Receptors In Human Neutrophils Research Articles

Pharmacological evidence that the inhibitory effects of prostaglandin E2 are mediated by the EP2 and EP4 receptors in human neutrophils.

Prostaglandin E2 (PGE2) is a recognized inhibitor of granulocyte functions. However, most of the data supporting this was obtained when available pharmacological tools mainly targeted the EP2 receptor. Herein, we revisited the inhibitory effect of PGE2 on reactive oxygen species production, leukotriene biosynthesis, and migration in human neutrophils. Our data confirm the inhibitory effect of PGE2 on these functions and unravel that the effect of PGE2 on human neutrophils is obtained by the combined action of EP2 and EP4 agonism. Accordingly, we also demonstrate that the inhibitory effect of PGE2 is fully prevented only by the combination of EP2 and EP4 receptor antagonists, underscoring the importance of targeting both receptors in the effect of PGE2. Conversely, we also show that the inhibition of ROS production by human eosinophils only involves the EP4 receptor, despite the fact that they also express the EP2 receptor.

Larixol inhibits fMLP-induced superoxide anion production and chemotaxis by targeting the βγ subunit of Gi-protein of fMLP receptor in human neutrophils

The over-activated neutrophils through G-protein-coupled receptors (GPCRs) caused inflammation or tissue damage. Therefore, GPCRs or their downstream molecules are major targets for inhibiting uncontrolled neutrophil activation. Our studies investigate the action and underlying mechanism of larixol, a diterpene extract from the root of euphorbia formosana, on fMLP-induced neutrophil respiratory burst, chemotaxis, and granular release. The immunoprecipitation assay was performed to investigate whether larixol inhibits fMLP-induced respiratory burst by interfering with the interaction of fMLP receptor Gi-protein βγ subunits with its downstream molecules. Briefly, larixol inhibited fMLP (0.1 μM)-induced superoxide anion production (IC50:1.98 ± 0.14 μM), the release of cathepsin G (IC50:2.76 ± 0.15 μM) and chemotaxis in a concentration-dependent manner; however, larixol did not inhibit these functions induced by PMA (100 nM). Larixol inhibited fMLP-induced Src kinase phosphorylation. Therefore, larixol attenuated the downstream signaling of Src kinases, ERK1/2, p38, and AKT phosphorylation. Moreover, larixol inhibited fMLP-induced intracellular calcium mobilization, PKC phosphorylation, and p47phox translocation from the cytosol to the plasma membrane. Larixol inhibited the interaction of the βγ subunits of Gi-protein of fMLP receptor with Src kinase or with PLCβ by the immunoprecipitation and duolink assay. Furthermore, larixol did not antagonize the formyl peptide receptors. Larixol did not increase cyclic nucleotide levels in neutrophils. These results suggest that larixol modulated fMLP-induced neutrophils superoxide anion production, chemotaxis, and granular releases by interrupting the interaction of the βγ subunits of Gi-protein with downstream signaling of the fMLP receptor.

Larixol Inhibits Fmlp-Induced Superoxide Anion Production and Chemotaxis by Targeting the Βγ Subunit of Gi-Protein of Fmlp Receptor in Human Neutrophils

Larixol Inhibits Fmlp-Induced Superoxide Anion Production and Chemotaxis by Targeting the Βγ Subunit of Gi-Protein of Fmlp Receptor in Human Neutrophils

CD200R1L is a functional evolutionary conserved activating receptor in human neutrophils.

Inhibitory and activating immune receptors play a key role in modulating the amplitude and duration of immune responses during infection and in maintaining immune balance in homeostatic conditions. The CD200 Receptor (CD200R) gene family in humans encodes one inhibitory receptor, CD200R1, and one putative activating member, CD200R1 Like (CD200R1L). It is demonstrated that CD200R1L is endogenously expressed by human neutrophils and activates cellular functions such as reactive oxygen species (ROS) production via Syk, PI3Kβ, PI3Kδ, and Rac GTPase signaling. Phylogenetic analysis shows that CD200R1L is present in many species among vertebrates, ranging from birds to primates, suggesting that evolutionary conservation of this receptor is critical for protection against co‐evolving pathogens. The duplication event that generated CD200R1L from CD200R occurred several times throughout evolution, supporting convergent evolution of CD200R1L. In our phylogenetic trees, CD200R1L has longer branch lengths than CD200R1 in most species, suggesting that CD200R1L is evolving faster than CD200R1. It is proposed that CD200R1L represents a hitherto uncharacterized activating receptor on human neutrophils.

The GPR40 Agonist GW9508 Enhances Neutrophil Function to Aid Bacterial Clearance During E. coli Infections.

G-protein-coupled receptor 40 (GPR40) is known to play a role in the regulation of fatty acids, insulin secretion, and inflammation. However, the function of this receptor in human neutrophils, one of the first leukocytes to arrive at the site of infection, remains to be fully elucidated. In the present study, we demonstrate that GPR40 is upregulated on activated human neutrophils and investigated the functional effects upon treatment with a selective agonist; GW9508. Interestingly, GPR40 expression was up-regulated after neutrophil stimulation with platelet-activating factor (10 nM) or leukotriene B4 (LTB4, 10 nM) suggesting potential regulatory roles for this receptor during inflammation. Indeed, GW9508 (1 and 10 μM) increased neutrophil chemotaxis in response to the chemokine IL-8 (30 ng/ml) and enhanced phagocytosis of Escherichia coli by approximately 50% when tested at 0.1 and 1 μM. These results were translated in vivo whereby administration of GW9508 (10 mg/kg, i.p.) during E. coli infections resulted in elevated peritoneal leukocyte infiltration with a higher phagocytic capacity. Importantly, GW9508 administration also modulated the lipid mediator profile, with increased levels of the pro-resolving mediators resolvin D3 and lipoxins. In conclusion, GPR40 is expressed by activated neutrophils and plays an important host protective role to aid clearance of bacterial infections.

Applying label-free dynamic mass redistribution assay for studying endogenous FPR1 receptor signalling in human neutrophils

IntroductionThe label-free dynamic mass redistribution-based assay (DMR) is a powerful method for studying signalling pathways of G protein-coupled receptors (GPCRs). Herein we present the label-free DMR assay as a robust readout for pharmacological characterization of formyl peptide receptors (FPRs) in human neutrophils. MethodsNeutrophils were isolated from fresh human blood and their responses to FPR1 and FPR2 agonists, i.e. compound 43, fMLF and WKYMVm were measured in a label-free DMR assay using Epic Benchtop System from Corning®. Obtained DMR traces were used to calculate agonist potencies. ResultsThe potencies (pEC50) of fMLF, WKYMVm and compound 43, determined on human neutrophils using the label-free DMR assay were 8.63, 7.76 and 5.92, respectively. The DMR response to fMLF, but not WKYMVm and compound 43 could be blocked by the FPR1-specific antagonist cyclosporin H. DiscussionWe conclude that the DMR assay can be used, and complements more traditional methods, to study the signalling and pharmacology of endogenous FPR receptors in human neutrophils.

Early tyrosine phosphorylation events following adenosine A2A receptor in human neutrophils: identification of regulated pathways.

Activation of the adenosine 2A receptor (A2AR) elevates intracellular levels of cAMP and acts as a physiologic inhibitor of inflammatory neutrophil functions. In this study, we looked into the impact of A2AR engagement on early phosphorylation events. Neutrophils were stimulated with well-characterized proinflammatory agonists in the absence or presence of an A2AR agonist {3-[4-[2-[ [6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino] ethyl] phenyl] propanoic acid (CGS 21680)}, PGE2, or a mixture of the compounds RO 20-1724 and forskolin. As assessed by immunoblotting, several proteins were tyrosine phosphorylated; CGS 21680 markedly decreased tyrosine phosphorylation levels of 4 regions (37-45, 50-55, 60, and 70 kDa). Key signaling protein kinases-p38 MAPK, Erk-1/2, PI3K/Akt, Hck, and Syk-showed decreased phosphorylation, whereas Lyn, SHIP-1, or phosphatase and tensin homolog (PTEN) was spared. PGE2 or the intracellular cAMP-elevating combination of RO 20-1724 and forskolin mostly mimicked the effect of CGS 21680. Together, results unveil intracellular signaling pathways targeted by the A2AR, some of which might be key in modulating neutrophil functions.

Inhibitory Effects of Areca Nut Extract on Expression of Complement Receptors and Fc Receptors in Human Neutrophils

Chewing of areca quid increases the prevalence of periodontal diseases. Areca nut extract (ANE) inhibits the phagocytic activity of human neutrophils. This in vitro study investigates the effects of ANE on complement- and antibody-opsonized phagocytosis by neutrophils. Expression of complement receptors, Fc receptors, and F-actin in ANE-treated neutrophils is also analyzed. The viability of ANE-treated neutrophils was determined using the propidium iodide staining method. The possible effects of ANE on the expression of complement receptors and Fc receptors were examined using an immunofluorescence staining method followed by flow cytometry and confocal laser scanning microscopy. The phagocytic activity of neutrophils against complement or immunoglobulin (Ig)G-opsonized fluorescent beads was analyzed using flow cytometry. Expression of F-actin was determined using confocal laser scanning microscopy. ANE significantly inhibited the production of complement receptors (CR1, CR3, and CR4) and Fc receptors (FcγRII and FcγRIII) in a concentration-dependent manner. Treatment of neutrophils with ANE significantly impaired their ability to phagocytose fluorescent beads. ANE also inhibited phagocytosis of fluorescent beads that were opsonized by complement or IgG. Moreover, expression of F-actin was inhibited after ANE treatment. ANE inhibits the complement- and IgG-mediated neutrophil phagocytosis that may result from reduction of the expression of complement receptors, Fc receptors, and F-actin formation after ANE treatment. The findings suggest that areca nut chewing may jeopardize the defensive functions of neutrophils and affect periodontal health.

Modulation of monosodium urate crystal-induced responses in neutrophils by the myeloid inhibitory C-type lectin-like receptor: potential therapeutic implications

IntroductionMonosodium urate crystals (MSU), the etiological agent of gout, are one of the most potent proinflammatory stimuli for neutrophils. The modulation of MSU-induced neutrophil activation by inhibitory receptors remains poorly characterized. The expression of the myeloid inhibitory C-type lectin-like receptor (MICL) in neutrophils is downregulated by several proinflammatory stimuli, suggestive of a role for this receptor in neutrophil function. We thus investigated the potential role of MICL in MSU-induced neutrophil activation.MethodsThe expression of MICL was monitored in human neutrophils by flow cytometry and Western blot analysis after stimulation with MSU. Protein tyrosine phosphorylation was also assessed by Western blot analysis and the production of IL-1 and IL-8 by enzyme-linked immunosorbent assay. Changes in the concentration of cytoplasmic free calcium were monitored with the Fura-2-acetoxymethyl ester calcium indicator. MICL expression was modulated with an anti-MICL antibody in neutrophils and siRNA in the PLB-985 neutrophil-like cell line.ResultsMSU induced the downregulation of MICL expression in neutrophils. A diminution in the expression of MICL induced by antibody cross-linking or siRNA enhanced the MSU-dependent increase in cytoplasmic calcium levels, protein tyrosine phosphorylation and IL-8 but not IL-1 production. Pretreatment of neutrophils with colchicine inhibited the MSU-induced downregulation of MICL expression.ConclusionsOur findings strongly suggest that MICL acts as an inhibitory receptor in human neutrophils since the downregulation of MICL expression enhances MSU-induced neutrophil activation. Since MSU downregulates the expression of MICL, MICL may play a pathogenic role in gout by enhancing neutrophil effector functions. In support of this notion, colchicine counteracts the MSU-induced loss of MICL expression. Our findings thus also provide further insight into the potential molecular mechanisms behind the anti-inflammatory properties of this drug.

What Formyl Peptide Receptors, if Any, Are Triggered by Compound 43 and Lipoxin A4?

In this study, we determined receptor preferences for compound 43, a nitrosylated pyrazolone derivative, and the eicosanoid lipoxin A(4) (LXA(4)), potent anti-inflammatory mediators in many experimental in vivo models. Their effects have been suggested to be mediated through binding to formyl peptide receptor (FPR)2 [earlier known as formyl peptide receptor-like 1 or the lipoxin A(4) receptor (ALXR)], one of the two members of the FPR family expressed in neutrophils. Compound 43 activates all neutrophil functions investigated, whereas LXA(4) induces a unique inhibiting pathway suggested to involve β-arrestin binding as an early signalling step, but not a transient rise in intracellular Ca(2+). We show that compound 43 can activate not only FPR2 but also FPR1, the other neutrophil receptor in the FPR family, and FPR1 is actually the preferred receptor in human neutrophils and possibly also in the murine equivalent. LXA(4) analogues from two commercial sources were used, and neither of these induced any translocation of β-arrestin as measured in an enzyme fragment complementation assay. The conclusions drawn from these experiments are that neither compound 43 nor LXA(4) works as FPR2 agonists in neutrophils, findings of importance for a proper interpretation of results obtained with these compounds as regulators of inflammation.

Characterization of FGF receptor expression in human neutrophils and their contribution to chemotaxis

Several members of the fibroblast growth factor (FGF) family are potent endothelial cell (EC) mitogens and angiogenic factors, and their activities can be mediated by four tyrosine kinase receptors (FGFR1-4). In addition, FGFs can induce the release of inflammatory mediators by ECs and the expression of adhesion molecules at their surface, thereby favoring the recruitment and transvascular migration of inflammatory cells such as neutrophils. Neither the expression nor the biological activities that could be mediated by FGFRs have been investigated in human neutrophils. By biochemical and cytological analyses, we observed that purified circulating human neutrophils from healthy individuals expressed varying levels of FGFRs in their cytosol and at their cytoplasmic membrane. FGFR-2 was identified as the sole cell surface receptor, with FGFR-1 and -4 localizing in the cytosol and FGFR-3 being undetectable. We assessed the capacity of FGF-1 and FGF-2 to induce neutrophil chemotaxis in a modified Boyden microchamber and observed that they increase neutrophil transmigration at 10(-10) and 10(-9) M and by 1.77- and 2.34-fold, respectively, as compared with PBS-treated cells. Treatment with a selective anti-FGFR-2 antibody reduced FGF-1-mediated chemotaxis by 75% and abrogated the effect of FGF-2, while the blockade of FGFR-1 and -4 partially inhibited (15-40%) FGF-chemotactic activities. In summary, our data are the first to report the expression of FGF receptors in human neutrophils, with FGF-1 and FGF-2 promoting neutrophil chemotaxis mainly through FGFR-2 activation.

A sulphonoglycolipid from the fern Polypodium decumanum and its effect on the platelet activating-factor receptor in human neutrophils.

The South American fern Polypodium decumanum, traditional name calaguala, has documented clinical use in oral treatment of skin disorders, including psoriasis. The inflammatory mediator platelet-activating factor (PAF), has been implicated in the pathogenesis of psoriasis. A constituent of a calaguala extract has been shown to have inhibitory activity in a PAF-induced exocytosis model in human neutrophils. The compound was identified as the sulphoquinovosyl diacylglycerol 1,2-di-O-palmitoyl-3-O-(6-sulpho-alpha-D-quinovopyranosyl)-glycero l by spectroscopic means. When subsequently studied in an in-vitro model for [3H]PAF binding in neutrophils from man the compound caused dose-dependent displacement of [3H]PAF from its receptor with an IC50 value of 2 microM. It is suggested that the compound acts through PAF receptor antagonism in intact human neutrophils.

Formyl Peptide Receptor 1 and 2 Dual Agonist Inhibits Human Neutrophil Chemotaxis by the Induction of Chemoattractant Receptor Cross-desensitization

Formyl peptide receptor 1 (FPR1) and FPR2/ALX are known to control neutrophil chemotaxis in response to various ligands. In this study, we investigated the inhibitory mechanism of compound 43 (Cpd43), an FPR1 and FPR2/ALX dual agonist, on human neutrophil chemotaxis. Precedent stimulation of human peripheral blood neutrophils with Cpd43 rendered the cells unresponsive in calcium mobilization induced by interleukin-8, C5a, or leukotriene B4. In addition, neutrophils pretreated with Cpd43 lost their chemotactic responses against these chemoattractants, wherein the expressions of chemoattractant receptors CXCR1, CXCR2, C5a receptor, and leukotriene B4 receptor 1 on the surface of neutrophils were all diminished significantly by treatment with Cpd43. By evaluating its pharmacological effect on 341 molecules, including receptors and enzymes, we also confirmed that Cpd43 has a highly specific affinity to FPR1 and FPR2/ALX and does not show binding affinity to the other chemoattractant receptors. These results indicate a previously unrecognized inhibitory mechanism of Cpd43 on neutrophil chemotaxis: the induction of cross-desensitization of multiple chemoattractant receptors in human neutrophils through its FPR1 and FPR2/ALX dual agonism.

Remote Ischemic Preconditioning Stimulus Decreases the Expression of Kinin Receptors in Human Neutrophils

Remote ischemic preconditioning (RIPC) has been shown to reduce ischemic-reperfusion injury and is induced by brief forearm ischemia. Kinins are known to be involved in RIPC and act via the G protein coupled B1 and B2 receptors. Interaction of the kinins with their respective receptors causes receptor internalization, thereby reducing the potential for further activation. This may be critical for the protective effect of RIPC and if so, we hypothesized, would significantly decrease the expression of kinin receptors on the surface of neutrophils. The study was performed on five healthy human volunteers. The left forearm was rendered ischemic for three 5-min periods, each separated by 5 min of reperfusion. Three venous blood samples were taken from the right arm, one before and two after RIPC. Neutrophil isolation, immunofluorescence labeling, and confocal microscopy were performed. Mean pixel intensity data were generated using a fixed circular area of interest (AOI, 40×40 μm). For every image, the AOI was placed over a cell and the mean pixel intensity was recorded. The mean intensity was expressed as pixel×10(2)/μm(2) and presented as mean±SEM. Immunofluorescence at the different time points was compared by one way analysis of variance with Bonferroni's post-hoc test. A P value<0.05 was considered significant. The mean pixel intensity for kinin B1 receptors was decreased at 24 h after RIPC compared with both baseline and 15 min after RIPC (P<0.001). Similarly, the intensity for B2 receptor labeling on neutrophils was significantly decreased 24 h after RIPC compared with the baseline value (P<0.001). RIPC decreases expression of kinin receptors on circulating human neutrophils. Reduction in kinin surface receptors suggests internalization of receptors and is consistent with the concepts of kinin receptor activation and their role in RIPC.

Cholesterol is obligatory for polarization and chemotaxis but not for endocytosis and associated signaling from chemoattractant receptors in human neutrophils

Plasma membrane cholesterol is critical for neutrophil chemotaxis, although how cholesterol affects chemotactic signaling pathway has not been clearly delineated. Here we demonstrate that cholesterol was absolutely required for polarized redistribution of key chemotactic mediators in human neutrophils in response to all chemoattractants tested (fMet-Leu-Phe, and the chemokines CXCL1, CXCL8 and CXCL12). In particular, PI3K and phosphatidylinositol-3,4,5 triphosphate (PIP(3)) failed to accumulate at the front and phosphatase and tensin homolog (PTEN) at the back of chemoattractant-stimulated neutrophils after cholesterol depletion. Cholesterol depletion did not affect early chemoattractant signaling events such as G-protein activation, intracellular calcium flux or G-protein-independent endocytosis-linked signaling, including the activation of mitogen-activated protein kinase (MAPK), Hck and Fgr transduced by beta-arrestin. During cell polarization, F-actin assemblies redistributed the cholesterol-rich microdomains and cytoskeleton-anchored proteins, including CD16 and CD44 from the leading edge. These data suggest that spatial polarization of chemotactic mediators is orchestrated by protein:protein interactions that organize cholesterol-rich domains of the plasma membrane.

Signaling pathways downstream of P2 receptors in human neutrophils

Extracellular nucleotides stimulate human neutrophils by activating the purinergic P2Y2 receptor. However, it is not completely understood which types of G proteins are activated downstream of this P2 receptor subtype. We investigated the G-protein coupling to P2Y2 receptors and several subsequent signaling events. Treatment of neutrophils with pertussis toxin (PTX), a Gi protein inhibitor, caused only ∼75% loss of nucleotide-induced Ca2+ mobilization indicating that nucleotides cause Ca2+ mobilization both through Gi-dependent and Gi-independent pathways. However, the PLC inhibitor U73122 almost completely inhibited Ca2+ mobilization in both nucleotide- and fMLP-stimulated neutrophils, strongly supporting the view that both the PTX-sensitive and the PTX-insensitive mechanism of Ca2+ increase require activation of PLC. We investigated the dependence of ERK phosphorylation on the Gi pathway. Treatment of neutrophils with PTX caused almost complete inhibition of ERK phosphorylation in nucleotide or fMLP activated neutrophils. U73122 caused inhibition of nucleotide- or fMLP-stimulated ERK phosphorylation, suggesting that although pertussis toxin-insensitive pathways cause measurable Ca2+ mobilization, they are not sufficient for causing ERK phosphorylation. Since PLC activation leads to intracellular Ca2+ increase and PKC activation, we investigated if these intracellular events are necessary for ERK phosphorylation. Exposure of cells to the Ca2+ chelator BAPTA had no effect on nucleotide- or fMLP-induced ERK phosphorylation. However, the PKC inhibitor GF109203X was able to almost completely inhibit nucleotide- or fMLP-induced ERK phosphorylation. We conclude that the P2Y2 receptor can cause Ca2+ mobilization through a PTX-insensitive but PLC-dependent pathway and ERK phosphorylation is highly dependent on activation of the Gi proteins.

Functional expression of the P2Y 14 receptor in human neutrophils

Previous studies using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis have shown that the P2Y 14 receptor is expressed at high levels in human neutrophils. Therefore the primary aim of this study was to determine whether the P2Y 14 receptor is functionally expressed in human neutrophils. In agreement with previous studies RT-PCR analysis detected the expression of P2Y 14 receptor mRNA in human neutrophils. UDP-glucose (IC 50 = 1 μM) induced a small but significant inhibition ( circa 30%) of forskolin-stimulated cAMP accumulation suggesting functional coupling of endogenously expressed P2Y 14 receptors to the inhibition of adenylyl cyclase activity in human neutrophils. In contrast, the other putative P2Y 14 receptor agonists UDP-galactose and UDP-glucuronic acid (at concentrations up to 100 μM) had no significant effect, whereas 100 μM UDP- N-acetylglucosamine-induced a small but significant inhibition of forskolin-stimulated cAMP accumulation (20% inhibition). UDP-galactose, UDP-glucuronic acid and UDP- N-acetylglucosamine behaved as partial agonists by blocking UDP-glucose mediated inhibition of forskolin-induced cAMP accumulation. Treatment of neutrophils with pertussis toxin (G i/o blocker) abolished the inhibitory effects of UDP-glucose on forskolin-stimulated cAMP accumulation. UDP-glucose (100 μM) also induced a modest increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, whereas the other sugar nucleotides had no effect on ERK1/2 activation. Finally, UDP-glucose and related sugar nucleotides had no significant effect on N-formyl-methionyl-leucyl-phenylalanine-induced elastase release from neutrophils. In summary, although we have shown that the P2Y 14 receptor is functionally expressed in human neutrophils (coupling to inhibition of forskolin-induced cAMP and ERK1/2 activation) it does not modulate neutrophil degranulation (assessed by monitoring elastase release). Clearly further studies are required in order to establish the functional role of the P2Y 14 receptor expressed in human neutrophils.

Signaling Pathways Involved in Leukocyte Adhesiveness and Migration during Inflammation: Potential Targets for Therapeutic Interventions?

In response to a chemotactic gradient of inflammatory mediators and chemokines, neutrophils adhere to vascular endothelium and directly migrate, leaving blood vessels, toward inflamed tissue areas, to exert their primary defense function. These events are mediated by distinct classes of cell surface receptors in human neutrophils, that not only drive cell adhesion and motility, but also interfere with the cells activation status, modulating different functions and survival. In this review we summarize the current understanding of the series of events that begins at the level of G-protein coupled receptor activation by chemoattractants, and the signaling pathways triggered by cell adhesion molecule interactions that lead to neutrophil adhesion, migration and activation during inflammation. Integrins, as adhesion receptors able to act as anchoring molecules (allowing firm cellular attachment to the ECM) and signaling receptors (transducing signals in both directions, outside-in and inside-out) are targets that potentially provide both therapeutic and diagnostic opportunities. We also present data obtained with integrin-selective ligands, the disintegrins, which could be useful tools to understand cellular processes as adhesion, migration, proliferation, activation and cell survival and may be also suggested as prototypes for designing therapeutic agents for the prevention or activation of integrin-mediated effects.

Differential expression of adenosine receptors in human neutrophils: up-regulation by specific Th1 cytokines and lipopolysaccharide

Four types of adenosine receptors have been identified in different tissues and cell types, namely, A1, A2A, A2B, and A3 receptors. We report that A2AR but not A2BR mRNA in freshly isolated polymorphonuclear neutrophils (PMN) is maximally up-regulated after 4 h stimulation with lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-alpha) and to a lesser extent, with interleukin (IL)-1beta. These effects were maintained up to 21 h. Consistent with changes in A2AR mRNA expression, up-regulation of A2AR protein was also detected after 4 h of LPS or TNF-alpha exposure. Up-regulation of A2AR protein expression was transient and returned to near basal levels after 12 h or 16 h stimulation with TNF-alpha or LPS, respectively. Conversely, IL-1beta failed to promote A2AR protein expression. Suppression of thapsigargin-induced leukotriene synthesis by the selective A2AR agonist CGS-21680 was found to be more pronounced when PMN were cultured for 4 h with LPS or TNF-alpha. In contrast, the up-regulation of A2AR has no impact on CGS-21680-induced inhibition of phospholipase D activation and superoxide production in response to formyl-Met-Leu-Phe. These results demonstrate that the A2AR is up-regulated by specific T helper cell type 1 cytokines and LPS. Although this could represent a potential feedback mechanism to control inflammation, the effect of A2AR up-regulation varied depending on the stimulus used to stimulate PMN functional responses after their incubation with proinflammatory mediators.

Extracellular Nucleotide UTP Regulate Lipopolisaccharide-Induced Cytokine Production in Human Neutrophils.

Extracellular nucleotides can function as paracrine and autocrine mediators that signal through P2Y nucleotide receptors. Human polymorphonuclear (PMN) cells express P2Y receptors on the cell surface but the role of these G protein-coupled receptors in human neutrophils is not well established. Interleukin-8 (IL-8), which is a CXC-chemokine that induces adherence of neutrophils to vascular endothelium and extravasation into tissue, is produced by macrophages and endothelium. Additionally, neutrophils can synthesize and release interleukin-8 upon stimulation with lipopolisacharides (LPS) but the intracellular signaling pathways involved in this process are not clearly understood. LPS is a potent activator of macrophages and is responsible for the initiation the septic shock, increasing the secretion of various cytokines. Here we present evidence that LPS (1 ng/ml to 100 ng/ml) causes IL-8 production in a dose- and time- dependent manner and demonstrate that LPS itself is responsible for mitogen-activated protein kinase (MAPK) ERK1/2 phosphorylation. We used an enzyme immunosorbent-linked assay (ELISA) to determinate the amount of IL-8 released by isolated human neutrophils and present evidence that extracellular nucleotide uridine triphosphate (UTP) (1 μM/ml to 100 μM/ml) can inhibit IL-8 production in human neutrophils in a dose- and time- dependent manner. The addition of ATP had a similar inhibitory effect suggesting that nucleotides act on neutrophils via the P2Y2 receptor, which is equally activated by UTP and ATP. A cytokine array was used to detect the release of 42 different cytokines in the extracellular medium. IL-8 and IL-6 ere the major cytokines detected in the medium, though other cytokines were detected in lower concentrations. In the current study, we demonstrated that extracellular nucleotides may have an important role in regulating IL-8 production in neutrophils and potentially modulating the inflammatory response.