transmigration. All data are mean±SEM of four experiments.

ANOVA showed significant effects of treatments in both (A and (p<0.01). In

(A), Dunnett test showed significant effects of individual treatments compared

to untreated control, *p<0.05. In (, Dunnett's test showed significant

difference between EPA-treated and untreated control (**p<0.01), but not between

AA+EPA and control.

> >

> > doi:10.1371/journal.pbio.1000177.g006

> > Prostaglandin D2 Is Required for Neutrophil Migration across Endothelial

Cells Stimulated with TNF-Î±

> >

> > Endothelial cells generate COX products constitutively. For example,

prostacyclin (PGI2) and prostaglandin D2 (PGD2) are endothelial cell-derived

vasoactive prostanoids that are also involved in the regulation of haemostasis,

being antagonists of platelet activation [17],[25]. These prostanoids are

difficult to measure in the systemic blood, as they have reported half-lives in

plasma that are measured in minutes [26],[27]. However, the constitutive nature

of their production is demonstrated by the presence in urine and body fluids of

their downstream metabolic products, PGF1Î± and delta-PGJ2, respectively

[28],[29].

> >

> > Prostanoids have a documented ability to regulate the migration of a number

of leukocyte subsets. For example, PGD2 induces chemotaxis of eosinophils and

T-lymphocytes [30] in vitro. Interestingly, the migration of monocyte-derived

migratory dendritic cells (DCs) may be tightly regulated by interplay between

different prostanoids. Thus, the ability of these cells to traffic out of tissue

into lymph nodes via the lymphatic circulation is dependent upon the presence of

PGE2 as a differentiation signal [31]–[34]. In this context PGE2 promotes the

function (but not the expression) of the chemokine receptor CCR7, so that DCs

efficiently respond to the chemokines CCL19 and CCL21. Importantly, a number of

studies have shown that the presence of PGD2 can in turn inhibit the ability of

DCs to migrate out of the lungs or the skin during an inflammatory response,

although the molecular mechanism by which this inhibition is achieved remains

undescribed [35],[36].

> >

> > As prostanoids are well documented to regulate leukocyte migration [37], we

tested the hypothesis that PGD2 was the endothelial cell-derived agent providing

the stimulus for neutrophil migration across the monolayer. We established a

population of surface adherent neutrophils on TNF-treated endothelial cells

supplemented with EPA and perfused synthetic PGD2 across the endothelial cells

and neutrophils to see whether this would reintroduce transmigration. The

provision of exogenous PGD2 but not PGD3 (which could be derived from EPA),

fully restored the ability of neutrophils to cross the endothelial cell

monolayer (Figure 7A). Prostaglandin D2 has two receptors.

Chemoattractant-receptor homologous molecule expressed on Th2 cells receptor

(CRTH2 or DP-2) is not expressed in neutrophils [35], whereas the DP-1 receptor

has been reported in these cells [38]. Here, neutrophils perfused across

TNF-stimulated endothelial cells in the presence of a DP-1 receptor antagonist

(BW868C) showed a greatly diminished efficiency of endothelial cell

transmigration (Figure 7B). Conversely, the transmigration of neutrophils was

re-established on EPA-supplemented endothelial cells when a synthetic DP-1

receptor agonist (BW245C) was perfused across cells adherent to the surface of

the monolayer (Figure 7C). Importantly, we could demonstrate that PGD2 was

operating directly on the neutrophils, because when these cells were harvested

after migrating across a TNF-stimulated endothelial cells they had up-regulated

CD11b (the alpha subunit of the Î²2 integrin CD11b/CD18 that is required for

efficient neutrophil transmigration) and proteolytically shed L-selectin (Figure

8). However, neutrophils harvested from the surface of EPA-treated endothelial

cells did not shed L-selectin or up-regulate CD11b. Thus, in this assay system,

the delivery of a PGD2-mediated signal to the neutrophils was necessary for full

cellular activation and efficient transendothelial migration.

> >

> > Figure 7. The role of AA- and EPA-derived eicosanoids in neutrophil

transmigration.

> >

> > (A) When PDG2 was perfused across a population of neutrophils adherent to

EPA-treated endothelial cells, neutrophil transmigration was restored. However,

PGD3 had no significant effect on neutrophil behaviour. All data are mean±SEM of

four experiments. ANOVA showed significant effect of treatment on

transmigration. *p<0.05 compared to EPA-supplemented endothelial cells in the

absence of PGD2 by Dunnett test. ( The DP1 receptor antagonist BW868c

dose-dependently inhibited neutrophil transmigration on TNF-stimulated

endothelial cells. All data are mean±SEM of five experiments; ANOVA showed a

significant effect of BW868c concentration (p<0.01). © Perfusion of the DP-1

receptor agonist, BW245C, across neutrophils adherent to EPA-supplemented

endothelial cells restored neutrophil migration to control levels. Data are

mean±SEM of five experiments. ANOVA showed a significant effect of treatment on

transmigration. **p<0.01 compared to EPA-supplemented endothelial cells in the

absence of BW868C by Dunnett test. (D) Neutrophils perfused across

TNF-stimulated endothelial cells in the presence of PGD3 showed a significantly

reduced ability to migrate across the monolayer. Data are mean±SEM of three

experiments; ANOVA showed a significant effect of PGD3 concentration on

transmigration (p<0.01). (E) The levels of PGD3 released from endothelial cells

are increased after EPA supplementation. Data are mean±SEM of three experiments.

ANOVA showed a significant effect of treatment (p<0.05). **p<0.01 for PGD3

production compared to endothelial cells activated with TNF without EPA

supplementation by Dunnett's test.

> >

> > doi:10.1371/journal.pbio.1000177.g007

> >

> > Figure 8. The effect of adhesion on the expression of L-selectin and CD11b

on neutrophils.

> >

> > Neutrophils that were freshly isolated, migrated across endothelial cells

stimulated with TNF or adherent to the surface of EPA-supplemented endothelial

cells (EC) activated with TNF were harvested and the expression of (A) CD11b or

( L-selectin (CD62L) assessed by flow cytometry. Data are mean±SEM of four

experiments; ANOVA showed significant effect of treatments in (A) (p<0.01) and

( (p<0.05). In (A and , Dunnett's test showed significant difference between

âˆ'EPA and freshly isolated cells (*p<0.01 and *p<0.05, respectively), but not

between +EPA and freshly isolated cells.

> >

> > doi:10.1371/journal.pbio.1000177.g008

> > PGD3 Generated from EPA Antagonises the PGD2-Mediated Migration of

Neutrophils across Endothelial Cells

> >

> > Taken together, these data show that PGD2 operates through the neutrophil

DP-1 receptor to provide a signal that is essential for transendothelial cell

migration. Moreover, the ability of neutrophils to respond to this signal is

abolished when endothelial cells are preloaded with EPA from which PGD3 is

generated. This implies that PGD3 may be an effective antagonist of the DP-1

receptor. To test this hypothesis, we perfused neutrophils across TNF-stimulated

endothelial cells in the presence of exogenous PGD3. Neutrophil transmigration

was inhibited in a dose-dependent manner by PGD3, showing that in the presence

of endogenously generated PGD2 it could effectively antagonise this process

(Figure 7D). Importantly, we were able to show that cells supplemented with

EPA-generated an increased amount of PGD3 (Figure 7E).

> >

> > The absolute levels of PGD2 and PGD3 that are generated in our assay system,

and thus the ratio of their abundance, are not easily assessed. The levels of

PGD2 measured as an endothelial cell releasate were at the limits of detection

(Table 3), presumably due to the short half-life of this prostanoid under

physiological conditions; however, significant levels of PGD3 were assayed upon

EPA supplementation, and concentrations in the order of 5–10 nM had accumulated

over the 24 h of the EPA supplementation period. The half-life of PGD3 in serum

or albumin-containing medium has not been reported to our knowledge; thus it is

not clear whether the measurements made here report the true levels of PGD3

production, or if metabolic degradation of this prostanoid renders these

measurements underestimates. Irrespective of this, our experiments utilising

synthetic PGD2 demonstrate that concentrations on the order of 1 nM were

sufficient to fully recapitulate the process of migration on EPA-supplemented

endothelial cells, and when using PGD3 as an antagonist of TNF-induced

transmigration marked effects were evident at 10–100 pM. Thus, the measurable

levels of PGD3 that we report would certainly be sufficient to effectively

antagonise the transmigration of neutrophils in our experimental model.

> >

> > Table 3. Analysis of eicosanoids by liquid chromatography tandem mass

spectrometry.

> >

> > doi:10.1371/journal.pbio.1000177.t003

> > Conclusions

> >

> > Our study demonstrates a hitherto unknown regulatory step in the recruitment

of neutrophils by cytokine-stimulated endothelial cells (refer to Figure 1 to

see how this new step fits with the known regulatory mechanisms of neutrophil

recruitment). After initial tethering by selectin molecules, a chemokine signal

induces arrest of the cell on the endothelial cell surface. However, the

chemokine signal is not sufficient to support transmigration across the

endothelial cell monolayer, and the arachidonic acid-derived prostanoid, PGD2,

is an essential downstream regulator of this process. PGD2 operates through the

DP-1 receptor and this signal can be effectively antagonised by PGD3 which is

generated from EPA released by the action of phospholipase enzymes on the

phospholipids of EPA supplemented cells. Not only does this study reveal a new

step in the recruitment of neutrophil recruitment during inflammation, it also

reveals a novel anti-inflammatory mechanism of action of the dietary n-3-PUFA,

EPA.

> >

> > Materials and Methods Top

> >

> > Cell Culture and Neutrophil Isolation

> >

> > Endothelial cells (EC) were isolated and cultured, as previously described

[39], with or without 0–5 µM EPA (Sigma) for 24 h. TNF-Î± (100 U/ml; R & D

systems) was added to the EC for the final 4 h of culture. In some experiments,

EC were treated with 100 U/ml TNF-Î± in the presence of 10 µM indomethacin or

0.1 µM SC-560 or 1 µM NS-398 (Sigma). In the AA reconstitution experiments, 5 µM

AA (Sigma) was added to the culture medium simultaneously with TNF-Î±.

> >

> > Blood from healthy adult volunteers was collected into tubes coated with

EDTA (1.6 mg/ml), and neutrophils were separated using 2-step density gradients

of Histopaque 1119 and 1077 (Sigma), as previously described [40]. After washing

in 0.15% BSA in PBS, cells were counted using a Coulter Multisizer (Coulter

Electronics), and neutrophils, which were >95% pure, were resuspended at a

concentration of 1×106/ml in PBS/Alb with calcium and magnesium.

> >

> > RNA Extraction and Real-Time PCR

> >

> > mRNA was isolated from EC using the Qiagen RNEasy Mini Kit 50 (Qiagen)

following the manufacturer's instructions. Real-time PCR (RT-PCR) was performed

using QuantiTect probe RT-PCR kit according to the manufacturer's instructions

(Qiagen). Primers were purchased from Applied Biosystems. The expression of each

target gene was normalised to Î²-actin expression, and the data presented

represent fold change compared to untreated EC.

> >

> > Luminex for Secreted Cytokines and Chemokines

> >

> > Supernatants were collected from EC that had been incubated with 0–5 µM EPA

for 24 h and had 100 U/ml TNF added to the culture for the final 4 h of culture.

The Luminex kit was purchased from Upstate/Chemicon, and the experiment

performed to the manufacturer's instructions. Data were collected and analysed

from the samples using a Luminex100 machine (Luminex).

> >

> > Flow-Based Adhesion Assay

> >

> > Glass capillaries (microslides) containing treated EC monolayers were

incorporated into a flow-based adhesion assay as described [39]. Briefly,

microslides were attached to cell and fluid reservoirs by silicon tubing at one

end and to a withdrawal syringe pump at the other end. After mounting on the

stage of a phase contrast microscope, EC were washed for 2 min with PBS

containing 0.15% BSA. Neutrophils were then perfused (1×106/ml) at 0.1 Pa for 4

min, followed by 15 min of wash. Video images were recorded throughout the

experiment and neutrophil behaviour analysed offline using Image Pro software

(Image-Pro Plus). In some experiments, neutrophils were perfused across EPA- and

TNF-treated EC, allowed to adhere, and subsequently perfused with 1 nM PGD2 or 1

nM PGD3 (both from Cayman Chemicals). Alternatively, neutrophils were treated

with BW868c (Cayman Chemicals), a DP1 receptor antagonist, for 10 min prior to

perfusion over TNF-Î±-stimulated EC. Neutrophils were also treated with the DP1

receptor agonist BW245c (Cayman Chemicals) prior to perfusion over EPA- and

TNF-Î±-treated EC. Neutrophils were also treated with 0–1 nM PGD3, immediately

prior to perfusion across TNF-stimulated EC. In all experiments, lipid reagents,

agonists, and antagonists were stored in 100% ethanol under nitrogen gas. Once

diluted, the medium contained <0.01% ethanol. All controls contained equivalent

concentrations of ethanol.

> >

> > Analysis of EPA Incorporation into Cellular Phospholipids

> >

> > First-passage confluent EC were treated with 5 µM of EPA for 24 h. EC were

removed from the plastic culture dish by scraping and stored in 0.88% KCl

solution at âˆ'20°C until analysis. Total lipid was extracted with

chloroform:methanol (2:1, v/v) containing butylated hydroxytoluene (50 mg/l) as

antioxidant. Fatty acids were subsequently hydrolysed from the lipid and

simultaneously methylated by incubation with methylation reagent (methanol

containing 2% v/v H2SO4) at 50°C for 2 h. Fatty acid methyl esters were

separated and identified using a Hewlett Packard 6890 gas chromatograph (Hewlett

Packard) fitted with a 30 mm×32 mm BPX 70 capillary column, film thickness 0.25

µm. Helium, at the initial flow rate of 1.0 ml/min, was used as the carrier gas.

Injector and detector temperatures were 275°C, and the column oven temperature

was maintained at 170°C for 12 min after sample injection. The oven temperature

was programmed to increase from 170 to 210°C at 5°C/min. Fatty acid methyl

esters were identified by comparison with authentic standards. Peak areas were

quantified using ChemStation software (Hewlett Packard). Each fatty acid was

expressed as weight percent of total fatty acids present.

> >

> > Eicosanoid Extraction and Reverse-Phase High-Performance Liquid

Chromatography Analysis

> >

> > Eicosanoids were extracted from control EC using C18 Sep-Pak cartridges

(Waters). Briefly, cartridges were conditioned with 5 ml of high-performance

liquid chromatography (HPLC)-grade MeOH and rinsed twice with water. Reactions

were terminated with addition of 100% ice-cold MeOH, containing 2 ng of internal

standard PGE2-d4. Sample volume was adjusted to 10% MeOH with water and applied

to the column. The column was rinsed with water and the sample eluted with 2 ml

of MeOH. Nitrogen gas was used to dry the sample, which was resuspended in 100

µl of MeOH and stored at âˆ'80°C until analysis.

> >

> > Prostaglandin Quantitation using Liquid Chromatography Tandem Mass

Spectrometry

> >

> > Prostaglandins were separated on a C18 ODS2, 5 µm, 150×4.6-mm column

(Waters) using a gradient of 50%–90% B over 20 min (A, water:acetonitrile:acetic

acid, 75:25:0.1; B, methanol:acetonitrile:acetic acid, 60:40:0.1) at 1 ml/min.

Products were quantitated by directing the HPLC output directly into the

electrospray source of a Q-Trap mass spectrometer (Applied Biosystems 4000

Q-Trap) operating in the negative mode (âˆ'4,500 V). Individual prostaglandins

were monitored in the Multiple Reaction Monitoring (MRM) mode using specific

parent to daughter transitions of m/z 349–269 for PGD3 with collision energies

of âˆ'26 V. Products were identified and quantified with reference to the

appropriate standards run in parallel under the same conditions, with 2 ng of

PGE2-d4 (m/z 355–275) added as an internal standard.

> >

> > Analysis of Neutrophil Adhesion Molecule Expression by Flow Cytometry

> >

> > Neutrophils for analysis were isolated from whole blood as described above.

Control cells were stained for flow cytometry directly after isolation. Cells

rolling on EPA-treated endothelial cells in microslides were harvested by

elution with 0.02% EDTA after 4 min of neutrophil perfusion and 11 min of wash

buffer perfusion (totalling 15 min of contact with endothelial cells). In order

to harvest neutrophils that had migrated across endothelial cells, endothelium

was cultured on 1.5 mg/ml type I collagen gels (Becton Dickinson). After

endothelial cell stimulation with TNF for 4 h, neutrophils were added for 15

min. Surface-adherent cells were washed off with 0.02% EDTA, and the gel was

dissolved using type VII collagenase (Sigma). Neutrophils were harvested from

the dissolved gel by centrifugation. All neutrophil populations were stained for

surface expression of CD11b (PE-conjugated, clone 2LPM19c, 2 µg/ml; DAKO) or

CD62L (FITC-conjugated, clone Dreg56,10 µg/ml, Beckton Dickinson). Expression

was analysed by flow cytometry using a DAKO CyAn, and data analysed using Summit

software (Becton Dickinson). Data are expressed as mean fluorescent intensity.

> >

> > Statistical Analysis

> >

> > Data were analysed using Prism software (GraphPad software). Results are

presented as means±standard error of the mean (SEM). Comparisons between

individual treatments were by paired t-test where appropriate. ANOVA was

performed to assess the effect of EPA concentration on PMN transmigration.

Significant findings were investigated further using Bonferroni multiple

comparison test or Dunnett's test.

> >

> > Author Contributions Top

> >

> > The author(s) have made the following declarations about their

contributions: Conceived and designed the experiments: VBO RFG PCC GBN GER.

Performed the experiments: SPT CMY BHM JM. Analyzed the data: SPT CMY BHM VBO JM

GER. Contributed reagents/materials/analysis tools: VBO RFG PCC GBN. Wrote the

paper: GER.

> >

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November 5, 2011

Fish oil could also worsen Samter's related asthma. Effects of a fish oil enriched diet on aspirin intolerant asthmatic patients: a pilot study.C Picado, J A Castillo, N Schinca, M Pujades, A Ordinas, A Coronas, and A Agusti-VidalServei de Pneumologia, Hospital Clinic, Facultad de Medicina, Barcelona, Spain.AbstractThe effect of a fish oil enriched diet containing about 3 g of eicosapentaenoic acid was studied in 10 patients with aspirin intolerant asthma. Subjects were studied during six weeks on a control diet followed by six weeks on the fish oil diet in a single blind study design. They were asked to record their peak expiratory flow (PEF) twice daily, bronchodilator and steroid doses, and subjective ratings of pulmonary symptoms on diary cards. There were no significant changes in symptom scores over the six weeks of either the control diet or the fish oil diet. PEF values, however, were significantly lower during the fifth and sixth week of the fish oil diet than during the control diet (308 v 262 l/min week 5 and 306 v 256 l/min week 6). Bronchodilator usage was also greater during the fifth and sixth week of the fish oil diet than during the control period (12.0 v 7.4 and 13.0 v 7.4 puffs a day in weeks 5 and 6). This pilot study suggests that fish diets may have a deleterious effect on patients with aspirin intolerant asthma.> > > > > > > > > > > > > > > > > PLoS Biology> > > Login |> > > Create Account |> > > Feedback> > > Browse> > > RSS> > > Search> > > Search> > > > > > > > > Advanced Search> > > Home> > > Browse Articles> > > Current Issue> > > Journal Archive> > > By Publication Date> > > By Subject> > > Collections> > > About> > > Journal Information> > > Editors> > > Academic Editor-in-Chief> > > Editorial Board> > > Article-Level Metrics> > > Open-Access License> > > Media Inquiries> > > PLoS in Print> > > Contact Us> > > For Readers> > > Guidelines for Notes, Comments, and Corrections> > > Guidelines for Rating> > > Help Using this Site> > > Site Map> > > For Authors and Reviewers> > > Publish with PLoS> > > Editorial and Publishing Policies> > > Author Guidelines> > > Figure and Table Guidelines> > > Reviewer Guidelines> > > Submit Your Paper> > > PLoS.org> > > Open Access> > > Join PLoS> > > PLoS Blogs> > > PLoS Store> > > PLoS Currents> > > Stay Connected> > > Hubs> > > Biodiversity> > > Clinical Trials> > > Journals> > > PLoS Biology> > > PLoS Medicine> > > PLoS Computational Biology> > > PLoS Genetics> > > PLoS Pathogens> > > PLoS ONE> > > PLoS Neglected Tropical Diseases> > > Download: PDF | Citation | XML> > > Print article> > > EzReprint New & improved!> > > Published in the August 2009 Issue of PLoS Biology> > > > > > Metrics info> > > > > > Total Article Views: 5530> > > > > > Cited in> > > CrossRef (12)> > > PubMed Central (8)> > > Scopus (20)> > > Web of Science® (17)> > > Average Rating (1 User Rating)> > > > > > Currently 3.5/5 Stars.> > > See all categories> > > Insight> > > Currently 4/5 Stars.> > > Reliability> > > Currently 3/5 Stars.> > > Style> > > Currently 4/5 Stars.> > > Rate This Article> > > More> > > Related Content> > > > > > Related Articles on the Web> > > Google Scholar> > > PubMed> > > More> > > Share this Article info> > > > > > 0diggsdigg> > > Email this article> > > Public Library of Science> > > Open Access> > > > > > Research Article> > > Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a New Step in Neutrophil Recruitment> > > > > > While investigating new mechanisms by which the dietary omega-3 fatty acids regulate inflammation, the authors have identified a new step in the regulation of neutrophil migration across vascular endothelial cells.> > > > > > Article> > > Metrics> > > Related Content> > > Comments: 0> > > To add a note, highlight some text. Hide notes> > > Make a general comment> > > Jump to> > > > > > Abstract> > > Author Summary> > > Introduction> > > Results/Discussion> > > Materials and Methods> > > Author Contributions> > > References> > > P. Tull1, Clara M. Yates1, H. Maskrey2, B. O'Donnell2, Jackie Madden3, F. Grimble3, Philip C. Calder3, Gerard B. Nash1, G. Ed. Rainger1*> > > > > > 1 Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, The Medical School, The University of Birmingham, Birmingham, United Kingdom, 2 Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff, United Kingdom, 3 Institute of Human Nutrition, School of Medicine, University of Southampton, Southampton, United Kingdom> > > > > > Abstract Top> > > > > > Inflammation is a physiological response to tissue trauma or infection, but leukocytes, which are the effector cells of the inflammatory process, have powerful tissue remodelling capabilities. Thus, to ensure their precise localisation, passage of leukocytes from the blood into inflamed tissue is tightly regulated. Recruitment of blood borne neutrophils to the tissue stroma occurs during early inflammation. In this process, peptide agonists of the chemokine family are assumed to provide a chemotactic stimulus capable of supporting the migration of neutrophils across vascular endothelial cells, through the basement membrane of the vessel wall, and out into the tissue stroma. Here, we show that, although an initial chemokine stimulus is essential for the recruitment of flowing neutrophils by endothelial cells stimulated with the inflammatory cytokine tumour necrosis factor-Î±, transit of the endothelial monolayer is regulated by an additional and downstream stimulus. This signal is supplied by the metabolism of the omega-6-polyunsaturated fatty acid (n-6-PUFA), arachidonic acid, into the eicosanoid prostaglandin-D2 (PGD2) by cyclooxygenase (COX) enzymes. This new step in the neutrophil recruitment process was revealed when the dietary n-3-PUFA, eicosapentaenoic acid (EPA), was utilised as an alternative substrate for COX enzymes, leading to the generation of PGD3. This alternative series eicosanoid inhibited the migration of neutrophils across endothelial cells by antagonising the PGD2 receptor. Here, we describe a new step in the neutrophil recruitment process that relies upon a lipid-mediated signal to regulate the migration of neutrophils across endothelial cells. PGD2 signalling is subordinate to the chemokine-mediated activation of neutrophils, but without the sequential delivery of this signal, neutrophils fail to penetrate the endothelial cell monolayer. Importantly, the ability of the dietary n-3-PUFA, EPA, to inhibit this process not only revealed an unsuspected level of regulation in the migration of inflammatory leukocytes, it also contributes to our understanding of the interactions of this bioactive lipid with the inflammatory system. Moreover, it indicates the potential for novel therapeutics that target the inflammatory system with greater affinity and/or specificity than supplementing the diet with n-3-PUFAs.> > > > > > Author Summary Top> > > > > > Inflammation is a physiological response to tissue trauma or infection. Neutrophils, which circulate in the blood stream, are the first inflammatory cells to be recruited to a site of tissue inflammation. In response to recruitment signals provided by chemotactic peptides called chemokines, neutrophils traverse the endothelial cell lining of blood vessels. This process involves a multistep cascade of neutrophil adhesion and activation events on the endothelial barrier. While investigating the anti-inflammatory functions of the omega-3 fatty acid , EPA, which is found, for instance, in dietary fish oil, we identified an additional unexpected lipid-derived signal that is essential for neutrophil migration across the endothelium. Our experiments show that a chemokine delivered the first signal needed to bind neutrophils firmly to the endothelial surface. However, in order to traverse the endothelium, a subsequent signal delivered by prostaglandin-D2 (PGD2), a lipid derived from the omega-6 fatty acid arachidonic acid, was essential. When EPA, was introduced into the experiment, it was used to form PGD3. This alternative lipid blocked interactions between PGD2 and its receptor on neutrophils, preventing the process of migration across the endothelial barrier. Thus, we reveal a new step in the recruitment of neutrophils during inflammation, and a novel anti-inflammatory mechanism of action of dietary EPA.> > > > > > Citation: Tull SP, Yates CM, Maskrey BH, O'Donnell VB, Madden J, et al. (2009) Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a New Step in Neutrophil Recruitment. PLoS Biol 7(8): e1000177. doi:10.1371/journal.pbio.1000177> > > > > > Academic Editor: Philip M. , NIH/NIAID, United States of America> > > > > > > > > Received: February 6, 2009; Accepted: July 14, 2009; Published: August 25, 2009> > > > > > Copyright: © 2009 Tull et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.> > > > > > Funding: This work was supported by the Biotechnology and Biological Sciences Research Council, United Kingdom (grant BBS/B/0105) and by the British Heart Foundation (grant BS/97001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.> > > > > > Competing interests: The authors have declared that no competing interests exist.> > > > > > Abbreviations: DC, dendritic cell> > > > > > * E-mail: g.e.rainger@> > > > > > Introduction Top> > > > > > In vertebrates, tissue trauma or infection causes the rapid initiation of an inflammatory reaction. The early phase of this phylactic response results in the localised recruitment of cells of the innate immune system from the blood, a tissue infiltrate that is dominated by neutrophils. The molecular processes that support the initial interactions between blood-borne neutrophils and the endothelial cells lining postcapillary venules (the site in the blood vasculature where leukocytes are recruited during inflammation) are well described (see Figure 1 for a schematic representation of the steps in the neutrophil recruitment process). In response to the localised production of inflammatory mediators, such as the cytokine tumour necrosis factor-Î± (TNF), activated endothelial cells decorate themselves with specialised adhesion receptors of the selectin family [1]–[3]. Due to their ability to rapidly form strong but short-lived bonds with carbohydrate counter-ligands on the neutrophil surface, E- and P-selectin are capable of tethering neutrophils from rapidly flowing blood [4]. The sequential formation and dissolution of selectin bonds also support a characteristic and dynamic form of adhesion, referred to as rolling [5]. Rolling adhesion does not require neutrophil activation. However, neutrophil migration through the vessel wall and into the inflamed tissue is dependent upon the receipt of an activating stimulus [5],[6]. We have previously shown that endothelial cells can present peptide agonists of the CXC-chemokine family to rolling neutrophils and thus stabilise adhesion [7],[8]. Ligation of the neutrophil CXC-Receptor-2 (CXCR2) by these agents is essential for activation of the Î²2-intergrin adhesion receptors and the reorganisation of the actin cytoskeleton that support transendothelial cell migration [8]. Here, we show that in the presence of an antibody that blocks chemokine interactions with CXCR2, neutrophils roll on the endothelial cell surface indefinitely (Figure 2), demonstrating that a chemokine signal is essential for neutrophil activation on TNF-stimulated endothelial cells. However, the removal of this primary activating stimulus provides no information on the requirement for additional, downstream signals, which might coordinate transit of the vessel wall and onward migration into stromal tissues.> > > > > > > > > Figure 1. Steps in the process of neutrophil recruitment.> > > > > > Step 1: Neutrophils are captured from flow and tether and roll on tumour necrosis factor-Î± (TNF)-stimulated endothelial cells. Step 2: Neutrophils are then activated by the action of CXC-chemokines on the chemokine receptor, CXCR2, a process resulting in the activation of neutrophil Î²2-integrins. Step 3: Î²2-integrins engage counter receptors on the endothelial cell surface, and the neutrophil becomes stationary. Step 4: Prostaglandin-D2 (PGD2), generated by the action of cyclooxygenase enzymes on the n-6-PUFA arachidonic acid (AA), binds the PGD2 receptor, DP-1. DP-1 generates signals that stabilise neutrophil adhesion, induce neutrophil shape change, and support the process of transmigration across the endothelial cell monolayer. Step 5: If the endothelial cells have been supplemented with the n-3-PUFA, eicosapentaenoic acid (EPA), the alternative series prostanoid, PGD3, is generated, and this antagonises PGD2-mediated neutrophil responses.> > > > > > doi:10.1371/journal.pbio.1000177.g001> > > > > > Figure 2. Chemokine signalling through CXCR2 is essential for neutrophil activation on TNF-stimulated endothelial cells.> > > > > > Using a flow-based adhesion assay, neutrophils isolated from whole blood were perfused across endothelial cells. Neutrophils were recruited to endothelial cells treated with TNF, but did not adhere to unstimulated endothelium. Detailed analysis of neutrophil behaviour showed that on TNF-stimulated endothelial cells, the majority of recruited cells transmigrated across the monolayer. When neutrophils were treated with anti-CXCR2, but not anti-CXCR1, neutrophil activation was inhibited so that nearly all of the recruited neutrophils rolled indefinitely on the monolayer and could not migrate; data are mean±SEM of four experiments **p<0.01 for comparison by paired t-test of neutrophil behaviour on TNF stimulated endothelial cells in the presence or absence of anti-CXCR2.> > > > > > doi:10.1371/journal.pbio.1000177.g002> > > Results/Discussion Top> > > > > > EPA Released from Endothelial Cell Membrane Phospholipids Inhibits the Recruitment of Neutrophils> > > > > > Dietary omega-3 polyunsaturated fatty acids (n-3-PUFAs) have anti-inflammatory properties. For example, their inclusion in the diet in the form of n-3-PUFA–rich fish oil reduces the symptoms of disease as well as the use of nonsteroidal anti-inflammatory drugs in arthritis patients with severe inflammatory joint disease [9]. In addition, dietary n-3-PUFAs may offer protection against vascular pathology associated with atherosclerosis, having been reported to be efficacious in epidemiological studies [10]. By modulating inflammation within the artery wall, n-3-PUFAS also alter the cellular and the structural composition of advanced atherosclerotic plaques in a manner that could reduce the incidence of plaque rupture or ulceration, a process that precedes tissue infarction (e.g., heart attack or stroke) [11]. Despite these documented anti-inflammatory benefits, the mode(s) of interaction of these lipids with the immune and inflammatory systems are not well understood. When we supplemented endothelial cell culture medium with the major n-3 -PUFA found in dietary fish oil supplements, eicosapentaenoic acid (EPA; 20:5n-3), it was incorporated into cellular phospholipids so that upon withdrawal of the free fatty acid from the culture medium, a pool of esterified EPA remained localised within endothelial cell membranes (Figure 3A and 3B). After withdrawal of free fatty acid and stimulation with TNF, EPA-treated cells were able to support similar levels of neutrophil adhesion to those that had received no lipid supplement (Figure 4A). However, detailed analysis showed that the behaviour of neutrophils on the two populations of endothelial cells was very different. In response to TNF, but in the absence of EPA, a small population of adherent neutrophils (~20%) rolled throughout the duration of a flow adhesion assay (Figure 4B). The remaining cells became activated on the endothelial cell surface, and this population of neutrophils became progressively smaller as cells migrated across the endothelial cell monolayer (Figure 4B). In contrast, when endothelial cells had been treated with EPA prior to TNF stimulation, there was a marked reduction in the number of cells undergoing transendothelial cell migration (Figure 4B). This was reduced at the earliest time point and did not increase over the duration of the experiment. Interestingly however, many of the cells that did become activated on the surface of endothelial cells reverted to a rolling form of adhesion (Figure 4B), implying that after the receipt of an initial chemokine stimulus, a second signal was required to allow prolonged adhesion and migration across the endothelial cell monolayer. Experiments using different concentrations of EPA showed that these inhibitory effects were evident at concentrations as low as 50 nM (Figure 4C).> > > > > > > > > Figure 3. The effects of EPA supplementation on the fatty acid content of endothelial cell phospholoipds.> > > > > > (A and The concentration of EPA in membrane phospholipids was increased after supplementation of culture medium with EPA for 24 h. Error bars indicate SEM. AA, arachidonic acid; FA, fatty acid; PA, palmitic acid; SA, stearic acid.> > > > > > doi:10.1371/journal.pbio.1000177.g003> > > > > > Figure 4. The effects of EPA supplementation on the adhesive behaviour of neutrophils.> > > > > > (A) EPA supplementation did not affect the number of neutrophils initially adhering to endothelial cells from flow. ( However, the time course of neutrophil behaviour on TNF-stimulated endothelial cells showed that EPA supplementation drastically altered neutrophil behaviour. Thus, the number of activated and surface adherent neutrophils decreased with time in the presence or absence of EPA. However, on endothelium that had not been supplemented with EPA, this was because neutrophils transmigrated across the monolayer, whereas on EPA-supplemented endothelium this was because activated cells reverted back to a rolling form of adhesion. ANOVA showed that there was a significant effect of treatment (i.e., ±EPA) on the percentage of cells that were rolling, surface adherent, or transmigrated (p<0.01). In addition, there was a significant effect of time on each form of behaviour for the EPA-treated cells (p<0.05âˆ'0.01). Bonferroni tests showed significant differences at specific time points as marked; **p<0.01. © Inhibition of neutrophil transmigration was evident at levels of EPA supplementation as low as 50 nM. Data are mean±SEM of five experiments. ANOVA showed significant effects of treatment (p<0.05); *p<0.05 compared to untreated control by Dunnett's test. Error bars indicate SEM.> > > > > > doi:10.1371/journal.pbio.1000177.g004> > > EPA Does Not Inhibit the Expression of Inflammatory Genes in Endothelial Cells> > > > > > To define the molecular mechanisms underlying these observations, we tested two prevalent hypotheses. The first, predicated on the observation that n-3-PUFAs regulate the transcription of endothelial cell inflammatory genes by down-regulating the activity of the nuclear factor-ÎºB, predicts changes in the levels of adhesion receptor and chemokine expression after supplementation with EPA [12]–[14]. However, previous studies have investigated the regulation of gene expression in the presence of up to 100 µM of n-3-PUFAs, whereas physiological blood plasma levels of free fatty acid are of the order of 1 µM [15], a level that can be increased several fold upon supplementation. Using 5 µM EPA as an approximation of the highest concentration achievable in vivo, we did not see changes in the expression of endothelial cell adhesion molecules when assessed for RNA transcripts or protein expression (Figure 5 and Table 1). Neither was there any alteration in the levels of cytokines and chemokines secreted by endothelial cells (Table 2). Thus, in this model, we were able to discount the gene regulation hypothesis.> > > > > > > > > Figure 5. The effect of EPA on TNF-induced surface adhesion molecule expression.> > > > > > The expression of (A) E-selectin and ( VCAM-1 was measured by ELISA in TNF-stimulated endothelial cells that were either unsupplemented or supplemented with EPA. Data are mean±SEM of seven experiments. ANOVA showed that there was a significant effect of the dose of TNF (p<0.01), but not of EPA, on the expression of both receptors.> > > > > > doi:10.1371/journal.pbio.1000177.g005> > > > > > Table 1. Effect of EPA treatment on mRNA expression of endothelial cell adhesion molecules and chemokines induced by TNF.> > > > > > doi:10.1371/journal.pbio.1000177.t001> > > > > > Table 2. Effect of EPA treatment of endothelial cells on the TNF-Î±-induced secretion of cytokines and chemokines.> > > > > > doi:10.1371/journal.pbio.1000177.t002> > > The Anti-Inflammatory Effects of EPA Supplementation Require the Metabolic Activity of Cyclooxygenase Enzymes> > > > > > The second hypothesis proposes that upon endothelial cell activation, EPA may compete with the n-6-PUFA arachidonic acid (AA; 20:4n-6) for cyclooxygenase enzymes (COX1 and COX2) after both fatty acids are liberated from membrane phospholipids by endogenous phospholipases [16]. Ordinarily, AA is metabolised by COX into 2-series endoperoxides, with downstream synthases converting these into the biologically active 2-series prostanoids [17]. However, when utilised as a COX substrate, the metabolism of EPA generates alternative 3-series prostanoids [16]. The 2-series prostanoids generated by COX activity are known to regulate aspects of the inflammatory response. For example, in the COX2 knockout mouse, neutrophil recruitment is dramatically reduced upon ischaemia and reperfusion-induced injury of the liver when compared to wild-type control animals [18]. In a murine model of lipopolysaccharide-induced lung inflammation, inhibitors of COX function (indomethacin and aspirin) modestly increased neutrophil recruitment [19]. Conversely in vitro, inhibition of COX function by aspirin can inhibit neutrophil migration [20] while having no effect on the levels of neutrophil adhesion to the endothelial cell monolayer [21]. Moreover, in murine models of acute and chronic inflammation, a reduction in PGE2 production by genetic deletion of its membrane-bound synthase, moderates the formation of inflammation-associated granulation tissue and angiogenesis, as well as decreasing the nociception of pain, indicating that PGE2 is proinflammatory in these models [22]. PGD2 also appears to have proinflammatory functions, as overexpression of the synthase generating this prostanoid increases production of inflammatory cytokines and chemokines, leading to exaggerated levels of eosinophil and lymphocyte recruitment [23], a mechanism that operates through the PGD2 receptor DP-2. In contrast, in acute peritoneal inflammation, knockout of PGD2 synthase increased inflammatory cytokine production and retarded the rate of inflammatory resolution by a mechanism that operated through the DP-1 receptor [24]. Thus, PGD2 appears to have pro- or anti-inflammatory capabilities depending on the nature of the inflammatory insult. The inflammatory activity of the equivalent 3-series prostanoids is not known. Here, by introducing a panel of COX inhibitors into endothelial cell cultures at the same time as they were activated with TNF, the effects of EPA supplementation could be replicated, with neutrophil transmigration being dramatically inhibited (Figure 6). Moreover, adding a molecular excess of AA to EPA-supplemented endothelial cells at the point of TNF activation could reverse the blockade of migration (Figure 6). Taken together, these data imply that a COX-derived product of AA is required for the transmigration of neutrophils across TNF-stimulated endothelial cells, and that in the presence of EPA, this pathway is efficiently antagonised.> > > > > > > > > Figure 6. A cyclooxygenase (COX)-derived eicosanoid is required for neutrophil transmigration across TNF-stimulated endothelial cells.> > > > > > (A) Inhibition of COX-1 (SC-560), COX-2 (NS-398), or both (indomethacin) caused inhibition of neutrophil transmigration, *p<0.05 by t-test. ( Addition of AA to EPA-supplemented endothelial cells during the period of TNF stimulation restored neutrophil transmigration. All data are mean±SEM of four experiments. ANOVA showed significant effects of treatments in both (A and (p<0.01). In (A), Dunnett test showed significant effects of individual treatments compared to untreated control, *p<0.05. In (, Dunnett's test showed significant difference between EPA-treated and untreated control (**p<0.01), but not between AA+EPA and control.> > > > > > doi:10.1371/journal.pbio.1000177.g006> > > Prostaglandin D2 Is Required for Neutrophil Migration across Endothelial Cells Stimulated with TNF-Î±> > > > > > Endothelial cells generate COX products constitutively. For example, prostacyclin (PGI2) and prostaglandin D2 (PGD2) are endothelial cell-derived vasoactive prostanoids that are also involved in the regulation of haemostasis, being antagonists of platelet activation [17],[25]. These prostanoids are difficult to measure in the systemic blood, as they have reported half-lives in plasma that are measured in minutes [26],[27]. However, the constitutive nature of their production is demonstrated by the presence in urine and body fluids of their downstream metabolic products, PGF1Î± and delta-PGJ2, respectively [28],[29].> > > > > > Prostanoids have a documented ability to regulate the migration of a number of leukocyte subsets. For example, PGD2 induces chemotaxis of eosinophils and T-lymphocytes [30] in vitro. Interestingly, the migration of monocyte-derived migratory dendritic cells (DCs) may be tightly regulated by interplay between different prostanoids. Thus, the ability of these cells to traffic out of tissue into lymph nodes via the lymphatic circulation is dependent upon the presence of PGE2 as a differentiation signal [31]–[34]. In this context PGE2 promotes the function (but not the expression) of the chemokine receptor CCR7, so that DCs efficiently respond to the chemokines CCL19 and CCL21. Importantly, a number of studies have shown that the presence of PGD2 can in turn inhibit the ability of DCs to migrate out of the lungs or the skin during an inflammatory response, although the molecular mechanism by which this inhibition is achieved remains undescribed [35],[36].> > > > > > As prostanoids are well documented to regulate leukocyte migration [37], we tested the hypothesis that PGD2 was the endothelial cell-derived agent providing the stimulus for neutrophil migration across the monolayer. We established a population of surface adherent neutrophils on TNF-treated endothelial cells supplemented with EPA and perfused synthetic PGD2 across the endothelial cells and neutrophils to see whether this would reintroduce transmigration. The provision of exogenous PGD2 but not PGD3 (which could be derived from EPA), fully restored the ability of neutrophils to cross the endothelial cell monolayer (Figure 7A). Prostaglandin D2 has two receptors. Chemoattractant-receptor homologous molecule expressed on Th2 cells receptor (CRTH2 or DP-2) is not expressed in neutrophils [35], whereas the DP-1 receptor has been reported in these cells [38]. Here, neutrophils perfused across TNF-stimulated endothelial cells in the presence of a DP-1 receptor antagonist (BW868C) showed a greatly diminished efficiency of endothelial cell transmigration (Figure 7B). Conversely, the transmigration of neutrophils was re-established on EPA-supplemented endothelial cells when a synthetic DP-1 receptor agonist (BW245C) was perfused across cells adherent to the surface of the monolayer (Figure 7C). Importantly, we could demonstrate that PGD2 was operating directly on the neutrophils, because when these cells were harvested after migrating across a TNF-stimulated endothelial cells they had up-regulated CD11b (the alpha subunit of the Î²2 integrin CD11b/CD18 that is required for efficient neutrophil transmigration) and proteolytically shed L-selectin (Figure 8). However, neutrophils harvested from the surface of EPA-treated endothelial cells did not shed L-selectin or up-regulate CD11b. Thus, in this assay system, the delivery of a PGD2-mediated signal to the neutrophils was necessary for full cellular activation and efficient transendothelial migration.> > > > > > > > > Figure 7. The role of AA- and EPA-derived eicosanoids in neutrophil transmigration.> > > > > > (A) When PDG2 was perfused across a population of neutrophils adherent to EPA-treated endothelial cells, neutrophil transmigration was restored. However, PGD3 had no significant effect on neutrophil behaviour. All data are mean±SEM of four experiments. ANOVA showed significant effect of treatment on transmigration. *p<0.05 compared to EPA-supplemented endothelial cells in the absence of PGD2 by Dunnett test. ( The DP1 receptor antagonist BW868c dose-dependently inhibited neutrophil transmigration on TNF-stimulated endothelial cells. All data are mean±SEM of five experiments; ANOVA showed a significant effect of BW868c concentration (p<0.01). © Perfusion of the DP-1 receptor agonist, BW245C, across neutrophils adherent to EPA-supplemented endothelial cells restored neutrophil migration to control levels. Data are mean±SEM of five experiments. ANOVA showed a significant effect of treatment on transmigration. **p<0.01 compared to EPA-supplemented endothelial cells in the absence of BW868C by Dunnett test. (D) Neutrophils perfused across TNF-stimulated endothelial cells in the presence of PGD3 showed a significantly reduced ability to migrate across the monolayer. Data are mean±SEM of three experiments; ANOVA showed a significant effect of PGD3 concentration on transmigration (p<0.01). (E) The levels of PGD3 released from endothelial cells are increased after EPA supplementation. Data are mean±SEM of three experiments. ANOVA showed a significant effect of treatment (p<0.05). **p<0.01 for PGD3 production compared to endothelial cells activated with TNF without EPA supplementation by Dunnett's test.> > > > > > doi:10.1371/journal.pbio.1000177.g007> > > > > > Figure 8. The effect of adhesion on the expression of L-selectin and CD11b on neutrophils.> > > > > > Neutrophils that were freshly isolated, migrated across endothelial cells stimulated with TNF or adherent to the surface of EPA-supplemented endothelial cells (EC) activated with TNF were harvested and the expression of (A) CD11b or ( L-selectin (CD62L) assessed by flow cytometry. Data are mean±SEM of four experiments; ANOVA showed significant effect of treatments in (A) (p<0.01) and ( (p<0.05). In (A and , Dunnett's test showed significant difference between âˆ'EPA and freshly isolated cells (*p<0.01 and *p<0.05, respectively), but not between +EPA and freshly isolated cells.> > > > > > doi:10.1371/journal.pbio.1000177.g008> > > PGD3 Generated from EPA Antagonises the PGD2-Mediated Migration of Neutrophils across Endothelial Cells> > > > > > Taken together, these data show that PGD2 operates through the neutrophil DP-1 receptor to provide a signal that is essential for transendothelial cell migration. Moreover, the ability of neutrophils to respond to this signal is abolished when endothelial cells are preloaded with EPA from which PGD3 is generated. This implies that PGD3 may be an effective antagonist of the DP-1 receptor. To test this hypothesis, we perfused neutrophils across TNF-stimulated endothelial cells in the presence of exogenous PGD3. Neutrophil transmigration was inhibited in a dose-dependent manner by PGD3, showing that in the presence of endogenously generated PGD2 it could effectively antagonise this process (Figure 7D). Importantly, we were able to show that cells supplemented with EPA-generated an increased amount of PGD3 (Figure 7E).> > > > > > The absolute levels of PGD2 and PGD3 that are generated in our assay system, and thus the ratio of their abundance, are not easily assessed. The levels of PGD2 measured as an endothelial cell releasate were at the limits of detection (Table 3), presumably due to the short half-life of this prostanoid under physiological conditions; however, significant levels of PGD3 were assayed upon EPA supplementation, and concentrations in the order of 5–10 nM had accumulated over the 24 h of the EPA supplementation period. The half-life of PGD3 in serum or albumin-containing medium has not been reported to our knowledge; thus it is not clear whether the measurements made here report the true levels of PGD3 production, or if metabolic degradation of this prostanoid renders these measurements underestimates. Irrespective of this, our experiments utilising synthetic PGD2 demonstrate that concentrations on the order of 1 nM were sufficient to fully recapitulate the process of migration on EPA-supplemented endothelial cells, and when using PGD3 as an antagonist of TNF-induced transmigration marked effects were evident at 10–100 pM. Thus, the measurable levels of PGD3 that we report would certainly be sufficient to effectively antagonise the transmigration of neutrophils in our experimental model.> > > > > > > > > Table 3. Analysis of eicosanoids by liquid chromatography tandem mass spectrometry.> > > > > > doi:10.1371/journal.pbio.1000177.t003> > > Conclusions> > > > > > Our study demonstrates a hitherto unknown regulatory step in the recruitment of neutrophils by cytokine-stimulated endothelial cells (refer to Figure 1 to see how this new step fits with the known regulatory mechanisms of neutrophil recruitment). After initial tethering by selectin molecules, a chemokine signal induces arrest of the cell on the endothelial cell surface. However, the chemokine signal is not sufficient to support transmigration across the endothelial cell monolayer, and the arachidonic acid-derived prostanoid, PGD2, is an essential downstream regulator of this process. PGD2 operates through the DP-1 receptor and this signal can be effectively antagonised by PGD3 which is generated from EPA released by the action of phospholipase enzymes on the phospholipids of EPA supplemented cells. Not only does this study reveal a new step in the recruitment of neutrophil recruitment during inflammation, it also reveals a novel anti-inflammatory mechanism of action of the dietary n-3-PUFA, EPA.> > > > > > Materials and Methods Top> > > > > > Cell Culture and Neutrophil Isolation> > > > > > Endothelial cells (EC) were isolated and cultured, as previously described [39], with or without 0–5 µM EPA (Sigma) for 24 h. TNF-Î± (100 U/ml; R & D systems) was added to the EC for the final 4 h of culture. In some experiments, EC were treated with 100 U/ml TNF-Î± in the presence of 10 µM indomethacin or 0.1 µM SC-560 or 1 µM NS-398 (Sigma). In the AA reconstitution experiments, 5 µM AA (Sigma) was added to the culture medium simultaneously with TNF-Î±.> > > > > > Blood from healthy adult volunteers was collected into tubes coated with EDTA (1.6 mg/ml), and neutrophils were separated using 2-step density gradients of Histopaque 1119 and 1077 (Sigma), as previously described [40]. After washing in 0.15% BSA in PBS, cells were counted using a Coulter Multisizer (Coulter Electronics), and neutrophils, which were >95% pure, were resuspended at a concentration of 1×106/ml in PBS/Alb with calcium and magnesium.> > > > > > RNA Extraction and Real-Time PCR> > > > > > mRNA was isolated from EC using the Qiagen RNEasy Mini Kit 50 (Qiagen) following the manufacturer's instructions. Real-time PCR (RT-PCR) was performed using QuantiTect probe RT-PCR kit according to the manufacturer's instructions (Qiagen). Primers were purchased from Applied Biosystems. The expression of each target gene was normalised to Î²-actin expression, and the data presented represent fold change compared to untreated EC.> > > > > > Luminex for Secreted Cytokines and Chemokines> > > > > > Supernatants were collected from EC that had been incubated with 0–5 µM EPA for 24 h and had 100 U/ml TNF added to the culture for the final 4 h of culture. The Luminex kit was purchased from Upstate/Chemicon, and the experiment performed to the manufacturer's instructions. Data were collected and analysed from the samples using a Luminex100 machine (Luminex).> > > > > > Flow-Based Adhesion Assay> > > > > > Glass capillaries (microslides) containing treated EC monolayers were incorporated into a flow-based adhesion assay as described [39]. Briefly, microslides were attached to cell and fluid reservoirs by silicon tubing at one end and to a withdrawal syringe pump at the other end. After mounting on the stage of a phase contrast microscope, EC were washed for 2 min with PBS containing 0.15% BSA. Neutrophils were then perfused (1×106/ml) at 0.1 Pa for 4 min, followed by 15 min of wash. Video images were recorded throughout the experiment and neutrophil behaviour analysed offline using Image Pro software (Image-Pro Plus). In some experiments, neutrophils were perfused across EPA- and TNF-treated EC, allowed to adhere, and subsequently perfused with 1 nM PGD2 or 1 nM PGD3 (both from Cayman Chemicals). Alternatively, neutrophils were treated with BW868c (Cayman Chemicals), a DP1 receptor antagonist, for 10 min prior to perfusion over TNF-Î±-stimulated EC. Neutrophils were also treated with the DP1 receptor agonist BW245c (Cayman Chemicals) prior to perfusion over EPA- and TNF-Î±-treated EC. Neutrophils were also treated with 0–1 nM PGD3, immediately prior to perfusion across TNF-stimulated EC. In all experiments, lipid reagents, agonists, and antagonists were stored in 100% ethanol under nitrogen gas. Once diluted, the medium contained <0.01% ethanol. All controls contained equivalent concentrations of ethanol.> > > > > > Analysis of EPA Incorporation into Cellular Phospholipids> > > > > > First-passage confluent EC were treated with 5 µM of EPA for 24 h. EC were removed from the plastic culture dish by scraping and stored in 0.88% KCl solution at âˆ'20°C until analysis. Total lipid was extracted with chloroform:methanol (2:1, v/v) containing butylated hydroxytoluene (50 mg/l) as antioxidant. Fatty acids were subsequently hydrolysed from the lipid and simultaneously methylated by incubation with methylation reagent (methanol containing 2% v/v H2SO4) at 50°C for 2 h. Fatty acid methyl esters were separated and identified using a Hewlett Packard 6890 gas chromatograph (Hewlett Packard) fitted with a 30 mm×32 mm BPX 70 capillary column, film thickness 0.25 µm. Helium, at the initial flow rate of 1.0 ml/min, was used as the carrier gas. Injector and detector temperatures were 275°C, and the column oven temperature was maintained at 170°C for 12 min after sample injection. The oven temperature was programmed to increase from 170 to 210°C at 5°C/min. Fatty acid methyl esters were identified by comparison with authentic standards. Peak areas were quantified using ChemStation software (Hewlett Packard). Each fatty acid was expressed as weight percent of total fatty acids present.> > > > > > Eicosanoid Extraction and Reverse-Phase High-Performance Liquid Chromatography Analysis> > > > > > Eicosanoids were extracted from control EC using C18 Sep-Pak cartridges (Waters). Briefly, cartridges were conditioned with 5 ml of high-performance liquid chromatography (HPLC)-grade MeOH and rinsed twice with water. Reactions were terminated with addition of 100% ice-cold MeOH, containing 2 ng of internal standard PGE2-d4. Sample volume was adjusted to 10% MeOH with water and applied to the column. The column was rinsed with water and the sample eluted with 2 ml of MeOH. Nitrogen gas was used to dry the sample, which was resuspended in 100 µl of MeOH and stored at âˆ'80°C until analysis.> > > > > > Prostaglandin Quantitation using Liquid Chromatography Tandem Mass Spectrometry> > > > > > Prostaglandins were separated on a C18 ODS2, 5 µm, 150×4.6-mm column (Waters) using a gradient of 50%–90% B over 20 min (A, water:acetonitrile:acetic acid, 75:25:0.1; B, methanol:acetonitrile:acetic acid, 60:40:0.1) at 1 ml/min. Products were quantitated by directing the HPLC output directly into the electrospray source of a Q-Trap mass spectrometer (Applied Biosystems 4000 Q-Trap) operating in the negative mode (âˆ'4,500 V). Individual prostaglandins were monitored in the Multiple Reaction Monitoring (MRM) mode using specific parent to daughter transitions of m/z 349–269 for PGD3 with collision energies of âˆ'26 V. Products were identified and quantified with reference to the appropriate standards run in parallel under the same conditions, with 2 ng of PGE2-d4 (m/z 355–275) added as an internal standard.> > > > > > Analysis of Neutrophil Adhesion Molecule Expression by Flow Cytometry> > > > > > Neutrophils for analysis were isolated from whole blood as described above. Control cells were stained for flow cytometry directly after isolation. Cells rolling on EPA-treated endothelial cells in microslides were harvested by elution with 0.02% EDTA after 4 min of neutrophil perfusion and 11 min of wash buffer perfusion (totalling 15 min of contact with endothelial cells). In order to harvest neutrophils that had migrated across endothelial cells, endothelium was cultured on 1.5 mg/ml type I collagen gels (Becton Dickinson). After endothelial cell stimulation with TNF for 4 h, neutrophils were added for 15 min. Surface-adherent cells were washed off with 0.02% EDTA, and the gel was dissolved using type VII collagenase (Sigma). Neutrophils were harvested from the dissolved gel by centrifugation. All neutrophil populations were stained for surface expression of CD11b (PE-conjugated, clone 2LPM19c, 2 µg/ml; DAKO) or CD62L (FITC-conjugated, clone Dreg56,10 µg/ml, Beckton Dickinson). Expression was analysed by flow cytometry using a DAKO CyAn, and data analysed using Summit software (Becton Dickinson). Data are expressed as mean fluorescent intensity.> > > > > > Statistical Analysis> > > > > > Data were analysed using Prism software (GraphPad software). Results are presented as means±standard error of the mean (SEM). Comparisons between individual treatments were by paired t-test where appropriate. ANOVA was performed to assess the effect of EPA concentration on PMN transmigration. 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November 6, 2011

Well that's confusing... I wonder why it would have this effect.On Nov 5, 2011, at 9:52 PM, damianodavid wrote:

Fish oil could also worsen Samter's related asthma. Effects of a fish oil enriched diet on aspirin intolerant asthmatic patients: a pilot study.C Picado, J A Castillo, N Schinca, M Pujades, A Ordinas, A Coronas, and A Agusti-VidalServei de Pneumologia, Hospital Clinic, Facultad de Medicina, Barcelona, Spain.AbstractThe effect of a fish oil enriched diet containing about 3 g of eicosapentaenoic acid was studied in 10 patients with aspirin intolerant asthma. Subjects were studied during six weeks on a control diet followed by six weeks on the fish oil diet in a single blind study design. They were asked to record their peak expiratory flow (PEF) twice daily, bronchodilator and steroid doses, and subjective ratings of pulmonary symptoms on diary cards. There were no significant changes in symptom scores over the six weeks of either the control diet or the fish oil diet. PEF values, however, were significantly lower during the fifth and sixth week of the fish oil diet than during the control diet (308 v 262 l/min week 5 and 306 v 256 l/min week 6). Bronchodilator usage was also greater during the fifth and sixth week of the fish oil diet than during the control period (12.0 v 7.4 and 13.0 v 7.4 puffs a day in weeks 5 and 6). This pilot study suggests that fish diets may have a deleterious effect on patients with aspirin intolerant asthma.> > > > > > > > > > > > > > > > > PLoS Biology> > > Login |> > > Create Account |> > > Feedback> > > Browse> > > RSS> > > Search> > > Search> > > > > > > > > Advanced Search> > > Home> > > Browse Articles> > > Current Issue> > > Journal Archive> > > By Publication Date> > > By Subject> > > Collections> > > About> > > Journal Information> > > Editors> > > Academic Editor-in-Chief> > > Editorial Board> > > Article-Level Metrics> > > Open-Access License> > > Media Inquiries> > > PLoS in Print> > > Contact Us> > > For Readers> > > Guidelines for Notes, Comments, and Corrections> > > Guidelines for Rating> > > Help Using this Site> > > Site Map> > > For Authors and Reviewers> > > Publish with PLoS> > > Editorial and Publishing Policies> > > Author Guidelines> > > Figure and Table Guidelines> > > Reviewer Guidelines> > > Submit Your Paper> > > PLoS.org> > > Open Access> > > Join PLoS> > > PLoS Blogs> > > PLoS Store> > > PLoS Currents> > > Stay Connected> > > Hubs> > > Biodiversity> > > Clinical Trials> > > Journals> > > PLoS Biology> > > PLoS Medicine> > > PLoS Computational Biology> > > PLoS Genetics> > > PLoS Pathogens> > > PLoS ONE> > > PLoS Neglected Tropical Diseases> > > Download: PDF | Citation | XML> > > Print article> > > EzReprint New & improved!> > > Published in the August 2009 Issue of PLoS Biology> > > > > > Metrics info> > > > > > Total Article Views: 5530> > > > > > Cited in> > > CrossRef (12)> > > PubMed Central (8)> > > Scopus (20)> > > Web of Science® (17)> > > Average Rating (1 User Rating)> > > > > > Currently 3.5/5 Stars.> > > See all categories> > > Insight> > > Currently 4/5 Stars.> > > Reliability> > > Currently 3/5 Stars.> > > Style> > > Currently 4/5 Stars.> > > Rate This Article> > > More> > > Related Content> > > > > > Related Articles on the Web> > > Google Scholar> > > PubMed> > > More> > > Share this Article info> > > > > > 0diggsdigg> > > Email this article> > > Public Library of Science> > > Open Access> > > > > > Research Article> > > Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a New Step in Neutrophil Recruitment> > > > > > While investigating new mechanisms by which the dietary omega-3 fatty acids regulate inflammation, the authors have identified a new step in the regulation of neutrophil migration across vascular endothelial cells.> > > > > > Article> > > Metrics> > > Related Content> > > Comments: 0> > > To add a note, highlight some text. Hide notes> > > Make a general comment> > > Jump to> > > > > > Abstract> > > Author Summary> > > Introduction> > > Results/Discussion> > > Materials and Methods> > > Author Contributions> > > References> > > P. Tull1, Clara M. Yates1, H. Maskrey2, B. O'Donnell2, Jackie Madden3, F. Grimble3, Philip C. Calder3, Gerard B. Nash1, G. Ed. Rainger1*> > > > > > 1 Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, The Medical School, The University of Birmingham, Birmingham, United Kingdom, 2 Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff, United Kingdom, 3 Institute of Human Nutrition, School of Medicine, University of Southampton, Southampton, United Kingdom> > > > > > Abstract Top> > > > > > Inflammation is a physiological response to tissue trauma or infection, but leukocytes, which are the effector cells of the inflammatory process, have powerful tissue remodelling capabilities. Thus, to ensure their precise localisation, passage of leukocytes from the blood into inflamed tissue is tightly regulated. Recruitment of blood borne neutrophils to the tissue stroma occurs during early inflammation. In this process, peptide agonists of the chemokine family are assumed to provide a chemotactic stimulus capable of supporting the migration of neutrophils across vascular endothelial cells, through the basement membrane of the vessel wall, and out into the tissue stroma. Here, we show that, although an initial chemokine stimulus is essential for the recruitment of flowing neutrophils by endothelial cells stimulated with the inflammatory cytokine tumour necrosis factor-Î±, transit of the endothelial monolayer is regulated by an additional and downstream stimulus. This signal is supplied by the metabolism of the omega-6-polyunsaturated fatty acid (n-6-PUFA), arachidonic acid, into the eicosanoid prostaglandin-D2 (PGD2) by cyclooxygenase (COX) enzymes. This new step in the neutrophil recruitment process was revealed when the dietary n-3-PUFA, eicosapentaenoic acid (EPA), was utilised as an alternative substrate for COX enzymes, leading to the generation of PGD3. This alternative series eicosanoid inhibited the migration of neutrophils across endothelial cells by antagonising the PGD2 receptor. Here, we describe a new step in the neutrophil recruitment process that relies upon a lipid-mediated signal to regulate the migration of neutrophils across endothelial cells. PGD2 signalling is subordinate to the chemokine-mediated activation of neutrophils, but without the sequential delivery of this signal, neutrophils fail to penetrate the endothelial cell monolayer. Importantly, the ability of the dietary n-3-PUFA, EPA, to inhibit this process not only revealed an unsuspected level of regulation in the migration of inflammatory leukocytes, it also contributes to our understanding of the interactions of this bioactive lipid with the inflammatory system. Moreover, it indicates the potential for novel therapeutics that target the inflammatory system with greater affinity and/or specificity than supplementing the diet with n-3-PUFAs.> > > > > > Author Summary Top> > > > > > Inflammation is a physiological response to tissue trauma or infection. Neutrophils, which circulate in the blood stream, are the first inflammatory cells to be recruited to a site of tissue inflammation. In response to recruitment signals provided by chemotactic peptides called chemokines, neutrophils traverse the endothelial cell lining of blood vessels. This process involves a multistep cascade of neutrophil adhesion and activation events on the endothelial barrier. While investigating the anti-inflammatory functions of the omega-3 fatty acid , EPA, which is found, for instance, in dietary fish oil, we identified an additional unexpected lipid-derived signal that is essential for neutrophil migration across the endothelium. Our experiments show that a chemokine delivered the first signal needed to bind neutrophils firmly to the endothelial surface. However, in order to traverse the endothelium, a subsequent signal delivered by prostaglandin-D2 (PGD2), a lipid derived from the omega-6 fatty acid arachidonic acid, was essential. When EPA, was introduced into the experiment, it was used to form PGD3. This alternative lipid blocked interactions between PGD2 and its receptor on neutrophils, preventing the process of migration across the endothelial barrier. Thus, we reveal a new step in the recruitment of neutrophils during inflammation, and a novel anti-inflammatory mechanism of action of dietary EPA.> > > > > > Citation: Tull SP, Yates CM, Maskrey BH, O'Donnell VB, Madden J, et al. (2009) Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a New Step in Neutrophil Recruitment. PLoS Biol 7(8): e1000177. doi:10.1371/journal.pbio.1000177> > > > > > Academic Editor: Philip M. , NIH/NIAID, United States of America> > > > > > > > > Received: February 6, 2009; Accepted: July 14, 2009; Published: August 25, 2009> > > > > > Copyright: © 2009 Tull et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.> > > > > > Funding: This work was supported by the Biotechnology and Biological Sciences Research Council, United Kingdom (grant BBS/B/0105) and by the British Heart Foundation (grant BS/97001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.> > > > > > Competing interests: The authors have declared that no competing interests exist.> > > > > > Abbreviations: DC, dendritic cell> > > > > > * E-mail: g.e.rainger@> > > > > > Introduction Top> > > > > > In vertebrates, tissue trauma or infection causes the rapid initiation of an inflammatory reaction. The early phase of this phylactic response results in the localised recruitment of cells of the innate immune system from the blood, a tissue infiltrate that is dominated by neutrophils. The molecular processes that support the initial interactions between blood-borne neutrophils and the endothelial cells lining postcapillary venules (the site in the blood vasculature where leukocytes are recruited during inflammation) are well described (see Figure 1 for a schematic representation of the steps in the neutrophil recruitment process). In response to the localised production of inflammatory mediators, such as the cytokine tumour necrosis factor-Î± (TNF), activated endothelial cells decorate themselves with specialised adhesion receptors of the selectin family [1]–[3]. Due to their ability to rapidly form strong but short-lived bonds with carbohydrate counter-ligands on the neutrophil surface, E- and P-selectin are capable of tethering neutrophils from rapidly flowing blood [4]. The sequential formation and dissolution of selectin bonds also support a characteristic and dynamic form of adhesion, referred to as rolling [5]. Rolling adhesion does not require neutrophil activation. However, neutrophil migration through the vessel wall and into the inflamed tissue is dependent upon the receipt of an activating stimulus [5],[6]. We have previously shown that endothelial cells can present peptide agonists of the CXC-chemokine family to rolling neutrophils and thus stabilise adhesion [7],[8]. Ligation of the neutrophil CXC-Receptor-2 (CXCR2) by these agents is essential for activation of the Î²2-intergrin adhesion receptors and the reorganisation of the actin cytoskeleton that support transendothelial cell migration [8]. Here, we show that in the presence of an antibody that blocks chemokine interactions with CXCR2, neutrophils roll on the endothelial cell surface indefinitely (Figure 2), demonstrating that a chemokine signal is essential for neutrophil activation on TNF-stimulated endothelial cells. However, the removal of this primary activating stimulus provides no information on the requirement for additional, downstream signals, which might coordinate transit of the vessel wall and onward migration into stromal tissues.> > > > > > > > > Figure 1. Steps in the process of neutrophil recruitment.> > > > > > Step 1: Neutrophils are captured from flow and tether and roll on tumour necrosis factor-Î± (TNF)-stimulated endothelial cells. Step 2: Neutrophils are then activated by the action of CXC-chemokines on the chemokine receptor, CXCR2, a process resulting in the activation of neutrophil Î²2-integrins. Step 3: Î²2-integrins engage counter receptors on the endothelial cell surface, and the neutrophil becomes stationary. Step 4: Prostaglandin-D2 (PGD2), generated by the action of cyclooxygenase enzymes on the n-6-PUFA arachidonic acid (AA), binds the PGD2 receptor, DP-1. DP-1 generates signals that stabilise neutrophil adhesion, induce neutrophil shape change, and support the process of transmigration across the endothelial cell monolayer. Step 5: If the endothelial cells have been supplemented with the n-3-PUFA, eicosapentaenoic acid (EPA), the alternative series prostanoid, PGD3, is generated, and this antagonises PGD2-mediated neutrophil responses.> > > > > > doi:10.1371/journal.pbio.1000177.g001> > > > > > Figure 2. Chemokine signalling through CXCR2 is essential for neutrophil activation on TNF-stimulated endothelial cells.> > > > > > Using a flow-based adhesion assay, neutrophils isolated from whole blood were perfused across endothelial cells. Neutrophils were recruited to endothelial cells treated with TNF, but did not adhere to unstimulated endothelium. Detailed analysis of neutrophil behaviour showed that on TNF-stimulated endothelial cells, the majority of recruited cells transmigrated across the monolayer. When neutrophils were treated with anti-CXCR2, but not anti-CXCR1, neutrophil activation was inhibited so that nearly all of the recruited neutrophils rolled indefinitely on the monolayer and could not migrate; data are mean±SEM of four experiments **p<0.01 for comparison by paired t-test of neutrophil behaviour on TNF stimulated endothelial cells in the presence or absence of anti-CXCR2.> > > > > > doi:10.1371/journal.pbio.1000177.g002> > > Results/Discussion Top> > > > > > EPA Released from Endothelial Cell Membrane Phospholipids Inhibits the Recruitment of Neutrophils> > > > > > Dietary omega-3 polyunsaturated fatty acids (n-3-PUFAs) have anti-inflammatory properties. For example, their inclusion in the diet in the form of n-3-PUFA–rich fish oil reduces the symptoms of disease as well as the use of nonsteroidal anti-inflammatory drugs in arthritis patients with severe inflammatory joint disease [9]. In addition, dietary n-3-PUFAs may offer protection against vascular pathology associated with atherosclerosis, having been reported to be efficacious in epidemiological studies [10]. By modulating inflammation within the artery wall, n-3-PUFAS also alter the cellular and the structural composition of advanced atherosclerotic plaques in a manner that could reduce the incidence of plaque rupture or ulceration, a process that precedes tissue infarction (e.g., heart attack or stroke) [11]. Despite these documented anti-inflammatory benefits, the mode(s) of interaction of these lipids with the immune and inflammatory systems are not well understood. When we supplemented endothelial cell culture medium with the major n-3 -PUFA found in dietary fish oil supplements, eicosapentaenoic acid (EPA; 20:5n-3), it was incorporated into cellular phospholipids so that upon withdrawal of the free fatty acid from the culture medium, a pool of esterified EPA remained localised within endothelial cell membranes (Figure 3A and 3B). After withdrawal of free fatty acid and stimulation with TNF, EPA-treated cells were able to support similar levels of neutrophil adhesion to those that had received no lipid supplement (Figure 4A). However, detailed analysis showed that the behaviour of neutrophils on the two populations of endothelial cells was very different. In response to TNF, but in the absence of EPA, a small population of adherent neutrophils (~20%) rolled throughout the duration of a flow adhesion assay (Figure 4B). The remaining cells became activated on the endothelial cell surface, and this population of neutrophils became progressively smaller as cells migrated across the endothelial cell monolayer (Figure 4B). In contrast, when endothelial cells had been treated with EPA prior to TNF stimulation, there was a marked reduction in the number of cells undergoing transendothelial cell migration (Figure 4B). This was reduced at the earliest time point and did not increase over the duration of the experiment. Interestingly however, many of the cells that did become activated on the surface of endothelial cells reverted to a rolling form of adhesion (Figure 4B), implying that after the receipt of an initial chemokine stimulus, a second signal was required to allow prolonged adhesion and migration across the endothelial cell monolayer. Experiments using different concentrations of EPA showed that these inhibitory effects were evident at concentrations as low as 50 nM (Figure 4C).> > > > > > > > > Figure 3. The effects of EPA supplementation on the fatty acid content of endothelial cell phospholoipds.> > > > > > (A and The concentration of EPA in membrane phospholipids was increased after supplementation of culture medium with EPA for 24 h. Error bars indicate SEM. AA, arachidonic acid; FA, fatty acid; PA, palmitic acid; SA, stearic acid.> > > > > > doi:10.1371/journal.pbio.1000177.g003> > > > > > Figure 4. The effects of EPA supplementation on the adhesive behaviour of neutrophils.> > > > > > (A) EPA supplementation did not affect the number of neutrophils initially adhering to endothelial cells from flow. ( However, the time course of neutrophil behaviour on TNF-stimulated endothelial cells showed that EPA supplementation drastically altered neutrophil behaviour. Thus, the number of activated and surface adherent neutrophils decreased with time in the presence or absence of EPA. However, on endothelium that had not been supplemented with EPA, this was because neutrophils transmigrated across the monolayer, whereas on EPA-supplemented endothelium this was because activated cells reverted back to a rolling form of adhesion. ANOVA showed that there was a significant effect of treatment (i.e., ±EPA) on the percentage of cells that were rolling, surface adherent, or transmigrated (p<0.01). In addition, there was a significant effect of time on each form of behaviour for the EPA-treated cells (p<0.05âˆ'0.01). Bonferroni tests showed significant differences at specific time points as marked; **p<0.01. © Inhibition of neutrophil transmigration was evident at levels of EPA supplementation as low as 50 nM. Data are mean±SEM of five experiments. ANOVA showed significant effects of treatment (p<0.05); *p<0.05 compared to untreated control by Dunnett's test. Error bars indicate SEM.> > > > > > doi:10.1371/journal.pbio.1000177.g004> > > EPA Does Not Inhibit the Expression of Inflammatory Genes in Endothelial Cells> > > > > > To define the molecular mechanisms underlying these observations, we tested two prevalent hypotheses. The first, predicated on the observation that n-3-PUFAs regulate the transcription of endothelial cell inflammatory genes by down-regulating the activity of the nuclear factor-ÎºB, predicts changes in the levels of adhesion receptor and chemokine expression after supplementation with EPA [12]–[14]. However, previous studies have investigated the regulation of gene expression in the presence of up to 100 µM of n-3-PUFAs, whereas physiological blood plasma levels of free fatty acid are of the order of 1 µM [15], a level that can be increased several fold upon supplementation. Using 5 µM EPA as an approximation of the highest concentration achievable in vivo, we did not see changes in the expression of endothelial cell adhesion molecules when assessed for RNA transcripts or protein expression (Figure 5 and Table 1). Neither was there any alteration in the levels of cytokines and chemokines secreted by endothelial cells (Table 2). Thus, in this model, we were able to discount the gene regulation hypothesis.> > > > > > > > > Figure 5. The effect of EPA on TNF-induced surface adhesion molecule expression.> > > > > > The expression of (A) E-selectin and ( VCAM-1 was measured by ELISA in TNF-stimulated endothelial cells that were either unsupplemented or supplemented with EPA. Data are mean±SEM of seven experiments. ANOVA showed that there was a significant effect of the dose of TNF (p<0.01), but not of EPA, on the expression of both receptors.> > > > > > doi:10.1371/journal.pbio.1000177.g005> > > > > > Table 1. Effect of EPA treatment on mRNA expression of endothelial cell adhesion molecules and chemokines induced by TNF.> > > > > > doi:10.1371/journal.pbio.1000177.t001> > > > > > Table 2. Effect of EPA treatment of endothelial cells on the TNF-Î±-induced secretion of cytokines and chemokines.> > > > > > doi:10.1371/journal.pbio.1000177.t002> > > The Anti-Inflammatory Effects of EPA Supplementation Require the Metabolic Activity of Cyclooxygenase Enzymes> > > > > > The second hypothesis proposes that upon endothelial cell activation, EPA may compete with the n-6-PUFA arachidonic acid (AA; 20:4n-6) for cyclooxygenase enzymes (COX1 and COX2) after both fatty acids are liberated from membrane phospholipids by endogenous phospholipases [16]. Ordinarily, AA is metabolised by COX into 2-series endoperoxides, with downstream synthases converting these into the biologically active 2-series prostanoids [17]. However, when utilised as a COX substrate, the metabolism of EPA generates alternative 3-series prostanoids [16]. The 2-series prostanoids generated by COX activity are known to regulate aspects of the inflammatory response. For example, in the COX2 knockout mouse, neutrophil recruitment is dramatically reduced upon ischaemia and reperfusion-induced injury of the liver when compared to wild-type control animals [18]. In a murine model of lipopolysaccharide-induced lung inflammation, inhibitors of COX function (indomethacin and aspirin) modestly increased neutrophil recruitment [19]. Conversely in vitro, inhibition of COX function by aspirin can inhibit neutrophil migration [20] while having no effect on the levels of neutrophil adhesion to the endothelial cell monolayer [21]. Moreover, in murine models of acute and chronic inflammation, a reduction in PGE2 production by genetic deletion of its membrane-bound synthase, moderates the formation of inflammation-associated granulation tissue and angiogenesis, as well as decreasing the nociception of pain, indicating that PGE2 is proinflammatory in these models [22]. PGD2 also appears to have proinflammatory functions, as overexpression of the synthase generating this prostanoid increases production of inflammatory cytokines and chemokines, leading to exaggerated levels of eosinophil and lymphocyte recruitment [23], a mechanism that operates through the PGD2 receptor DP-2. In contrast, in acute peritoneal inflammation, knockout of PGD2 synthase increased inflammatory cytokine production and retarded the rate of inflammatory resolution by a mechanism that operated through the DP-1 receptor [24]. Thus, PGD2 appears to have pro- or anti-inflammatory capabilities depending on the nature of the inflammatory insult. The inflammatory activity of the equivalent 3-series prostanoids is not known. Here, by introducing a panel of COX inhibitors into endothelial cell cultures at the same time as they were activated with TNF, the effects of EPA supplementation could be replicated, with neutrophil transmigration being dramatically inhibited (Figure 6). Moreover, adding a molecular excess of AA to EPA-supplemented endothelial cells at the point of TNF activation could reverse the blockade of migration (Figure 6). Taken together, these data imply that a COX-derived product of AA is required for the transmigration of neutrophils across TNF-stimulated endothelial cells, and that in the presence of EPA, this pathway is efficiently antagonised.> > > > > > > > > Figure 6. A cyclooxygenase (COX)-derived eicosanoid is required for neutrophil transmigration across TNF-stimulated endothelial cells.> > > > > > (A) Inhibition of COX-1 (SC-560), COX-2 (NS-398), or both (indomethacin) caused inhibition of neutrophil transmigration, *p<0.05 by t-test. ( Addition of AA to EPA-supplemented endothelial cells during the period of TNF stimulation restored neutrophil transmigration. All data are mean±SEM of four experiments. ANOVA showed significant effects of treatments in both (A and (p<0.01). In (A), Dunnett test showed significant effects of individual treatments compared to untreated control, *p<0.05. In (, Dunnett's test showed significant difference between EPA-treated and untreated control (**p<0.01), but not between AA+EPA and control.> > > > > > doi:10.1371/journal.pbio.1000177.g006> > > Prostaglandin D2 Is Required for Neutrophil Migration across Endothelial Cells Stimulated with TNF-Î±> > > > > > Endothelial cells generate COX products constitutively. For example, prostacyclin (PGI2) and prostaglandin D2 (PGD2) are endothelial cell-derived vasoactive prostanoids that are also involved in the regulation of haemostasis, being antagonists of platelet activation [17],[25]. These prostanoids are difficult to measure in the systemic blood, as they have reported half-lives in plasma that are measured in minutes [26],[27]. However, the constitutive nature of their production is demonstrated by the presence in urine and body fluids of their downstream metabolic products, PGF1Î± and delta-PGJ2, respectively [28],[29].> > > > > > Prostanoids have a documented ability to regulate the migration of a number of leukocyte subsets. For example, PGD2 induces chemotaxis of eosinophils and T-lymphocytes [30] in vitro. Interestingly, the migration of monocyte-derived migratory dendritic cells (DCs) may be tightly regulated by interplay between different prostanoids. Thus, the ability of these cells to traffic out of tissue into lymph nodes via the lymphatic circulation is dependent upon the presence of PGE2 as a differentiation signal [31]–[34]. In this context PGE2 promotes the function (but not the expression) of the chemokine receptor CCR7, so that DCs efficiently respond to the chemokines CCL19 and CCL21. Importantly, a number of studies have shown that the presence of PGD2 can in turn inhibit the ability of DCs to migrate out of the lungs or the skin during an inflammatory response, although the molecular mechanism by which this inhibition is achieved remains undescribed [35],[36].> > > > > > As prostanoids are well documented to regulate leukocyte migration [37], we tested the hypothesis that PGD2 was the endothelial cell-derived agent providing the stimulus for neutrophil migration across the monolayer. We established a population of surface adherent neutrophils on TNF-treated endothelial cells supplemented with EPA and perfused synthetic PGD2 across the endothelial cells and neutrophils to see whether this would reintroduce transmigration. The provision of exogenous PGD2 but not PGD3 (which could be derived from EPA), fully restored the ability of neutrophils to cross the endothelial cell monolayer (Figure 7A). Prostaglandin D2 has two receptors. Chemoattractant-receptor homologous molecule expressed on Th2 cells receptor (CRTH2 or DP-2) is not expressed in neutrophils [35], whereas the DP-1 receptor has been reported in these cells [38]. Here, neutrophils perfused across TNF-stimulated endothelial cells in the presence of a DP-1 receptor antagonist (BW868C) showed a greatly diminished efficiency of endothelial cell transmigration (Figure 7B). Conversely, the transmigration of neutrophils was re-established on EPA-supplemented endothelial cells when a synthetic DP-1 receptor agonist (BW245C) was perfused across cells adherent to the surface of the monolayer (Figure 7C). Importantly, we could demonstrate that PGD2 was operating directly on the neutrophils, because when these cells were harvested after migrating across a TNF-stimulated endothelial cells they had up-regulated CD11b (the alpha subunit of the Î²2 integrin CD11b/CD18 that is required for efficient neutrophil transmigration) and proteolytically shed L-selectin (Figure 8). However, neutrophils harvested from the surface of EPA-treated endothelial cells did not shed L-selectin or up-regulate CD11b. Thus, in this assay system, the delivery of a PGD2-mediated signal to the neutrophils was necessary for full cellular activation and efficient transendothelial migration.> > > > > > > > > Figure 7. The role of AA- and EPA-derived eicosanoids in neutrophil transmigration.> > > > > > (A) When PDG2 was perfused across a population of neutrophils adherent to EPA-treated endothelial cells, neutrophil transmigration was restored. However, PGD3 had no significant effect on neutrophil behaviour. All data are mean±SEM of four experiments. ANOVA showed significant effect of treatment on transmigration. *p<0.05 compared to EPA-supplemented endothelial cells in the absence of PGD2 by Dunnett test. ( The DP1 receptor antagonist BW868c dose-dependently inhibited neutrophil transmigration on TNF-stimulated endothelial cells. All data are mean±SEM of five experiments; ANOVA showed a significant effect of BW868c concentration (p<0.01). © Perfusion of the DP-1 receptor agonist, BW245C, across neutrophils adherent to EPA-supplemented endothelial cells restored neutrophil migration to control levels. Data are mean±SEM of five experiments. ANOVA showed a significant effect of treatment on transmigration. **p<0.01 compared to EPA-supplemented endothelial cells in the absence of BW868C by Dunnett test. (D) Neutrophils perfused across TNF-stimulated endothelial cells in the presence of PGD3 showed a significantly reduced ability to migrate across the monolayer. Data are mean±SEM of three experiments; ANOVA showed a significant effect of PGD3 concentration on transmigration (p<0.01). (E) The levels of PGD3 released from endothelial cells are increased after EPA supplementation. Data are mean±SEM of three experiments. ANOVA showed a significant effect of treatment (p<0.05). **p<0.01 for PGD3 production compared to endothelial cells activated with TNF without EPA supplementation by Dunnett's test.> > > > > > doi:10.1371/journal.pbio.1000177.g007> > > > > > Figure 8. The effect of adhesion on the expression of L-selectin and CD11b on neutrophils.> > > > > > Neutrophils that were freshly isolated, migrated across endothelial cells stimulated with TNF or adherent to the surface of EPA-supplemented endothelial cells (EC) activated with TNF were harvested and the expression of (A) CD11b or ( L-selectin (CD62L) assessed by flow cytometry. Data are mean±SEM of four experiments; ANOVA showed significant effect of treatments in (A) (p<0.01) and ( (p<0.05). In (A and , Dunnett's test showed significant difference between âˆ'EPA and freshly isolated cells (*p<0.01 and *p<0.05, respectively), but not between +EPA and freshly isolated cells.> > > > > > doi:10.1371/journal.pbio.1000177.g008> > > PGD3 Generated from EPA Antagonises the PGD2-Mediated Migration of Neutrophils across Endothelial Cells> > > > > > Taken together, these data show that PGD2 operates through the neutrophil DP-1 receptor to provide a signal that is essential for transendothelial cell migration. Moreover, the ability of neutrophils to respond to this signal is abolished when endothelial cells are preloaded with EPA from which PGD3 is generated. This implies that PGD3 may be an effective antagonist of the DP-1 receptor. To test this hypothesis, we perfused neutrophils across TNF-stimulated endothelial cells in the presence of exogenous PGD3. Neutrophil transmigration was inhibited in a dose-dependent manner by PGD3, showing that in the presence of endogenously generated PGD2 it could effectively antagonise this process (Figure 7D). Importantly, we were able to show that cells supplemented with EPA-generated an increased amount of PGD3 (Figure 7E).> > > > > > The absolute levels of PGD2 and PGD3 that are generated in our assay system, and thus the ratio of their abundance, are not easily assessed. The levels of PGD2 measured as an endothelial cell releasate were at the limits of detection (Table 3), presumably due to the short half-life of this prostanoid under physiological conditions; however, significant levels of PGD3 were assayed upon EPA supplementation, and concentrations in the order of 5–10 nM had accumulated over the 24 h of the EPA supplementation period. The half-life of PGD3 in serum or albumin-containing medium has not been reported to our knowledge; thus it is not clear whether the measurements made here report the true levels of PGD3 production, or if metabolic degradation of this prostanoid renders these measurements underestimates. Irrespective of this, our experiments utilising synthetic PGD2 demonstrate that concentrations on the order of 1 nM were sufficient to fully recapitulate the process of migration on EPA-supplemented endothelial cells, and when using PGD3 as an antagonist of TNF-induced transmigration marked effects were evident at 10–100 pM. Thus, the measurable levels of PGD3 that we report would certainly be sufficient to effectively antagonise the transmigration of neutrophils in our experimental model.> > > > > > > > > Table 3. Analysis of eicosanoids by liquid chromatography tandem mass spectrometry.> > > > > > doi:10.1371/journal.pbio.1000177.t003> > > Conclusions> > > > > > Our study demonstrates a hitherto unknown regulatory step in the recruitment of neutrophils by cytokine-stimulated endothelial cells (refer to Figure 1 to see how this new step fits with the known regulatory mechanisms of neutrophil recruitment). After initial tethering by selectin molecules, a chemokine signal induces arrest of the cell on the endothelial cell surface. However, the chemokine signal is not sufficient to support transmigration across the endothelial cell monolayer, and the arachidonic acid-derived prostanoid, PGD2, is an essential downstream regulator of this process. PGD2 operates through the DP-1 receptor and this signal can be effectively antagonised by PGD3 which is generated from EPA released by the action of phospholipase enzymes on the phospholipids of EPA supplemented cells. Not only does this study reveal a new step in the recruitment of neutrophil recruitment during inflammation, it also reveals a novel anti-inflammatory mechanism of action of the dietary n-3-PUFA, EPA.> > > > > > Materials and Methods Top> > > > > > Cell Culture and Neutrophil Isolation> > > > > > Endothelial cells (EC) were isolated and cultured, as previously described [39], with or without 0–5 µM EPA (Sigma) for 24 h. TNF-Î± (100 U/ml; R & D systems) was added to the EC for the final 4 h of culture. In some experiments, EC were treated with 100 U/ml TNF-Î± in the presence of 10 µM indomethacin or 0.1 µM SC-560 or 1 µM NS-398 (Sigma). In the AA reconstitution experiments, 5 µM AA (Sigma) was added to the culture medium simultaneously with TNF-Î±.> > > > > > Blood from healthy adult volunteers was collected into tubes coated with EDTA (1.6 mg/ml), and neutrophils were separated using 2-step density gradients of Histopaque 1119 and 1077 (Sigma), as previously described [40]. After washing in 0.15% BSA in PBS, cells were counted using a Coulter Multisizer (Coulter Electronics), and neutrophils, which were >95% pure, were resuspended at a concentration of 1×106/ml in PBS/Alb with calcium and magnesium.> > > > > > RNA Extraction and Real-Time PCR> > > > > > mRNA was isolated from EC using the Qiagen RNEasy Mini Kit 50 (Qiagen) following the manufacturer's instructions. Real-time PCR (RT-PCR) was performed using QuantiTect probe RT-PCR kit according to the manufacturer's instructions (Qiagen). Primers were purchased from Applied Biosystems. The expression of each target gene was normalised to Î²-actin expression, and the data presented represent fold change compared to untreated EC.> > > > > > Luminex for Secreted Cytokines and Chemokines> > > > > > Supernatants were collected from EC that had been incubated with 0–5 µM EPA for 24 h and had 100 U/ml TNF added to the culture for the final 4 h of culture. The Luminex kit was purchased from Upstate/Chemicon, and the experiment performed to the manufacturer's instructions. Data were collected and analysed from the samples using a Luminex100 machine (Luminex).> > > > > > Flow-Based Adhesion Assay> > > > > > Glass capillaries (microslides) containing treated EC monolayers were incorporated into a flow-based adhesion assay as described [39]. Briefly, microslides were attached to cell and fluid reservoirs by silicon tubing at one end and to a withdrawal syringe pump at the other end. After mounting on the stage of a phase contrast microscope, EC were washed for 2 min with PBS containing 0.15% BSA. Neutrophils were then perfused (1×106/ml) at 0.1 Pa for 4 min, followed by 15 min of wash. Video images were recorded throughout the experiment and neutrophil behaviour analysed offline using Image Pro software (Image-Pro Plus). In some experiments, neutrophils were perfused across EPA- and TNF-treated EC, allowed to adhere, and subsequently perfused with 1 nM PGD2 or 1 nM PGD3 (both from Cayman Chemicals). Alternatively, neutrophils were treated with BW868c (Cayman Chemicals), a DP1 receptor antagonist, for 10 min prior to perfusion over TNF-Î±-stimulated EC. Neutrophils were also treated with the DP1 receptor agonist BW245c (Cayman Chemicals) prior to perfusion over EPA- and TNF-Î±-treated EC. Neutrophils were also treated with 0–1 nM PGD3, immediately prior to perfusion across TNF-stimulated EC. In all experiments, lipid reagents, agonists, and antagonists were stored in 100% ethanol under nitrogen gas. Once diluted, the medium contained <0.01% ethanol. All controls contained equivalent concentrations of ethanol.> > > > > > Analysis of EPA Incorporation into Cellular Phospholipids> > > > > > First-passage confluent EC were treated with 5 µM of EPA for 24 h. EC were removed from the plastic culture dish by scraping and stored in 0.88% KCl solution at âˆ'20°C until analysis. Total lipid was extracted with chloroform:methanol (2:1, v/v) containing butylated hydroxytoluene (50 mg/l) as antioxidant. Fatty acids were subsequently hydrolysed from the lipid and simultaneously methylated by incubation with methylation reagent (methanol containing 2% v/v H2SO4) at 50°C for 2 h. Fatty acid methyl esters were separated and identified using a Hewlett Packard 6890 gas chromatograph (Hewlett Packard) fitted with a 30 mm×32 mm BPX 70 capillary column, film thickness 0.25 µm. Helium, at the initial flow rate of 1.0 ml/min, was used as the carrier gas. Injector and detector temperatures were 275°C, and the column oven temperature was maintained at 170°C for 12 min after sample injection. The oven temperature was programmed to increase from 170 to 210°C at 5°C/min. Fatty acid methyl esters were identified by comparison with authentic standards. Peak areas were quantified using ChemStation software (Hewlett Packard). Each fatty acid was expressed as weight percent of total fatty acids present.> > > > > > Eicosanoid Extraction and Reverse-Phase High-Performance Liquid Chromatography Analysis> > > > > > Eicosanoids were extracted from control EC using C18 Sep-Pak cartridges (Waters). Briefly, cartridges were conditioned with 5 ml of high-performance liquid chromatography (HPLC)-grade MeOH and rinsed twice with water. Reactions were terminated with addition of 100% ice-cold MeOH, containing 2 ng of internal standard PGE2-d4. Sample volume was adjusted to 10% MeOH with water and applied to the column. The column was rinsed with water and the sample eluted with 2 ml of MeOH. Nitrogen gas was used to dry the sample, which was resuspended in 100 µl of MeOH and stored at âˆ'80°C until analysis.> > > > > > Prostaglandin Quantitation using Liquid Chromatography Tandem Mass Spectrometry> > > > > > Prostaglandins were separated on a C18 ODS2, 5 µm, 150×4.6-mm column (Waters) using a gradient of 50%–90% B over 20 min (A, water:acetonitrile:acetic acid, 75:25:0.1; B, methanol:acetonitrile:acetic acid, 60:40:0.1) at 1 ml/min. Products were quantitated by directing the HPLC output directly into the electrospray source of a Q-Trap mass spectrometer (Applied Biosystems 4000 Q-Trap) operating in the negative mode (âˆ'4,500 V). Individual prostaglandins were monitored in the Multiple Reaction Monitoring (MRM) mode using specific parent to daughter transitions of m/z 349–269 for PGD3 with collision energies of âˆ'26 V. Products were identified and quantified with reference to the appropriate standards run in parallel under the same conditions, with 2 ng of PGE2-d4 (m/z 355–275) added as an internal standard.> > > > > > Analysis of Neutrophil Adhesion Molecule Expression by Flow Cytometry> > > > > > Neutrophils for analysis were isolated from whole blood as described above. Control cells were stained for flow cytometry directly after isolation. Cells rolling on EPA-treated endothelial cells in microslides were harvested by elution with 0.02% EDTA after 4 min of neutrophil perfusion and 11 min of wash buffer perfusion (totalling 15 min of contact with endothelial cells). In order to harvest neutrophils that had migrated across endothelial cells, endothelium was cultured on 1.5 mg/ml type I collagen gels (Becton Dickinson). After endothelial cell stimulation with TNF for 4 h, neutrophils were added for 15 min. Surface-adherent cells were washed off with 0.02% EDTA, and the gel was dissolved using type VII collagenase (Sigma). Neutrophils were harvested from the dissolved gel by centrifugation. All neutrophil populations were stained for surface expression of CD11b (PE-conjugated, clone 2LPM19c, 2 µg/ml; DAKO) or CD62L (FITC-conjugated, clone Dreg56,10 µg/ml, Beckton Dickinson). Expression was analysed by flow cytometry using a DAKO CyAn, and data analysed using Summit software (Becton Dickinson). Data are expressed as mean fluorescent intensity.> > > > > > Statistical Analysis> > > > > > Data were analysed using Prism software (GraphPad software). Results are presented as means±standard error of the mean (SEM). Comparisons between individual treatments were by paired t-test where appropriate. ANOVA was performed to assess the effect of EPA concentration on PMN transmigration. Significant findings were investigated further using Bonferroni multiple comparison test or Dunnett's test.> > > > > > Author Contributions Top> > > > > > The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: VBO RFG PCC GBN GER. Performed the experiments: SPT CMY BHM JM. Analyzed the data: SPT CMY BHM VBO JM GER. Contributed reagents/materials/analysis tools: VBO RFG PCC GBN. Wrote the paper: GER.> > > > > > References Top> > > > > > Springer T. A (1995) Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol 57: 827–872. Find this article online> > > Dunon D, Piali L, Imhof B. A (1996) To stick or not to stick: the new leukocyte homing paradigm. Curr Opin Cell Biol 8: 714–723. Find this article online> > > Liu Y, Shaw S. K, Ma S, Yang L, Luscinskas F. W, et al. (2004) Regulation of leukocyte transmigration: cell surface interactions and signaling events. 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Arterioscler Thromb Vasc Biol 15: 622–628. Find this article online> > > Collie-Duguid E. S. R, Wahle K. W. J (1996) Inhibitory effect of fish oil N-3 polyunsaturated fatty acids on the expression of endothelial cell adhesion molecules. Biochem Biophys Res Commun 220: 969–974. Find this article online> > > Shaw D, Hall W, Jeffs N, C. M (2007) Comparative effects of fatty acids on endothelial inflammatory gene expression. Eur J Nutr 46: 328. Find this article online> > > Kusunoki M, Tsutsumi K, Nakayama M, Kurokawa T, Nakamura T, et al. (2007) Relationship between serum concentrations of saturated fatty acids and unsaturated fatty acids and the homeostasis model insulin resistance index in Japanese patients with type 2 diabetes mellitus. J Med Invest 54: 243–247. Find this article online> > > Calder P. C (2001) N-3 polyunsaturated fatty acids, inflammation and immunity: pouring oil on troubled waters or another fishy tale? Nutr Res 21: 309–341. 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L (1979) Granulocyte adhesion to endothelium in culture. J Cell Sci 38: 225–235. Find this article online> > > Kamei D, Yamakawa K, Takegoshi Y, Mikami-Nakanishi M, Nakatani Y, et al. (2004) reduced pain hypersensitivity and inflammation in mice lacking microsomal prostaglandin E synthase-1. J Biol Chem 279: 33684–33695. Find this article online> > > Fujitani Y, Kanaoka Y, Aritake K, Uodome N, Okazaki-Hatake K, et al. (2002) Pronounced eosinophilic lung inflammation and th2 cytokine release in human lipocalin-type prostaglandin d synthase transgenic mice. J Immunol 168: 443–449. Find this article online> > > Rajakariar R, Hilliard M, Lawrence T, Trivedi S, Colville-Nash P, et al. (2007) From the cover: hematopoietic prostaglandin D2 synthase controls the onset and resolution of acute inflammation through PGD2 and 15-deoxy{Delta}12 14 PGJ2. Proc Natl Acad Sci U S A 104: 20979–20984. 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Blood 100: 1362–1372. Find this article online> > > Scandella E, Men Y, Legler D. F, Gillessen S, Prikler L, et al. (2004) CCL19/CCL21-triggered signal transduction and migration of dendritic cells requires prostaglandin E2. Blood 103: 1595–1601. Find this article online> > > Legler D. F, Krause P, Scandella E, Singer E, Groettrup M (2006) Prostaglandin E2 is generally required for human dendritic cell migration and exerts its effect via EP2 and EP4 receptors. J Immunol 176: 966–973. Find this article online> > > Angeli V, Faveeuw C, Roye O, Fontaine J, Teissier E, et al. (2001) Role of the parasite-derived prostaglandin D2in the inhibition of epidermal langerhans cell migration during schistosomiasis infection. J Exp Med 193: 1135–1147. Find this article online> > > Hammad H, Jan de Heer H, Soullie T, Hoogsteden H. C, Trottein F, et al. (2003) Prostaglandin D2 inhibits airway dendritic cell migration and function in steady state conditions by selective activation of the D prostanoid receptor 1. J Immunol 171: 3936–3940. Find this article online> > > Sandig H, Pease J. E, Sabroe I (2007) Contrary prostaglandins: the opposing roles of PGD2 and its metabolites in leukocyte function. J Leukocyte Biol 81: 372–382. Find this article online> > > Schratl P, Royer J. F, Kostenis E, Ulven T, Sturm E. M, et al. (2007) The role of the prostaglandin D2 receptor, DP, in eosinophil trafficking. J Immunol 179: 4792–4799. Find this article online> > > Bahra P, Rainger G. E, Wautier J. L, Nguyet-Thin L, Nash G. B (1998) Each step during transendothelial migration of flowing neutrophils is regulated by the stimulatory concentration of tumour necrosis factor-alpha. Cell Adhes Commu 6: 491–501. Find this article online> > > Cooke B. M, Usami S, I, Nash G. 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November 6, 2011

This is a good find, but a strange one too. I read the article, and the study,

although a bit old (1988), seems well-designed, however.

A possible explanation I see is that the subjects were fed too much Omega-3s

(150g of sardines per day + 2g of EPA from a supplement), to such a point that

the decrease in -2 products that was achieved proved more detrimental than

the switchover from series-4 to series-5 leukotrienes that took place was

beneficial.

In this hypothesis, Omegas-3s would still be beneficial, but in smaller

quantities, inasmuch as that they would contribute to reduce the Omega-6 /

Omega-3 imbalance of the diet while this imbalance still exists, but they might

turn less beneficial in higher quantities, once the imbalance is corrected.

Even this supposition, however, is not perfectly coherent with the reported

experiences of members of this group ; for instance, I remember Lori Baur told

us she had had excellent results with the fish oil concentrates that Dr. Floyd

Chilton used to sell several years ago.

So, as they often conclude abstracts, I am afraid that more studies are required

to help us get a clearer picture.

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> > > > Research Article

> > > > Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a

> New Step in Neutrophil Recruitment

> > > >

> > > > While investigating new mechanisms by which the dietary omega-3

> fatty acids regulate inflammation, the authors have identified a new

> step in the regulation of neutrophil migration across vascular

> endothelial cells.

> > > >

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> > > >

> > > > Abstract

> > > > Author Summary

> > > > Introduction

> > > > Results/Discussion

> > > > Materials and Methods

> > > > Author Contributions

> > > > References

> > > > P. Tull1, Clara M. Yates1, H. Maskrey2,

> B. O'Donnell2, Jackie Madden3, F. Grimble3, Philip C. Calder3,

> Gerard B. Nash1, G. Ed. Rainger1*

> > > >

> > > > 1 Centre for Cardiovascular Sciences, School of Clinical and

> Experimental Medicine, The Medical School, The University of Birmingham,

> Birmingham, United Kingdom, 2 Department of Medical Biochemistry and

> Immunology, School of Medicine, Cardiff University, Cardiff, United

> Kingdom, 3 Institute of Human Nutrition, School of Medicine, University

> of Southampton, Southampton, United Kingdom

> > > >

> > > > Abstract Top

> > > >

> > > > Inflammation is a physiological response to tissue trauma or

> infection, but leukocytes, which are the effector cells of the

> inflammatory process, have powerful tissue remodelling capabilities.

> Thus, to ensure their precise localisation, passage of leukocytes from

> the blood into inflamed tissue is tightly regulated. Recruitment of

> blood borne neutrophils to the tissue stroma occurs during early

> inflammation. In this process, peptide agonists of the chemokine family

> are assumed to provide a chemotactic stimulus capable of supporting the

> migration of neutrophils across vascular endothelial cells, through the

> basement membrane of the vessel wall, and out into the tissue stroma.

> Here, we show that, although an initial chemokine stimulus is essential

> for the recruitment of flowing neutrophils by endothelial cells

> stimulated with the inflammatory cytokine tumour necrosis factor-Î±,

> transit of the endothelial monolayer is regulated by an additional and

> downstream stimulus. This signal is supplied by the metabolism of the

> omega-6-polyunsaturated fatty acid (n-6-PUFA), arachidonic acid, into

> the eicosanoid prostaglandin-D2 (PGD2) by cyclooxygenase (COX) enzymes.

> This new step in the neutrophil recruitment process was revealed when

> the dietary n-3-PUFA, eicosapentaenoic acid (EPA), was utilised as an

> alternative substrate for COX enzymes, leading to the generation of

> PGD3. This alternative series eicosanoid inhibited the migration of

> neutrophils across endothelial cells by antagonising the PGD2 receptor.

> Here, we describe a new step in the neutrophil recruitment process that

> relies upon a lipid-mediated signal to regulate the migration of

> neutrophils across endothelial cells. PGD2 signalling is subordinate to

> the chemokine-mediated activation of neutrophils, but without the

> sequential delivery of this signal, neutrophils fail to penetrate the

> endothelial cell monolayer. Importantly, the ability of the dietary

> n-3-PUFA, EPA, to inhibit this process not only revealed an unsuspected

> level of regulation in the migration of inflammatory leukocytes, it also

> contributes to our understanding of the interactions of this bioactive

> lipid with the inflammatory system. Moreover, it indicates the potential

> for novel therapeutics that target the inflammatory system with greater

> affinity and/or specificity than supplementing the diet with n-3-PUFAs.

> > > >

> > > > Author Summary Top

> > > >

> > > > Inflammation is a physiological response to tissue trauma or

> infection. Neutrophils, which circulate in the blood stream, are the

> first inflammatory cells to be recruited to a site of tissue

> inflammation. In response to recruitment signals provided by chemotactic

> peptides called chemokines, neutrophils traverse the endothelial cell

> lining of blood vessels. This process involves a multistep cascade of

> neutrophil adhesion and activation events on the endothelial barrier.

> While investigating the anti-inflammatory functions of the omega-3 fatty

> acid , EPA, which is found, for instance, in dietary fish oil, we

> identified an additional unexpected lipid-derived signal that is

> essential for neutrophil migration across the endothelium. Our

> experiments show that a chemokine delivered the first signal needed to

> bind neutrophils firmly to the endothelial surface. However, in order to

> traverse the endothelium, a subsequent signal delivered by

> prostaglandin-D2 (PGD2), a lipid derived from the omega-6 fatty acid

> arachidonic acid, was essential. When EPA, was introduced into the

> experiment, it was used to form PGD3. This alternative lipid blocked

> interactions between PGD2 and its receptor on neutrophils, preventing

> the process of migration across the endothelial barrier. Thus, we reveal

> a new step in the recruitment of neutrophils during inflammation, and a

> novel anti-inflammatory mechanism of action of dietary EPA.

> > > >

> > > > Citation: Tull SP, Yates CM, Maskrey BH, O'Donnell VB, Madden J,

> et al. (2009) Omega-3 Fatty Acids and Inflammation: Novel Interactions

> Reveal a New Step in Neutrophil Recruitment. PLoS Biol 7(8): e1000177.

> doi:10.1371/journal.pbio.1000177

> > > >

> > > > Academic Editor: Philip M. , NIH/NIAID, United States of

> America

> > > >

> > > > Received: February 6, 2009; Accepted: July 14, 2009; Published:

> August 25, 2009

> > > >

> distributed under the terms of the Creative Commons Attribution License,

> which permits unrestricted use, distribution, and reproduction in any

> medium, provided the original author and source are credited.

> > > >

> > > > Funding: This work was supported by the Biotechnology and

> Biological Sciences Research Council, United Kingdom (grant BBS/B/0105)

> and by the British Heart Foundation (grant BS/97001). The funders had no

> role in study design, data collection and analysis, decision to publish,

> or preparation of the manuscript.

> > > >

> > > > Competing interests: The authors have declared that no competing

> interests exist.

> > > >

> > > > Abbreviations: DC, dendritic cell

> > > >

> > > > * E-mail: g.e.rainger@

> > > >

> > > > Introduction Top

> > > >

> > > > In vertebrates, tissue trauma or infection causes the rapid

> initiation of an inflammatory reaction. The early phase of this

> phylactic response results in the localised recruitment of cells of the

> innate immune system from the blood, a tissue infiltrate that is

> dominated by neutrophils. The molecular processes that support the

> initial interactions between blood-borne neutrophils and the endothelial

> cells lining postcapillary venules (the site in the blood vasculature

> where leukocytes are recruited during inflammation) are well described

> (see Figure 1 for a schematic representation of the steps in the

> neutrophil recruitment process). In response to the localised production

> of inflammatory mediators, such as the cytokine tumour necrosis

> factor-Î± (TNF), activated endothelial cells decorate themselves with

> specialised adhesion receptors of the selectin family [1]–[3]. Due

> to their ability to rapidly form strong but short-lived bonds with

> carbohydrate counter-ligands on the neutrophil surface, E- and

> P-selectin are capable of tethering neutrophils from rapidly flowing

> blood [4]. The sequential formation and dissolution of selectin bonds

> also support a characteristic and dynamic form of adhesion, referred to

> as rolling [5]. Rolling adhesion does not require neutrophil activation.

> However, neutrophil migration through the vessel wall and into the

> inflamed tissue is dependent upon the receipt of an activating stimulus

> [5],[6]. We have previously shown that endothelial cells can present

> peptide agonists of the CXC-chemokine family to rolling neutrophils and

> thus stabilise adhesion [7],[8]. Ligation of the neutrophil

> CXC-Receptor-2 (CXCR2) by these agents is essential for activation of

> the Î²2-intergrin adhesion receptors and the reorganisation of the

> actin cytoskeleton that support transendothelial cell migration [8].

> Here, we show that in the presence of an antibody that blocks chemokine

> interactions with CXCR2, neutrophils roll on the endothelial cell

> surface indefinitely (Figure 2), demonstrating that a chemokine signal

> is essential for neutrophil activation on TNF-stimulated endothelial

> cells. However, the removal of this primary activating stimulus provides

> no information on the requirement for additional, downstream signals,

> which might coordinate transit of the vessel wall and onward migration

> into stromal tissues.

> > > >

> > > > Figure 1. Steps in the process of neutrophil recruitment.

> > > >

> > > > Step 1: Neutrophils are captured from flow and tether and roll on

> tumour necrosis factor-Î± (TNF)-stimulated endothelial cells. Step 2:

> Neutrophils are then activated by the action of CXC-chemokines on the

> chemokine receptor, CXCR2, a process resulting in the activation of

> neutrophil Î²2-integrins. Step 3: Î²2-integrins engage counter

> receptors on the endothelial cell surface, and the neutrophil becomes

> stationary. Step 4: Prostaglandin-D2 (PGD2), generated by the action of

> cyclooxygenase enzymes on the n-6-PUFA arachidonic acid (AA), binds the

> PGD2 receptor, DP-1. DP-1 generates signals that stabilise neutrophil

> adhesion, induce neutrophil shape change, and support the process of

> transmigration across the endothelial cell monolayer. Step 5: If the

> endothelial cells have been supplemented with the n-3-PUFA,

> eicosapentaenoic acid (EPA), the alternative series prostanoid, PGD3, is

> generated, and this antagonises PGD2-mediated neutrophil responses.

> > > >

> > > > doi:10.1371/journal.pbio.1000177.g001

> > > >

> > > > Figure 2. Chemokine signalling through CXCR2 is essential for

> neutrophil activation on TNF-stimulated endothelial cells.

> > > >

> > > > Using a flow-based adhesion assay, neutrophils isolated from whole

> blood were perfused across endothelial cells. Neutrophils were recruited

> to endothelial cells treated with TNF, but did not adhere to

> unstimulated endothelium. Detailed analysis of neutrophil behaviour

> showed that on TNF-stimulated endothelial cells, the majority of

> recruited cells transmigrated across the monolayer. When neutrophils

> were treated with anti-CXCR2, but not anti-CXCR1, neutrophil activation

> was inhibited so that nearly all of the recruited neutrophils rolled

> indefinitely on the monolayer and could not migrate; data are

> mean±SEM of four experiments **p<0.01 for comparison by paired t-test

> of neutrophil behaviour on TNF stimulated endothelial cells in the

> presence or absence of anti-CXCR2.

> > > >

> > > > doi:10.1371/journal.pbio.1000177.g002

> > > > Results/Discussion Top

> > > >

> > > > EPA Released from Endothelial Cell Membrane Phospholipids Inhibits

> the Recruitment of Neutrophils

> > > >

> > > > Dietary omega-3 polyunsaturated fatty acids (n-3-PUFAs) have

> anti-inflammatory properties. For example, their inclusion in the diet

> in the form of n-3-PUFA–rich fish oil reduces the symptoms of

> disease as well as the use of nonsteroidal anti-inflammatory drugs in

> arthritis patients with severe inflammatory joint disease [9]. In

> addition, dietary n-3-PUFAs may offer protection against vascular

> pathology associated with atherosclerosis, having been reported to be

> efficacious in epidemiological studies [10]. By modulating inflammation

> within the artery wall, n-3-PUFAS also alter the cellular and the

> structural composition of advanced atherosclerotic plaques in a manner

> that could reduce the incidence of plaque rupture or ulceration, a

> process that precedes tissue infarction (e.g., heart attack or stroke)

> [11]. Despite these documented anti-inflammatory benefits, the mode(s)

> of interaction of these lipids with the immune and inflammatory systems

> are not well understood. When we supplemented endothelial cell culture

> medium with the major n-3 -PUFA found in dietary fish oil supplements,

> eicosapentaenoic acid (EPA; 20:5n-3), it was incorporated into cellular

> phospholipids so that upon withdrawal of the free fatty acid from the

> culture medium, a pool of esterified EPA remained localised within

> endothelial cell membranes (Figure 3A and 3B). After withdrawal of free

> fatty acid and stimulation with TNF, EPA-treated cells were able to

> support similar levels of neutrophil adhesion to those that had received

> no lipid supplement (Figure 4A). However, detailed analysis showed that

> the behaviour of neutrophils on the two populations of endothelial cells

> was very different. In response to TNF, but in the absence of EPA, a

> small population of adherent neutrophils (~20%) rolled throughout the

> duration of a flow adhesion assay (Figure 4B). The remaining cells

> became activated on the endothelial cell surface, and this population of

> neutrophils became progressively smaller as cells migrated across the

> endothelial cell monolayer (Figure 4B). In contrast, when endothelial

> cells had been treated with EPA prior to TNF stimulation, there was a

> marked reduction in the number of cells undergoing transendothelial cell

> migration (Figure 4B). This was reduced at the earliest time point and

> did not increase over the duration of the experiment. Interestingly

> however, many of the cells that did become activated on the surface of

> endothelial cells reverted to a rolling form of adhesion (Figure 4B),

> implying that after the receipt of an initial chemokine stimulus, a

> second signal was required to allow prolonged adhesion and migration

> across the endothelial cell monolayer. Experiments using different

> concentrations of EPA showed that these inhibitory effects were evident

> at concentrations as low as 50 nM (Figure 4C).

> > > >

> > > > Figure 3. The effects of EPA supplementation on the fatty acid

> content of endothelial cell phospholoipds.

> > > >

> > > > (A and The concentration of EPA in membrane phospholipids was

> increased after supplementation of culture medium with EPA for 24 h.

> Error bars indicate SEM. AA, arachidonic acid; FA, fatty acid; PA,

> palmitic acid; SA, stearic acid.

> > > >

> > > > doi:10.1371/journal.pbio.1000177.g003

> > > >

> > > > Figure 4. The effects of EPA supplementation on the adhesive

> behaviour of neutrophils.

> > > >

> > > > (A) EPA supplementation did not affect the number of neutrophils

> initially adhering to endothelial cells from flow. ( However, the time

> course of neutrophil behaviour on TNF-stimulated endothelial cells

> showed that EPA supplementation drastically altered neutrophil

> behaviour. Thus, the number of activated and surface adherent

> neutrophils decreased with time in the presence or absence of EPA.

> However, on endothelium that had not been supplemented with EPA, this

> was because neutrophils transmigrated across the monolayer, whereas on

> EPA-supplemented endothelium this was because activated cells reverted

> back to a rolling form of adhesion. ANOVA showed that there was a

> significant effect of treatment (i.e., ±EPA) on the percentage of

> cells that were rolling, surface adherent, or transmigrated (p<0.01). In

> addition, there was a significant effect of time on each form of

> behaviour for the EPA-treated cells (p<0.05âˆ'0.01). Bonferroni

> tests showed significant differences at specific time points as marked;

> **p<0.01. © Inhibition of neutrophil transmigration was evident at

> levels of EPA supplementation as low as 50 nM. Data are mean±SEM of

> five experiments. ANOVA showed significant effects of treatment

> (p<0.05); *p<0.05 compared to untreated control by Dunnett's test. Error

> bars indicate SEM.

> > > >

> > > > doi:10.1371/journal.pbio.1000177.g004

> > > > EPA Does Not Inhibit the Expression of Inflammatory Genes in

> Endothelial Cells

> > > >

> > > > To define the molecular mechanisms underlying these observations,

> we tested two prevalent hypotheses. The first, predicated on the

> observation that n-3-PUFAs regulate the transcription of endothelial

> cell inflammatory genes by down-regulating the activity of the nuclear

> factor-ÎºB, predicts changes in the levels of adhesion receptor and

> chemokine expression after supplementation with EPA [12]–[14].

> However, previous studies have investigated the regulation of gene

> expression in the presence of up to 100 µM of n-3-PUFAs, whereas

> physiological blood plasma levels of free fatty acid are of the order of

> 1 µM [15], a level that can be increased several fold upon

> supplementation. Using 5 µM EPA as an approximation of the highest

> concentration achievable in vivo, we did not see changes in the

> expression of endothelial cell adhesion molecules when assessed for RNA

> transcripts or protein expression (Figure 5 and Table 1). Neither was

> there any alteration in the levels of cytokines and chemokines secreted

> by endothelial cells (Table 2). Thus, in this model, we were able to

> discount the gene regulation hypothesis.

> > > >

> > > > Figure 5. The effect of EPA on TNF-induced surface adhesion

> molecule expression.

> > > >

> > > > The expression of (A) E-selectin and ( VCAM-1 was measured by

> ELISA in TNF-stimulated endothelial cells that were either

> unsupplemented or supplemented with EPA. Data are mean±SEM of seven

> experiments. ANOVA showed that there was a significant effect of the

> dose of TNF (p<0.01), but not of EPA, on the expression of both

> receptors.

> > > >

> > > > doi:10.1371/journal.pbio.1000177.g005

> > > >

> > > > Table 1. Effect of EPA treatment on mRNA expression of endothelial

> cell adhesion molecules and chemokines induced by TNF.

> > > >

> > > > doi:10.1371/journal.pbio.1000177.t001

> > > >

> > > > Table 2. Effect of EPA treatment of endothelial cells on the

> TNF-Î±-induced secretion of cytokines and chemokines.

> > > >

> > > > doi:10.1371/journal.pbio.1000177.t002

> > > > The Anti-Inflammatory Effects of EPA Supplementation Require the

> Metabolic Activity of Cyclooxygenase Enzymes

> > > >

> > > > The second hypothesis proposes that upon endothelial cell

> activation, EPA may compete with the n-6-PUFA arachidonic acid (AA;

> 20:4n-6) for cyclooxygenase enzymes (COX1 and COX2) after both fatty

> acids are liberated from membrane phospholipids by endogenous

> phospholipases [16]. Ordinarily, AA is metabolised by COX into 2-series

> endoperoxides, with downstream synthases converting these into the

> biologically active 2-series prostanoids [17]. However, when utilised as

> a COX substrate, the metabolism of EPA generates alternative 3-series

> prostanoids [16]. The 2-series prostanoids generated by COX activity are

> known to regulate aspects of the inflammatory response. For example, in

> the COX2 knockout mouse, neutrophil recruitment is dramatically reduced

> upon ischaemia and reperfusion-induced injury of the liver when compared

> to wild-type control animals [18]. In a murine model of

> lipopolysaccharide-induced lung inflammation, inhibitors of COX function

> (indomethacin and aspirin) modestly increased neutrophil recruitment

> [19]. Conversely in vitro, inhibition of COX function by aspirin can

> inhibit neutrophil migration [20] while having no effect on the levels

> of neutrophil adhesion to the endothelial cell monolayer [21]. Moreover,

> in murine models of acute and chronic inflammation, a reduction in PGE2

> production by genetic deletion of its membrane-bound synthase, moderates

> the formation of inflammation-associated granulation tissue and

> angiogenesis, as well as decreasing the nociception of pain, indicating

> that PGE2 is proinflammatory in these models [22]. PGD2 also appears to

> have proinflammatory functions, as overexpression of the synthase

> generating this prostanoid increases production of inflammatory

> cytokines and chemokines, leading to exaggerated levels of eosinophil

> and lymphocyte recruitment [23], a mechanism that operates through the

> PGD2 receptor DP-2. In contrast, in acute peritoneal inflammation,

> knockout of PGD2 synthase increased inflammatory cytokine production and

> retarded the rate of inflammatory resolution by a mechanism that

> operated through the DP-1 receptor [24]. Thus, PGD2 appears to have pro-

> or anti-inflammatory capabilities depending on the nature of the

> inflammatory insult. The inflammatory activity of the equivalent

> 3-series prostanoids is not known. Here, by introducing a panel of COX

> inhibitors into endothelial cell cultures at the same time as they were

> activated with TNF, the effects of EPA supplementation could be

> replicated, with neutrophil transmigration being dramatically inhibited

> (Figure 6). Moreover, adding a molecular excess of AA to

> EPA-supplemented endothelial cells at the point of TNF activation could

> reverse the blockade of migration (Figure 6). Taken together, these data

> imply that a COX-derived product of AA is required for the

> transmigration of neutrophils across TNF-stimulated endothelial cells,

> and that in the presence of EPA, this pathway is efficiently

> antagonised.

> > > >

> > > > Figure 6. A cyclooxygenase (COX)-derived eicosanoid is required

> for neutrophil transmigration across TNF-stimulated endothelial cells.

> > > >

> > > > (A) Inhibition of COX-1 (SC-560), COX-2 (NS-398), or both

> (indomethacin) caused inhibition of neutrophil transmigration, *p<0.05

> by t-test. ( Addition of AA to EPA-supplemented endothelial cells

> during the period of TNF stimulation restored neutrophil transmigration.

> All data are mean±SEM of four experiments. ANOVA showed significant

> effects of treatments in both (A and (p<0.01). In (A), Dunnett test

> showed significant effects of individual treatments compared to

> untreated control, *p<0.05. In (, Dunnett's test showed significant

> difference between EPA-treated and untreated control (**p<0.01), but not

> between AA+EPA and control.

> > > >

> > > > doi:10.1371/journal.pbio.1000177.g006

> > > > Prostaglandin D2 Is Required for Neutrophil Migration across

> Endothelial Cells Stimulated with TNF-Î±

> > > >

> > > > Endothelial cells generate COX products constitutively. For

> example, prostacyclin (PGI2) and prostaglandin D2 (PGD2) are endothelial

> cell-derived vasoactive prostanoids that are also involved in the

> regulation of haemostasis, being antagonists of platelet activation

> [17],[25]. These prostanoids are difficult to measure in the systemic

> blood, as they have reported half-lives in plasma that are measured in

> minutes [26],[27]. However, the constitutive nature of their production

> is demonstrated by the presence in urine and body fluids of their

> downstream metabolic products, PGF1Î± and delta-PGJ2, respectively

> [28],[29].

> > > >

> > > > Prostanoids have a documented ability to regulate the migration of

> a number of leukocyte subsets. For example, PGD2 induces chemotaxis of

> eosinophils and T-lymphocytes [30] in vitro. Interestingly, the

> migration of monocyte-derived migratory dendritic cells (DCs) may be

> tightly regulated by interplay between different prostanoids. Thus, the

> ability of these cells to traffic out of tissue into lymph nodes via the

> lymphatic circulation is dependent upon the presence of PGE2 as a

> differentiation signal [31]–[34]. In this context PGE2 promotes the

> function (but not the expression) of the chemokine receptor CCR7, so

> that DCs efficiently respond to the chemokines CCL19 and CCL21.

> Importantly, a number of studies have shown that the presence of PGD2

> can in turn inhibit the ability of DCs to migrate out of the lungs or

> the skin during an inflammatory response, although the molecular

> mechanism by which this inhibition is achieved remains undescribed

> [35],[36].

> > > >

> > > > As prostanoids are well documented to regulate leukocyte migration

> [37], we tested the hypothesis that PGD2 was the endothelial

> cell-derived agent providing the stimulus for neutrophil migration

> across the monolayer. We established a population of surface adherent

> neutrophils on TNF-treated endothelial cells supplemented with EPA and

> perfused synthetic PGD2 across the endothelial cells and neutrophils to

> see whether this would reintroduce transmigration. The provision of

> exogenous PGD2 but not PGD3 (which could be derived from EPA), fully

> restored the ability of neutrophils to cross the endothelial cell

> monolayer (Figure 7A). Prostaglandin D2 has two receptors.

> Chemoattractant-receptor homologous molecule expressed on Th2 cells

> receptor (CRTH2 or DP-2) is not expressed in neutrophils [35], whereas

> the DP-1 receptor has been reported in these cells [38]. Here,

> neutrophils perfused across TNF-stimulated endothelial cells in the

> presence of a DP-1 receptor antagonist (BW868C) showed a greatly

> diminished efficiency of endothelial cell transmigration (Figure 7B).

> Conversely, the transmigration of neutrophils was re-established on

> EPA-supplemented endothelial cells when a synthetic DP-1 receptor

> agonist (BW245C) was perfused across cells adherent to the surface of

> the monolayer (Figure 7C). Importantly, we could demonstrate that PGD2

> was operating directly on the neutrophils, because when these cells were

> harvested after migrating across a TNF-stimulated endothelial cells they

> had up-regulated CD11b (the alpha subunit of the Î²2 integrin

> CD11b/CD18 that is required for efficient neutrophil transmigration) and

> proteolytically shed L-selectin (Figure 8). However, neutrophils

> harvested from the surface of EPA-treated endothelial cells did not shed

> L-selectin or up-regulate CD11b. Thus, in this assay system, the

> delivery of a PGD2-mediated signal to the neutrophils was necessary for

> full cellular activation and efficient transendothelial migration.

> > > >

> > > > Figure 7. The role of AA- and EPA-derived eicosanoids in

> neutrophil transmigration.

> > > >

> > > > (A) When PDG2 was perfused across a population of neutrophils

> adherent to EPA-treated endothelial cells, neutrophil transmigration was

> restored. However, PGD3 had no significant effect on neutrophil

> behaviour. All data are mean±SEM of four experiments. ANOVA showed

> significant effect of treatment on transmigration. *p<0.05 compared to

> EPA-supplemented endothelial cells in the absence of PGD2 by Dunnett

> test. ( The DP1 receptor antagonist BW868c dose-dependently inhibited

> neutrophil transmigration on TNF-stimulated endothelial cells. All data

> are mean±SEM of five experiments; ANOVA showed a significant effect

> of BW868c concentration (p<0.01). © Perfusion of the DP-1 receptor

> agonist, BW245C, across neutrophils adherent to EPA-supplemented

> endothelial cells restored neutrophil migration to control levels. Data

> are mean±SEM of five experiments. ANOVA showed a significant effect

> of treatment on transmigration. **p<0.01 compared to EPA-supplemented

> endothelial cells in the absence of BW868C by Dunnett test. (D)

> Neutrophils perfused across TNF-stimulated endothelial cells in the

> presence of PGD3 showed a significantly reduced ability to migrate

> across the monolayer. Data are mean±SEM of three experiments; ANOVA

> showed a significant effect of PGD3 concentration on transmigration

> (p<0.01). (E) The levels of PGD3 released from endothelial cells are

> increased after EPA supplementation. Data are mean±SEM of three

> experiments. ANOVA showed a significant effect of treatment (p<0.05).

> **p<0.01 for PGD3 production compared to endothelial cells activated

> with TNF without EPA supplementation by Dunnett's test.

> > > >

> > > > doi:10.1371/journal.pbio.1000177.g007

> > > >

> > > > Figure 8. The effect of adhesion on the expression of L-selectin

> and CD11b on neutrophils.

> > > >

> > > > Neutrophils that were freshly isolated, migrated across

> endothelial cells stimulated with TNF or adherent to the surface of

> EPA-supplemented endothelial cells (EC) activated with TNF were

> harvested and the expression of (A) CD11b or ( L-selectin (CD62L)

> assessed by flow cytometry. Data are mean±SEM of four experiments;

> ANOVA showed significant effect of treatments in (A) (p<0.01) and (

> (p<0.05). In (A and , Dunnett's test showed significant difference

> between âˆ'EPA and freshly isolated cells (*p<0.01 and *p<0.05,

> respectively), but not between +EPA and freshly isolated cells.

> > > >

> > > > doi:10.1371/journal.pbio.1000177.g008

> > > > PGD3 Generated from EPA Antagonises the PGD2-Mediated Migration of

> Neutrophils across Endothelial Cells

> > > >

> > > > Taken together, these data show that PGD2 operates through the

> neutrophil DP-1 receptor to provide a signal that is essential for

> transendothelial cell migration. Moreover, the ability of neutrophils to

> respond to this signal is abolished when endothelial cells are preloaded

> with EPA from which PGD3 is generated. This implies that PGD3 may be an

> effective antagonist of the DP-1 receptor. To test this hypothesis, we

> perfused neutrophils across TNF-stimulated endothelial cells in the

> presence of exogenous PGD3. Neutrophil transmigration was inhibited in a

> dose-dependent manner by PGD3, showing that in the presence of

> endogenously generated PGD2 it could effectively antagonise this process

> (Figure 7D). Importantly, we were able to show that cells supplemented

> with EPA-generated an increased amount of PGD3 (Figure 7E).

> > > >

> > > > The absolute levels of PGD2 and PGD3 that are generated in our

> assay system, and thus the ratio of their abundance, are not easily

> assessed. The levels of PGD2 measured as an endothelial cell releasate

> were at the limits of detection (Table 3), presumably due to the short

> half-life of this prostanoid under physiological conditions; however,

> significant levels of PGD3 were assayed upon EPA supplementation, and

> concentrations in the order of 5–10 nM had accumulated over the 24 h

> of the EPA supplementation period. The half-life of PGD3 in serum or

> albumin-containing medium has not been reported to our knowledge; thus

> it is not clear whether the measurements made here report the true

> levels of PGD3 production, or if metabolic degradation of this

> prostanoid renders these measurements underestimates. Irrespective of

> this, our experiments utilising synthetic PGD2 demonstrate that

> concentrations on the order of 1 nM were sufficient to fully

> recapitulate the process of migration on EPA-supplemented endothelial

> cells, and when using PGD3 as an antagonist of TNF-induced

> transmigration marked effects were evident at 10–100 pM. Thus, the

> measurable levels of PGD3 that we report would certainly be sufficient

> to effectively antagonise the transmigration of neutrophils in our

> experimental model.

> > > >

> > > > Table 3. Analysis of eicosanoids by liquid chromatography tandem

> mass spectrometry.

> > > >

> > > > doi:10.1371/journal.pbio.1000177.t003

> > > > Conclusions

> > > >

> > > > Our study demonstrates a hitherto unknown regulatory step in the

> recruitment of neutrophils by cytokine-stimulated endothelial cells

> (refer to Figure 1 to see how this new step fits with the known

> regulatory mechanisms of neutrophil recruitment). After initial

> tethering by selectin molecules, a chemokine signal induces arrest of

> the cell on the endothelial cell surface. However, the chemokine signal

> is not sufficient to support transmigration across the endothelial cell

> monolayer, and the arachidonic acid-derived prostanoid, PGD2, is an

> essential downstream regulator of this process. PGD2 operates through

> the DP-1 receptor and this signal can be effectively antagonised by PGD3

> which is generated from EPA released by the action of phospholipase

> enzymes on the phospholipids of EPA supplemented cells. Not only does

> this study reveal a new step in the recruitment of neutrophil

> recruitment during inflammation, it also reveals a novel

> anti-inflammatory mechanism of action of the dietary n-3-PUFA, EPA.

> > > >

> > > > Materials and Methods Top

> > > >

> > > > Cell Culture and Neutrophil Isolation

> > > >

> > > > Endothelial cells (EC) were isolated and cultured, as previously

> described [39], with or without 0–5 µM EPA (Sigma) for 24 h.

> TNF-Î± (100 U/ml; R & D systems) was added to the EC for the final 4 h

> of culture. In some experiments, EC were treated with 100 U/ml TNF-Î±

> in the presence of 10 µM indomethacin or 0.1 µM SC-560 or 1 µM

> NS-398 (Sigma). In the AA reconstitution experiments, 5 µM AA (Sigma)

> was added to the culture medium simultaneously with TNF-Î±.

> > > >

> > > > Blood from healthy adult volunteers was collected into tubes

> coated with EDTA (1.6 mg/ml), and neutrophils were separated using

> 2-step density gradients of Histopaque 1119 and 1077 (Sigma), as

> previously described [40]. After washing in 0.15% BSA in PBS, cells were

> counted using a Coulter Multisizer (Coulter Electronics), and

> neutrophils, which were >95% pure, were resuspended at a concentration

> of 1×106/ml in PBS/Alb with calcium and magnesium.

> > > >

> > > > RNA Extraction and Real-Time PCR

> > > >

> > > > mRNA was isolated from EC using the Qiagen RNEasy Mini Kit 50

> (Qiagen) following the manufacturer's instructions. Real-time PCR

> (RT-PCR) was performed using QuantiTect probe RT-PCR kit according to

> the manufacturer's instructions (Qiagen). Primers were purchased from

> Applied Biosystems. The expression of each target gene was normalised to

> Î²-actin expression, and the data presented represent fold change

> compared to untreated EC.

> > > >

> > > > Luminex for Secreted Cytokines and Chemokines

> > > >

> > > > Supernatants were collected from EC that had been incubated with

> 0–5 µM EPA for 24 h and had 100 U/ml TNF added to the culture for

> the final 4 h of culture. The Luminex kit was purchased from

> Upstate/Chemicon, and the experiment performed to the manufacturer's

> instructions. Data were collected and analysed from the samples using a

> Luminex100 machine (Luminex).

> > > >

> > > > Flow-Based Adhesion Assay

> > > >

> > > > Glass capillaries (microslides) containing treated EC monolayers

> were incorporated into a flow-based adhesion assay as described [39].

> Briefly, microslides were attached to cell and fluid reservoirs by

> silicon tubing at one end and to a withdrawal syringe pump at the other

> end. After mounting on the stage of a phase contrast microscope, EC were

> washed for 2 min with PBS containing 0.15% BSA. Neutrophils were then

> perfused (1×106/ml) at 0.1 Pa for 4 min, followed by 15 min of wash.

> Video images were recorded throughout the experiment and neutrophil

> behaviour analysed offline using Image Pro software (Image-Pro Plus). In

> some experiments, neutrophils were perfused across EPA- and TNF-treated

> EC, allowed to adhere, and subsequently perfused with 1 nM PGD2 or 1 nM

> PGD3 (both from Cayman Chemicals). Alternatively, neutrophils were

> treated with BW868c (Cayman Chemicals), a DP1 receptor antagonist, for

> 10 min prior to perfusion over TNF-Î±-stimulated EC. Neutrophils were

> also treated with the DP1 receptor agonist BW245c (Cayman Chemicals)

> prior to perfusion over EPA- and TNF-Î±-treated EC. Neutrophils were

> also treated with 0–1 nM PGD3, immediately prior to perfusion across

> TNF-stimulated EC. In all experiments, lipid reagents, agonists, and

> antagonists were stored in 100% ethanol under nitrogen gas. Once

> diluted, the medium contained <0.01% ethanol. All controls contained

> equivalent concentrations of ethanol.

> > > >

> > > > Analysis of EPA Incorporation into Cellular Phospholipids

> > > >

> > > > First-passage confluent EC were treated with 5 µM of EPA for 24

> h. EC were removed from the plastic culture dish by scraping and stored

> in 0.88% KCl solution at âˆ'20°C until analysis. Total lipid was

> extracted with chloroform:methanol (2:1, v/v) containing butylated

> hydroxytoluene (50 mg/l) as antioxidant. Fatty acids were subsequently

> hydrolysed from the lipid and simultaneously methylated by incubation

> with methylation reagent (methanol containing 2% v/v H2SO4) at 50°C

> for 2 h. Fatty acid methyl esters were separated and identified using a

> Hewlett Packard 6890 gas chromatograph (Hewlett Packard) fitted with a

> 30 mm×32 mm BPX 70 capillary column, film thickness 0.25 µm.

> Helium, at the initial flow rate of 1.0 ml/min, was used as the carrier

> gas. Injector and detector temperatures were 275°C, and the column

> oven temperature was maintained at 170°C for 12 min after sample

> injection. The oven temperature was programmed to increase from 170 to

> 210°C at 5°C/min. Fatty acid methyl esters were identified by

> comparison with authentic standards. Peak areas were quantified using

> ChemStation software (Hewlett Packard). Each fatty acid was expressed as

> weight percent of total fatty acids present.

> > > >

> > > > Eicosanoid Extraction and Reverse-Phase High-Performance Liquid

> Chromatography Analysis

> > > >

> > > > Eicosanoids were extracted from control EC using C18 Sep-Pak

> cartridges (Waters). Briefly, cartridges were conditioned with 5 ml of

> high-performance liquid chromatography (HPLC)-grade MeOH and rinsed

> twice with water. Reactions were terminated with addition of 100%

> ice-cold MeOH, containing 2 ng of internal standard PGE2-d4. Sample

> volume was adjusted to 10% MeOH with water and applied to the column.

> The column was rinsed with water and the sample eluted with 2 ml of

> MeOH. Nitrogen gas was used to dry the sample, which was resuspended in

> 100 µl of MeOH and stored at âˆ'80°C until analysis.

> > > >

> > > > Prostaglandin Quantitation using Liquid Chromatography Tandem Mass

> Spectrometry

> > > >

> > > > Prostaglandins were separated on a C18 ODS2, 5 µm,

> 150×4.6-mm column (Waters) using a gradient of 50%–90% B over 20

> min (A, water:acetonitrile:acetic acid, 75:25:0.1; B,

> methanol:acetonitrile:acetic acid, 60:40:0.1) at 1 ml/min. Products were

> quantitated by directing the HPLC output directly into the electrospray

> source of a Q-Trap mass spectrometer (Applied Biosystems 4000 Q-Trap)

> operating in the negative mode (âˆ'4,500 V). Individual

> prostaglandins were monitored in the Multiple Reaction Monitoring (MRM)

> mode using specific parent to daughter transitions of m/z 349–269

> for PGD3 with collision energies of âˆ'26 V. Products were

> identified and quantified with reference to the appropriate standards

> run in parallel under the same conditions, with 2 ng of PGE2-d4 (m/z

> 355–275) added as an internal standard.

> > > >

> > > > Analysis of Neutrophil Adhesion Molecule Expression by Flow

> Cytometry

> > > >

> > > > Neutrophils for analysis were isolated from whole blood as

> described above. Control cells were stained for flow cytometry directly

> after isolation. Cells rolling on EPA-treated endothelial cells in

> microslides were harvested by elution with 0.02% EDTA after 4 min of

> neutrophil perfusion and 11 min of wash buffer perfusion (totalling 15

> min of contact with endothelial cells). In order to harvest neutrophils

> that had migrated across endothelial cells, endothelium was cultured on

> 1.5 mg/ml type I collagen gels (Becton Dickinson). After endothelial

> cell stimulation with TNF for 4 h, neutrophils were added for 15 min.

> Surface-adherent cells were washed off with 0.02% EDTA, and the gel was

> dissolved using type VII collagenase (Sigma). Neutrophils were harvested

> from the dissolved gel by centrifugation. All neutrophil populations

> were stained for surface expression of CD11b (PE-conjugated, clone

> 2LPM19c, 2 µg/ml; DAKO) or CD62L (FITC-conjugated, clone Dreg56,10

> µg/ml, Beckton Dickinson). Expression was analysed by flow cytometry

> using a DAKO CyAn, and data analysed using Summit software (Becton

> Dickinson). Data are expressed as mean fluorescent intensity.

> > > >

> > > > Statistical Analysis

> > > >

> > > > Data were analysed using Prism software (GraphPad software).

> Results are presented as means±standard error of the mean (SEM).

> Comparisons between individual treatments were by paired t-test where

> appropriate. ANOVA was performed to assess the effect of EPA

> concentration on PMN transmigration. Significant findings were

> investigated further using Bonferroni multiple comparison test or

> Dunnett's test.

> > > >

> > > > Author Contributions Top

> > > >

> > > > The author(s) have made the following declarations about their

> contributions: Conceived and designed the experiments: VBO RFG PCC GBN

> GER. Performed the experiments: SPT CMY BHM JM. Analyzed the data: SPT

> CMY BHM VBO JM GER. Contributed reagents/materials/analysis tools: VBO

> RFG PCC GBN. Wrote the paper: GER.

> > > >

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November 7, 2011

Hi Asfy,I believe this article was published in Aug 2009 Issue of Plos

Biology.I think by adding omega-3 EPA and DHA you change the properties of the

cell membranes as well (phospholipid).Fish oil itself can cause Samter's to

worsen.A more sustainable source might be algae.Also adding toasted sesame seed

oil might help.Dr. Barry Sears has a website and in the resources section there

is an article called " Understanding Eicosanoids " As well as a Book " Toxic Fat "

which gives a good overview.Also the brain has a high DHA content.

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> > > > > Research Article

> > > > > Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a

> > New Step in Neutrophil Recruitment

> > > > >

> > > > > While investigating new mechanisms by which the dietary omega-3

> > fatty acids regulate inflammation, the authors have identified a new

> > step in the regulation of neutrophil migration across vascular

> > endothelial cells.

> > > > >

> > > > > Article

> > > > > Metrics

> > > > > Related Content

> > > > > Comments: 0

> > > > > To add a note, highlight some text. Hide notes

> > > > > Make a general comment

> > > > > Jump to

> > > > >

> > > > > Abstract

> > > > > Author Summary

> > > > > Introduction

> > > > > Results/Discussion

> > > > > Materials and Methods

> > > > > Author Contributions

> > > > > References

> > > > > P. Tull1, Clara M. Yates1, H. Maskrey2,

> > B. O'Donnell2, Jackie Madden3, F. Grimble3, Philip C. Calder3,

> > Gerard B. Nash1, G. Ed. Rainger1*

> > > > >

> > > > > 1 Centre for Cardiovascular Sciences, School of Clinical and

> > Experimental Medicine, The Medical School, The University of Birmingham,

> > Birmingham, United Kingdom, 2 Department of Medical Biochemistry and

> > Immunology, School of Medicine, Cardiff University, Cardiff, United

> > Kingdom, 3 Institute of Human Nutrition, School of Medicine, University

> > of Southampton, Southampton, United Kingdom

> > > > >

> > > > > Abstract Top

> > > > >

> > > > > Inflammation is a physiological response to tissue trauma or

> > infection, but leukocytes, which are the effector cells of the

> > inflammatory process, have powerful tissue remodelling capabilities.

> > Thus, to ensure their precise localisation, passage of leukocytes from

> > the blood into inflamed tissue is tightly regulated. Recruitment of

> > blood borne neutrophils to the tissue stroma occurs during early

> > inflammation. In this process, peptide agonists of the chemokine family

> > are assumed to provide a chemotactic stimulus capable of supporting the

> > migration of neutrophils across vascular endothelial cells, through the

> > basement membrane of the vessel wall, and out into the tissue stroma.

> > Here, we show that, although an initial chemokine stimulus is essential

> > for the recruitment of flowing neutrophils by endothelial cells

> > stimulated with the inflammatory cytokine tumour necrosis factor-Î±,

> > transit of the endothelial monolayer is regulated by an additional and

> > downstream stimulus. This signal is supplied by the metabolism of the

> > omega-6-polyunsaturated fatty acid (n-6-PUFA), arachidonic acid, into

> > the eicosanoid prostaglandin-D2 (PGD2) by cyclooxygenase (COX) enzymes.

> > This new step in the neutrophil recruitment process was revealed when

> > the dietary n-3-PUFA, eicosapentaenoic acid (EPA), was utilised as an

> > alternative substrate for COX enzymes, leading to the generation of

> > PGD3. This alternative series eicosanoid inhibited the migration of

> > neutrophils across endothelial cells by antagonising the PGD2 receptor.

> > Here, we describe a new step in the neutrophil recruitment process that

> > relies upon a lipid-mediated signal to regulate the migration of

> > neutrophils across endothelial cells. PGD2 signalling is subordinate to

> > the chemokine-mediated activation of neutrophils, but without the

> > sequential delivery of this signal, neutrophils fail to penetrate the

> > endothelial cell monolayer. Importantly, the ability of the dietary

> > n-3-PUFA, EPA, to inhibit this process not only revealed an unsuspected

> > level of regulation in the migration of inflammatory leukocytes, it also

> > contributes to our understanding of the interactions of this bioactive

> > lipid with the inflammatory system. Moreover, it indicates the potential

> > for novel therapeutics that target the inflammatory system with greater

> > affinity and/or specificity than supplementing the diet with n-3-PUFAs.

> > > > >

> > > > > Author Summary Top

> > > > >

> > > > > Inflammation is a physiological response to tissue trauma or

> > infection. Neutrophils, which circulate in the blood stream, are the

> > first inflammatory cells to be recruited to a site of tissue

> > inflammation. In response to recruitment signals provided by chemotactic

> > peptides called chemokines, neutrophils traverse the endothelial cell

> > lining of blood vessels. This process involves a multistep cascade of

> > neutrophil adhesion and activation events on the endothelial barrier.

> > While investigating the anti-inflammatory functions of the omega-3 fatty

> > acid , EPA, which is found, for instance, in dietary fish oil, we

> > identified an additional unexpected lipid-derived signal that is

> > essential for neutrophil migration across the endothelium. Our

> > experiments show that a chemokine delivered the first signal needed to

> > bind neutrophils firmly to the endothelial surface. However, in order to

> > traverse the endothelium, a subsequent signal delivered by

> > prostaglandin-D2 (PGD2), a lipid derived from the omega-6 fatty acid

> > arachidonic acid, was essential. When EPA, was introduced into the

> > experiment, it was used to form PGD3. This alternative lipid blocked

> > interactions between PGD2 and its receptor on neutrophils, preventing

> > the process of migration across the endothelial barrier. Thus, we reveal

> > a new step in the recruitment of neutrophils during inflammation, and a

> > novel anti-inflammatory mechanism of action of dietary EPA.

> > > > >

> > > > > Citation: Tull SP, Yates CM, Maskrey BH, O'Donnell VB, Madden J,

> > et al. (2009) Omega-3 Fatty Acids and Inflammation: Novel Interactions

> > Reveal a New Step in Neutrophil Recruitment. PLoS Biol 7(8): e1000177.

> > doi:10.1371/journal.pbio.1000177

> > > > >

> > > > > Academic Editor: Philip M. , NIH/NIAID, United States of

> > America

> > > > >

> > > > > Received: February 6, 2009; Accepted: July 14, 2009; Published:

> > August 25, 2009

> > > > >

> > distributed under the terms of the Creative Commons Attribution License,

> > which permits unrestricted use, distribution, and reproduction in any

> > medium, provided the original author and source are credited.

> > > > >

> > > > > Funding: This work was supported by the Biotechnology and

> > Biological Sciences Research Council, United Kingdom (grant BBS/B/0105)

> > and by the British Heart Foundation (grant BS/97001). The funders had no

> > role in study design, data collection and analysis, decision to publish,

> > or preparation of the manuscript.

> > > > >

> > > > > Competing interests: The authors have declared that no competing

> > interests exist.

> > > > >

> > > > > Abbreviations: DC, dendritic cell

> > > > >

> > > > > * E-mail: g.e.rainger@

> > > > >

> > > > > Introduction Top

> > > > >

> > > > > In vertebrates, tissue trauma or infection causes the rapid

> > initiation of an inflammatory reaction. The early phase of this

> > phylactic response results in the localised recruitment of cells of the

> > innate immune system from the blood, a tissue infiltrate that is

> > dominated by neutrophils. The molecular processes that support the

> > initial interactions between blood-borne neutrophils and the endothelial

> > cells lining postcapillary venules (the site in the blood vasculature

> > where leukocytes are recruited during inflammation) are well described

> > (see Figure 1 for a schematic representation of the steps in the

> > neutrophil recruitment process). In response to the localised production

> > of inflammatory mediators, such as the cytokine tumour necrosis

> > factor-Î± (TNF), activated endothelial cells decorate themselves with

> > specialised adhesion receptors of the selectin family [1]–[3]. Due

> > to their ability to rapidly form strong but short-lived bonds with

> > carbohydrate counter-ligands on the neutrophil surface, E- and

> > P-selectin are capable of tethering neutrophils from rapidly flowing

> > blood [4]. The sequential formation and dissolution of selectin bonds

> > also support a characteristic and dynamic form of adhesion, referred to

> > as rolling [5]. Rolling adhesion does not require neutrophil activation.

> > However, neutrophil migration through the vessel wall and into the

> > inflamed tissue is dependent upon the receipt of an activating stimulus

> > [5],[6]. We have previously shown that endothelial cells can present

> > peptide agonists of the CXC-chemokine family to rolling neutrophils and

> > thus stabilise adhesion [7],[8]. Ligation of the neutrophil

> > CXC-Receptor-2 (CXCR2) by these agents is essential for activation of

> > the Î²2-intergrin adhesion receptors and the reorganisation of the

> > actin cytoskeleton that support transendothelial cell migration [8].

> > Here, we show that in the presence of an antibody that blocks chemokine

> > interactions with CXCR2, neutrophils roll on the endothelial cell

> > surface indefinitely (Figure 2), demonstrating that a chemokine signal

> > is essential for neutrophil activation on TNF-stimulated endothelial

> > cells. However, the removal of this primary activating stimulus provides

> > no information on the requirement for additional, downstream signals,

> > which might coordinate transit of the vessel wall and onward migration

> > into stromal tissues.

> > > > >

> > > > > Figure 1. Steps in the process of neutrophil recruitment.

> > > > >

> > > > > Step 1: Neutrophils are captured from flow and tether and roll on

> > tumour necrosis factor-Î± (TNF)-stimulated endothelial cells. Step 2:

> > Neutrophils are then activated by the action of CXC-chemokines on the

> > chemokine receptor, CXCR2, a process resulting in the activation of

> > neutrophil Î²2-integrins. Step 3: Î²2-integrins engage counter

> > receptors on the endothelial cell surface, and the neutrophil becomes

> > stationary. Step 4: Prostaglandin-D2 (PGD2), generated by the action of

> > cyclooxygenase enzymes on the n-6-PUFA arachidonic acid (AA), binds the

> > PGD2 receptor, DP-1. DP-1 generates signals that stabilise neutrophil

> > adhesion, induce neutrophil shape change, and support the process of

> > transmigration across the endothelial cell monolayer. Step 5: If the

> > endothelial cells have been supplemented with the n-3-PUFA,

> > eicosapentaenoic acid (EPA), the alternative series prostanoid, PGD3, is

> > generated, and this antagonises PGD2-mediated neutrophil responses.

> > > > >

> > > > > doi:10.1371/journal.pbio.1000177.g001

> > > > >

> > > > > Figure 2. Chemokine signalling through CXCR2 is essential for

> > neutrophil activation on TNF-stimulated endothelial cells.

> > > > >

> > > > > Using a flow-based adhesion assay, neutrophils isolated from whole

> > blood were perfused across endothelial cells. Neutrophils were recruited

> > to endothelial cells treated with TNF, but did not adhere to

> > unstimulated endothelium. Detailed analysis of neutrophil behaviour

> > showed that on TNF-stimulated endothelial cells, the majority of

> > recruited cells transmigrated across the monolayer. When neutrophils

> > were treated with anti-CXCR2, but not anti-CXCR1, neutrophil activation

> > was inhibited so that nearly all of the recruited neutrophils rolled

> > indefinitely on the monolayer and could not migrate; data are

> > mean±SEM of four experiments **p<0.01 for comparison by paired t-test

> > of neutrophil behaviour on TNF stimulated endothelial cells in the

> > presence or absence of anti-CXCR2.

> > > > >

> > > > > doi:10.1371/journal.pbio.1000177.g002

> > > > > Results/Discussion Top

> > > > >

> > > > > EPA Released from Endothelial Cell Membrane Phospholipids Inhibits

> > the Recruitment of Neutrophils

> > > > >

> > > > > Dietary omega-3 polyunsaturated fatty acids (n-3-PUFAs) have

> > anti-inflammatory properties. For example, their inclusion in the diet

> > in the form of n-3-PUFA–rich fish oil reduces the symptoms of

> > disease as well as the use of nonsteroidal anti-inflammatory drugs in

> > arthritis patients with severe inflammatory joint disease [9]. In

> > addition, dietary n-3-PUFAs may offer protection against vascular

> > pathology associated with atherosclerosis, having been reported to be

> > efficacious in epidemiological studies [10]. By modulating inflammation

> > within the artery wall, n-3-PUFAS also alter the cellular and the

> > structural composition of advanced atherosclerotic plaques in a manner

> > that could reduce the incidence of plaque rupture or ulceration, a

> > process that precedes tissue infarction (e.g., heart attack or stroke)

> > [11]. Despite these documented anti-inflammatory benefits, the mode(s)

> > of interaction of these lipids with the immune and inflammatory systems

> > are not well understood. When we supplemented endothelial cell culture

> > medium with the major n-3 -PUFA found in dietary fish oil supplements,

> > eicosapentaenoic acid (EPA; 20:5n-3), it was incorporated into cellular

> > phospholipids so that upon withdrawal of the free fatty acid from the

> > culture medium, a pool of esterified EPA remained localised within

> > endothelial cell membranes (Figure 3A and 3B). After withdrawal of free

> > fatty acid and stimulation with TNF, EPA-treated cells were able to

> > support similar levels of neutrophil adhesion to those that had received

> > no lipid supplement (Figure 4A). However, detailed analysis showed that

> > the behaviour of neutrophils on the two populations of endothelial cells

> > was very different. In response to TNF, but in the absence of EPA, a

> > small population of adherent neutrophils (~20%) rolled throughout the

> > duration of a flow adhesion assay (Figure 4B). The remaining cells

> > became activated on the endothelial cell surface, and this population of

> > neutrophils became progressively smaller as cells migrated across the

> > endothelial cell monolayer (Figure 4B). In contrast, when endothelial

> > cells had been treated with EPA prior to TNF stimulation, there was a

> > marked reduction in the number of cells undergoing transendothelial cell

> > migration (Figure 4B). This was reduced at the earliest time point and

> > did not increase over the duration of the experiment. Interestingly

> > however, many of the cells that did become activated on the surface of

> > endothelial cells reverted to a rolling form of adhesion (Figure 4B),

> > implying that after the receipt of an initial chemokine stimulus, a

> > second signal was required to allow prolonged adhesion and migration

> > across the endothelial cell monolayer. Experiments using different

> > concentrations of EPA showed that these inhibitory effects were evident

> > at concentrations as low as 50 nM (Figure 4C).

> > > > >

> > > > > Figure 3. The effects of EPA supplementation on the fatty acid

> > content of endothelial cell phospholoipds.

> > > > >

> > > > > (A and The concentration of EPA in membrane phospholipids was

> > increased after supplementation of culture medium with EPA for 24 h.

> > Error bars indicate SEM. AA, arachidonic acid; FA, fatty acid; PA,

> > palmitic acid; SA, stearic acid.

> > > > >

> > > > > doi:10.1371/journal.pbio.1000177.g003

> > > > >

> > > > > Figure 4. The effects of EPA supplementation on the adhesive

> > behaviour of neutrophils.

> > > > >

> > > > > (A) EPA supplementation did not affect the number of neutrophils

> > initially adhering to endothelial cells from flow. ( However, the time

> > course of neutrophil behaviour on TNF-stimulated endothelial cells

> > showed that EPA supplementation drastically altered neutrophil

> > behaviour. Thus, the number of activated and surface adherent

> > neutrophils decreased with time in the presence or absence of EPA.

> > However, on endothelium that had not been supplemented with EPA, this

> > was because neutrophils transmigrated across the monolayer, whereas on

> > EPA-supplemented endothelium this was because activated cells reverted

> > back to a rolling form of adhesion. ANOVA showed that there was a

> > significant effect of treatment (i.e., ±EPA) on the percentage of

> > cells that were rolling, surface adherent, or transmigrated (p<0.01). In

> > addition, there was a significant effect of time on each form of

> > behaviour for the EPA-treated cells (p<0.05âˆ'0.01). Bonferroni

> > tests showed significant differences at specific time points as marked;

> > levels of EPA supplementation as low as 50 nM. Data are mean±SEM of

> > five experiments. ANOVA showed significant effects of treatment

> > (p<0.05); *p<0.05 compared to untreated control by Dunnett's test. Error

> > bars indicate SEM.

> > > > >

> > > > > doi:10.1371/journal.pbio.1000177.g004

> > > > > EPA Does Not Inhibit the Expression of Inflammatory Genes in

> > Endothelial Cells

> > > > >

> > > > > To define the molecular mechanisms underlying these observations,

> > we tested two prevalent hypotheses. The first, predicated on the

> > observation that n-3-PUFAs regulate the transcription of endothelial

> > cell inflammatory genes by down-regulating the activity of the nuclear

> > factor-ÎºB, predicts changes in the levels of adhesion receptor and

> > chemokine expression after supplementation with EPA [12]–[14].

> > However, previous studies have investigated the regulation of gene

> > expression in the presence of up to 100 µM of n-3-PUFAs, whereas

> > physiological blood plasma levels of free fatty acid are of the order of

> > 1 µM [15], a level that can be increased several fold upon

> > supplementation. Using 5 µM EPA as an approximation of the highest

> > concentration achievable in vivo, we did not see changes in the

> > expression of endothelial cell adhesion molecules when assessed for RNA

> > transcripts or protein expression (Figure 5 and Table 1). Neither was

> > there any alteration in the levels of cytokines and chemokines secreted

> > by endothelial cells (Table 2). Thus, in this model, we were able to

> > discount the gene regulation hypothesis.

> > > > >

> > > > > Figure 5. The effect of EPA on TNF-induced surface adhesion

> > molecule expression.

> > > > >

> > > > > The expression of (A) E-selectin and ( VCAM-1 was measured by

> > ELISA in TNF-stimulated endothelial cells that were either

> > unsupplemented or supplemented with EPA. Data are mean±SEM of seven

> > experiments. ANOVA showed that there was a significant effect of the

> > dose of TNF (p<0.01), but not of EPA, on the expression of both

> > receptors.

> > > > >

> > > > > doi:10.1371/journal.pbio.1000177.g005

> > > > >

> > > > > Table 1. Effect of EPA treatment on mRNA expression of endothelial

> > cell adhesion molecules and chemokines induced by TNF.

> > > > >

> > > > > doi:10.1371/journal.pbio.1000177.t001

> > > > >

> > > > > Table 2. Effect of EPA treatment of endothelial cells on the

> > TNF-Î±-induced secretion of cytokines and chemokines.

> > > > >

> > > > > doi:10.1371/journal.pbio.1000177.t002

> > > > > The Anti-Inflammatory Effects of EPA Supplementation Require the

> > Metabolic Activity of Cyclooxygenase Enzymes

> > > > >

> > > > > The second hypothesis proposes that upon endothelial cell

> > activation, EPA may compete with the n-6-PUFA arachidonic acid (AA;

> > 20:4n-6) for cyclooxygenase enzymes (COX1 and COX2) after both fatty

> > acids are liberated from membrane phospholipids by endogenous

> > phospholipases [16]. Ordinarily, AA is metabolised by COX into 2-series

> > endoperoxides, with downstream synthases converting these into the

> > biologically active 2-series prostanoids [17]. However, when utilised as

> > a COX substrate, the metabolism of EPA generates alternative 3-series

> > prostanoids [16]. The 2-series prostanoids generated by COX activity are

> > known to regulate aspects of the inflammatory response. For example, in

> > the COX2 knockout mouse, neutrophil recruitment is dramatically reduced

> > upon ischaemia and reperfusion-induced injury of the liver when compared

> > to wild-type control animals [18]. In a murine model of

> > lipopolysaccharide-induced lung inflammation, inhibitors of COX function

> > (indomethacin and aspirin) modestly increased neutrophil recruitment

> > [19]. Conversely in vitro, inhibition of COX function by aspirin can

> > inhibit neutrophil migration [20] while having no effect on the levels

> > of neutrophil adhesion to the endothelial cell monolayer [21]. Moreover,

> > in murine models of acute and chronic inflammation, a reduction in PGE2

> > production by genetic deletion of its membrane-bound synthase, moderates

> > the formation of inflammation-associated granulation tissue and

> > angiogenesis, as well as decreasing the nociception of pain, indicating

> > that PGE2 is proinflammatory in these models [22]. PGD2 also appears to

> > have proinflammatory functions, as overexpression of the synthase

> > generating this prostanoid increases production of inflammatory

> > cytokines and chemokines, leading to exaggerated levels of eosinophil

> > and lymphocyte recruitment [23], a mechanism that operates through the

> > PGD2 receptor DP-2. In contrast, in acute peritoneal inflammation,

> > knockout of PGD2 synthase increased inflammatory cytokine production and

> > retarded the rate of inflammatory resolution by a mechanism that

> > operated through the DP-1 receptor [24]. Thus, PGD2 appears to have pro-

> > or anti-inflammatory capabilities depending on the nature of the

> > inflammatory insult. The inflammatory activity of the equivalent

> > 3-series prostanoids is not known. Here, by introducing a panel of COX

> > inhibitors into endothelial cell cultures at the same time as they were

> > activated with TNF, the effects of EPA supplementation could be

> > replicated, with neutrophil transmigration being dramatically inhibited

> > (Figure 6). Moreover, adding a molecular excess of AA to

> > EPA-supplemented endothelial cells at the point of TNF activation could

> > reverse the blockade of migration (Figure 6). Taken together, these data

> > imply that a COX-derived product of AA is required for the

> > transmigration of neutrophils across TNF-stimulated endothelial cells,

> > and that in the presence of EPA, this pathway is efficiently

> > antagonised.

> > > > >

> > > > > Figure 6. A cyclooxygenase (COX)-derived eicosanoid is required

> > for neutrophil transmigration across TNF-stimulated endothelial cells.

> > > > >

> > > > > (A) Inhibition of COX-1 (SC-560), COX-2 (NS-398), or both

> > (indomethacin) caused inhibition of neutrophil transmigration, *p<0.05

> > by t-test. ( Addition of AA to EPA-supplemented endothelial cells

> > during the period of TNF stimulation restored neutrophil transmigration.

> > All data are mean±SEM of four experiments. ANOVA showed significant

> > effects of treatments in both (A and (p<0.01). In (A), Dunnett test

> > showed significant effects of individual treatments compared to

> > untreated control, *p<0.05. In (, Dunnett's test showed significant

> > difference between EPA-treated and untreated control (**p<0.01), but not

> > between AA+EPA and control.

> > > > >

> > > > > doi:10.1371/journal.pbio.1000177.g006

> > > > > Prostaglandin D2 Is Required for Neutrophil Migration across

> > Endothelial Cells Stimulated with TNF-Î±

> > > > >

> > > > > Endothelial cells generate COX products constitutively. For

> > example, prostacyclin (PGI2) and prostaglandin D2 (PGD2) are endothelial

> > cell-derived vasoactive prostanoids that are also involved in the

> > regulation of haemostasis, being antagonists of platelet activation

> > [17],[25]. These prostanoids are difficult to measure in the systemic

> > blood, as they have reported half-lives in plasma that are measured in

> > minutes [26],[27]. However, the constitutive nature of their production

> > is demonstrated by the presence in urine and body fluids of their

> > downstream metabolic products, PGF1Î± and delta-PGJ2, respectively

> > [28],[29].

> > > > >

> > > > > Prostanoids have a documented ability to regulate the migration of

> > a number of leukocyte subsets. For example, PGD2 induces chemotaxis of

> > eosinophils and T-lymphocytes [30] in vitro. Interestingly, the

> > migration of monocyte-derived migratory dendritic cells (DCs) may be

> > tightly regulated by interplay between different prostanoids. Thus, the

> > ability of these cells to traffic out of tissue into lymph nodes via the

> > lymphatic circulation is dependent upon the presence of PGE2 as a

> > differentiation signal [31]–[34]. In this context PGE2 promotes the

> > function (but not the expression) of the chemokine receptor CCR7, so

> > that DCs efficiently respond to the chemokines CCL19 and CCL21.

> > Importantly, a number of studies have shown that the presence of PGD2

> > can in turn inhibit the ability of DCs to migrate out of the lungs or

> > the skin during an inflammatory response, although the molecular

> > mechanism by which this inhibition is achieved remains undescribed

> > [35],[36].

> > > > >

> > > > > As prostanoids are well documented to regulate leukocyte migration

> > [37], we tested the hypothesis that PGD2 was the endothelial

> > cell-derived agent providing the stimulus for neutrophil migration

> > across the monolayer. We established a population of surface adherent

> > neutrophils on TNF-treated endothelial cells supplemented with EPA and

> > perfused synthetic PGD2 across the endothelial cells and neutrophils to

> > see whether this would reintroduce transmigration. The provision of

> > exogenous PGD2 but not PGD3 (which could be derived from EPA), fully

> > restored the ability of neutrophils to cross the endothelial cell

> > monolayer (Figure 7A). Prostaglandin D2 has two receptors.

> > Chemoattractant-receptor homologous molecule expressed on Th2 cells

> > receptor (CRTH2 or DP-2) is not expressed in neutrophils [35], whereas

> > the DP-1 receptor has been reported in these cells [38]. Here,

> > neutrophils perfused across TNF-stimulated endothelial cells in the

> > presence of a DP-1 receptor antagonist (BW868C) showed a greatly

> > diminished efficiency of endothelial cell transmigration (Figure 7B).

> > Conversely, the transmigration of neutrophils was re-established on

> > EPA-supplemented endothelial cells when a synthetic DP-1 receptor

> > agonist (BW245C) was perfused across cells adherent to the surface of

> > the monolayer (Figure 7C). Importantly, we could demonstrate that PGD2

> > was operating directly on the neutrophils, because when these cells were

> > harvested after migrating across a TNF-stimulated endothelial cells they

> > had up-regulated CD11b (the alpha subunit of the Î²2 integrin

> > CD11b/CD18 that is required for efficient neutrophil transmigration) and

> > proteolytically shed L-selectin (Figure 8). However, neutrophils

> > harvested from the surface of EPA-treated endothelial cells did not shed

> > L-selectin or up-regulate CD11b. Thus, in this assay system, the

> > delivery of a PGD2-mediated signal to the neutrophils was necessary for

> > full cellular activation and efficient transendothelial migration.

> > > > >

> > > > > Figure 7. The role of AA- and EPA-derived eicosanoids in

> > neutrophil transmigration.

> > > > >

> > > > > (A) When PDG2 was perfused across a population of neutrophils

> > adherent to EPA-treated endothelial cells, neutrophil transmigration was

> > restored. However, PGD3 had no significant effect on neutrophil

> > behaviour. All data are mean±SEM of four experiments. ANOVA showed

> > significant effect of treatment on transmigration. *p<0.05 compared to

> > EPA-supplemented endothelial cells in the absence of PGD2 by Dunnett

> > test. ( The DP1 receptor antagonist BW868c dose-dependently inhibited

> > neutrophil transmigration on TNF-stimulated endothelial cells. All data

> > are mean±SEM of five experiments; ANOVA showed a significant effect

> > agonist, BW245C, across neutrophils adherent to EPA-supplemented

> > endothelial cells restored neutrophil migration to control levels. Data

> > are mean±SEM of five experiments. ANOVA showed a significant effect

> > of treatment on transmigration. **p<0.01 compared to EPA-supplemented

> > endothelial cells in the absence of BW868C by Dunnett test. (D)

> > Neutrophils perfused across TNF-stimulated endothelial cells in the

> > presence of PGD3 showed a significantly reduced ability to migrate

> > across the monolayer. Data are mean±SEM of three experiments; ANOVA

> > showed a significant effect of PGD3 concentration on transmigration

> > (p<0.01). (E) The levels of PGD3 released from endothelial cells are

> > increased after EPA supplementation. Data are mean±SEM of three

> > experiments. ANOVA showed a significant effect of treatment (p<0.05).

> > **p<0.01 for PGD3 production compared to endothelial cells activated

> > with TNF without EPA supplementation by Dunnett's test.

> > > > >

> > > > > doi:10.1371/journal.pbio.1000177.g007

> > > > >

> > > > > Figure 8. The effect of adhesion on the expression of L-selectin

> > and CD11b on neutrophils.

> > > > >

> > > > > Neutrophils that were freshly isolated, migrated across

> > endothelial cells stimulated with TNF or adherent to the surface of

> > EPA-supplemented endothelial cells (EC) activated with TNF were

> > harvested and the expression of (A) CD11b or ( L-selectin (CD62L)

> > assessed by flow cytometry. Data are mean±SEM of four experiments;

> > ANOVA showed significant effect of treatments in (A) (p<0.01) and (

> > (p<0.05). In (A and , Dunnett's test showed significant difference

> > between âˆ'EPA and freshly isolated cells (*p<0.01 and *p<0.05,

> > respectively), but not between +EPA and freshly isolated cells.

> > > > >

> > > > > doi:10.1371/journal.pbio.1000177.g008

> > > > > PGD3 Generated from EPA Antagonises the PGD2-Mediated Migration of

> > Neutrophils across Endothelial Cells

> > > > >

> > > > > Taken together, these data show that PGD2 operates through the

> > neutrophil DP-1 receptor to provide a signal that is essential for

> > transendothelial cell migration. Moreover, the ability of neutrophils to

> > respond to this signal is abolished when endothelial cells are preloaded

> > with EPA from which PGD3 is generated. This implies that PGD3 may be an

> > effective antagonist of the DP-1 receptor. To test this hypothesis, we

> > perfused neutrophils across TNF-stimulated endothelial cells in the

> > presence of exogenous PGD3. Neutrophil transmigration was inhibited in a

> > dose-dependent manner by PGD3, showing that in the presence of

> > endogenously generated PGD2 it could effectively antagonise this process

> > (Figure 7D). Importantly, we were able to show that cells supplemented

> > with EPA-generated an increased amount of PGD3 (Figure 7E).

> > > > >

> > > > > The absolute levels of PGD2 and PGD3 that are generated in our

> > assay system, and thus the ratio of their abundance, are not easily

> > assessed. The levels of PGD2 measured as an endothelial cell releasate

> > were at the limits of detection (Table 3), presumably due to the short

> > half-life of this prostanoid under physiological conditions; however,

> > significant levels of PGD3 were assayed upon EPA supplementation, and

> > concentrations in the order of 5–10 nM had accumulated over the 24 h

> > of the EPA supplementation period. The half-life of PGD3 in serum or

> > albumin-containing medium has not been reported to our knowledge; thus

> > it is not clear whether the measurements made here report the true

> > levels of PGD3 production, or if metabolic degradation of this

> > prostanoid renders these measurements underestimates. Irrespective of

> > this, our experiments utilising synthetic PGD2 demonstrate that

> > concentrations on the order of 1 nM were sufficient to fully

> > recapitulate the process of migration on EPA-supplemented endothelial

> > cells, and when using PGD3 as an antagonist of TNF-induced

> > transmigration marked effects were evident at 10–100 pM. Thus, the

> > measurable levels of PGD3 that we report would certainly be sufficient

> > to effectively antagonise the transmigration of neutrophils in our

> > experimental model.

> > > > >

> > > > > Table 3. Analysis of eicosanoids by liquid chromatography tandem

> > mass spectrometry.

> > > > >

> > > > > doi:10.1371/journal.pbio.1000177.t003

> > > > > Conclusions

> > > > >

> > > > > Our study demonstrates a hitherto unknown regulatory step in the

> > recruitment of neutrophils by cytokine-stimulated endothelial cells

> > (refer to Figure 1 to see how this new step fits with the known

> > regulatory mechanisms of neutrophil recruitment). After initial

> > tethering by selectin molecules, a chemokine signal induces arrest of

> > the cell on the endothelial cell surface. However, the chemokine signal

> > is not sufficient to support transmigration across the endothelial cell

> > monolayer, and the arachidonic acid-derived prostanoid, PGD2, is an

> > essential downstream regulator of this process. PGD2 operates through

> > the DP-1 receptor and this signal can be effectively antagonised by PGD3

> > which is generated from EPA released by the action of phospholipase

> > enzymes on the phospholipids of EPA supplemented cells. Not only does

> > this study reveal a new step in the recruitment of neutrophil

> > recruitment during inflammation, it also reveals a novel

> > anti-inflammatory mechanism of action of the dietary n-3-PUFA, EPA.

> > > > >

> > > > > Materials and Methods Top

> > > > >

> > > > > Cell Culture and Neutrophil Isolation

> > > > >

> > > > > Endothelial cells (EC) were isolated and cultured, as previously

> > described [39], with or without 0–5 µM EPA (Sigma) for 24 h.

> > TNF-Î± (100 U/ml; R & D systems) was added to the EC for the final 4 h

> > of culture. In some experiments, EC were treated with 100 U/ml TNF-Î±

> > in the presence of 10 µM indomethacin or 0.1 µM SC-560 or 1 µM

> > NS-398 (Sigma). In the AA reconstitution experiments, 5 µM AA (Sigma)

> > was added to the culture medium simultaneously with TNF-Î±.

> > > > >

> > > > > Blood from healthy adult volunteers was collected into tubes

> > coated with EDTA (1.6 mg/ml), and neutrophils were separated using

> > 2-step density gradients of Histopaque 1119 and 1077 (Sigma), as

> > previously described [40]. After washing in 0.15% BSA in PBS, cells were

> > counted using a Coulter Multisizer (Coulter Electronics), and

> > neutrophils, which were >95% pure, were resuspended at a concentration

> > of 1×106/ml in PBS/Alb with calcium and magnesium.

> > > > >

> > > > > RNA Extraction and Real-Time PCR

> > > > >

> > > > > mRNA was isolated from EC using the Qiagen RNEasy Mini Kit 50

> > (Qiagen) following the manufacturer's instructions. Real-time PCR

> > (RT-PCR) was performed using QuantiTect probe RT-PCR kit according to

> > the manufacturer's instructions (Qiagen). Primers were purchased from

> > Applied Biosystems. The expression of each target gene was normalised to

> > Î²-actin expression, and the data presented represent fold change

> > compared to untreated EC.

> > > > >

> > > > > Luminex for Secreted Cytokines and Chemokines

> > > > >

> > > > > Supernatants were collected from EC that had been incubated with

> > 0–5 µM EPA for 24 h and had 100 U/ml TNF added to the culture for

> > the final 4 h of culture. The Luminex kit was purchased from

> > Upstate/Chemicon, and the experiment performed to the manufacturer's

> > instructions. Data were collected and analysed from the samples using a

> > Luminex100 machine (Luminex).

> > > > >

> > > > > Flow-Based Adhesion Assay

> > > > >

> > > > > Glass capillaries (microslides) containing treated EC monolayers

> > were incorporated into a flow-based adhesion assay as described [39].

> > Briefly, microslides were attached to cell and fluid reservoirs by

> > silicon tubing at one end and to a withdrawal syringe pump at the other

> > end. After mounting on the stage of a phase contrast microscope, EC were

> > washed for 2 min with PBS containing 0.15% BSA. Neutrophils were then

> > perfused (1×106/ml) at 0.1 Pa for 4 min, followed by 15 min of wash.

> > Video images were recorded throughout the experiment and neutrophil

> > behaviour analysed offline using Image Pro software (Image-Pro Plus). In

> > some experiments, neutrophils were perfused across EPA- and TNF-treated

> > EC, allowed to adhere, and subsequently perfused with 1 nM PGD2 or 1 nM

> > PGD3 (both from Cayman Chemicals). Alternatively, neutrophils were

> > treated with BW868c (Cayman Chemicals), a DP1 receptor antagonist, for

> > 10 min prior to perfusion over TNF-Î±-stimulated EC. Neutrophils were

> > also treated with the DP1 receptor agonist BW245c (Cayman Chemicals)

> > prior to perfusion over EPA- and TNF-Î±-treated EC. Neutrophils were

> > also treated with 0–1 nM PGD3, immediately prior to perfusion across

> > TNF-stimulated EC. In all experiments, lipid reagents, agonists, and

> > antagonists were stored in 100% ethanol under nitrogen gas. Once

> > diluted, the medium contained <0.01% ethanol. All controls contained

> > equivalent concentrations of ethanol.

> > > > >

> > > > > Analysis of EPA Incorporation into Cellular Phospholipids

> > > > >

> > > > > First-passage confluent EC were treated with 5 µM of EPA for 24

> > h. EC were removed from the plastic culture dish by scraping and stored

> > in 0.88% KCl solution at âˆ'20°C until analysis. Total lipid was

> > extracted with chloroform:methanol (2:1, v/v) containing butylated

> > hydroxytoluene (50 mg/l) as antioxidant. Fatty acids were subsequently

> > hydrolysed from the lipid and simultaneously methylated by incubation

> > with methylation reagent (methanol containing 2% v/v H2SO4) at 50°C

> > for 2 h. Fatty acid methyl esters were separated and identified using a

> > Hewlett Packard 6890 gas chromatograph (Hewlett Packard) fitted with a

> > 30 mm×32 mm BPX 70 capillary column, film thickness 0.25 µm.

> > Helium, at the initial flow rate of 1.0 ml/min, was used as the carrier

> > gas. Injector and detector temperatures were 275°C, and the column

> > oven temperature was maintained at 170°C for 12 min after sample

> > injection. The oven temperature was programmed to increase from 170 to

> > 210°C at 5°C/min. Fatty acid methyl esters were identified by

> > comparison with authentic standards. Peak areas were quantified using

> > ChemStation software (Hewlett Packard). Each fatty acid was expressed as

> > weight percent of total fatty acids present.

> > > > >

> > > > > Eicosanoid Extraction and Reverse-Phase High-Performance Liquid

> > Chromatography Analysis

> > > > >

> > > > > Eicosanoids were extracted from control EC using C18 Sep-Pak

> > cartridges (Waters). Briefly, cartridges were conditioned with 5 ml of

> > high-performance liquid chromatography (HPLC)-grade MeOH and rinsed

> > twice with water. Reactions were terminated with addition of 100%

> > ice-cold MeOH, containing 2 ng of internal standard PGE2-d4. Sample

> > volume was adjusted to 10% MeOH with water and applied to the column.

> > The column was rinsed with water and the sample eluted with 2 ml of

> > MeOH. Nitrogen gas was used to dry the sample, which was resuspended in

> > 100 µl of MeOH and stored at âˆ'80°C until analysis.

> > > > >

> > > > > Prostaglandin Quantitation using Liquid Chromatography Tandem Mass

> > Spectrometry

> > > > >

> > > > > Prostaglandins were separated on a C18 ODS2, 5 µm,

> > 150×4.6-mm column (Waters) using a gradient of 50%–90% B over 20

> > min (A, water:acetonitrile:acetic acid, 75:25:0.1; B,

> > methanol:acetonitrile:acetic acid, 60:40:0.1) at 1 ml/min. Products were

> > quantitated by directing the HPLC output directly into the electrospray

> > source of a Q-Trap mass spectrometer (Applied Biosystems 4000 Q-Trap)

> > operating in the negative mode (âˆ'4,500 V). Individual

> > prostaglandins were monitored in the Multiple Reaction Monitoring (MRM)

> > mode using specific parent to daughter transitions of m/z 349–269

> > for PGD3 with collision energies of âˆ'26 V. Products were

> > identified and quantified with reference to the appropriate standards

> > run in parallel under the same conditions, with 2 ng of PGE2-d4 (m/z

> > 355–275) added as an internal standard.

> > > > >

> > > > > Analysis of Neutrophil Adhesion Molecule Expression by Flow

> > Cytometry

> > > > >

> > > > > Neutrophils for analysis were isolated from whole blood as

> > described above. Control cells were stained for flow cytometry directly

> > after isolation. Cells rolling on EPA-treated endothelial cells in

> > microslides were harvested by elution with 0.02% EDTA after 4 min of

> > neutrophil perfusion and 11 min of wash buffer perfusion (totalling 15

> > min of contact with endothelial cells). In order to harvest neutrophils

> > that had migrated across endothelial cells, endothelium was cultured on

> > 1.5 mg/ml type I collagen gels (Becton Dickinson). After endothelial

> > cell stimulation with TNF for 4 h, neutrophils were added for 15 min.

> > Surface-adherent cells were washed off with 0.02% EDTA, and the gel was

> > dissolved using type VII collagenase (Sigma). Neutrophils were harvested

> > from the dissolved gel by centrifugation. All neutrophil populations

> > were stained for surface expression of CD11b (PE-conjugated, clone

> > 2LPM19c, 2 µg/ml; DAKO) or CD62L (FITC-conjugated, clone Dreg56,10

> > µg/ml, Beckton Dickinson). Expression was analysed by flow cytometry

> > using a DAKO CyAn, and data analysed using Summit software (Becton

> > Dickinson). Data are expressed as mean fluorescent intensity.

> > > > >

> > > > > Statistical Analysis

> > > > >

> > > > > Data were analysed using Prism software (GraphPad software).

> > Results are presented as means±standard error of the mean (SEM).

> > Comparisons between individual treatments were by paired t-test where

> > appropriate. ANOVA was performed to assess the effect of EPA

> > concentration on PMN transmigration. Significant findings were

> > investigated further using Bonferroni multiple comparison test or

> > Dunnett's test.

> > > > >

> > > > > Author Contributions Top

> > > > >

> > > > > The author(s) have made the following declarations about their

> > contributions: Conceived and designed the experiments: VBO RFG PCC GBN

> > GER. Performed the experiments: SPT CMY BHM JM. Analyzed the data: SPT

> > CMY BHM VBO JM GER. Contributed reagents/materials/analysis tools: VBO

> > RFG PCC GBN. Wrote the paper: GER.

> > > > >

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