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Published as doi: 10.1096/fj.06-7457com.
 (The FASEB Journal. 2007;21:1575-1585.)
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The Toll-like receptor 7/8-ligand resiquimod (R-848) primes human neutrophils for leukotriene B4, prostaglandin E2 and platelet-activating factor biosynthesis

Kim Hattermann, Serge Picard, Mathieu Borgeat, Patrick Leclerc, Marc Pouliot and Pierre Borgeat

Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Quebec Research Center and Faculty of Medicine, Laval University, Québec, Canada

1Correspondence: Centre de Recherche en Rhumatologie et Immunologie, CHUQ Research Center (CHUL) and Laval University, 2705, boul. Laurier, local T1–49, Québec, Canada, G1V 4G2, #46166. E-mail: pierre.borgeat{at}crchul.ulaval.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Toll-like receptors (TLR) recognize pathogen-associated molecular patterns and play important roles in the innate immune system. While single-stranded viral RNA is the natural ligand of TLR7/TLR8, the imidazoquinoline resiquimod (R-848) is recognized as a potent synthetic agonist of TLR7/TLR8. We investigated the effects of TLR7/8 activation on lipid mediator production in polymorphonuclear leukocytes exposed to R-848. Although R-848 had minimal effects by itself, it strongly enhanced leukotriene B4 formation on subsequent stimulation by fMLP, platelet-activating factor, and the ionophore A23187. R-848 acted via TLR8 but not TLR7 as shown by the lack of effect of the TLR7-specific ligand imiquimod. Priming with R-848 also resulted in enhanced arachidonic acid release and platelet-activating factor formation following fMLP stimulation, as well as enhanced prostaglandin E2 synthesis following the addition of arachidonic acid. Western blot analysis demonstrated that R-848 induced the phosphorylation of the cytosolic phospholipase A2{alpha}, promoted 5-lipoxygenase translocation and potently stimulated the expression of the type 2 cyclooxygenase. Bafilomycin A1, an inhibitor of endosomal acidification, efficiently inhibited all R-848-induced effects. These studies demonstrate that TLR8 signaling strongly promotes inflammatory lipid mediator biosynthesis and provide novel insights on innate immune response to viral infections.—Hattermann, K., Picard, S., Borgeat, M., Leclerc, P., Pouliot, M., Borgeat, P. The Toll-like receptor 7/8-ligand resiquimod (R-848) primes human neutrophils for leukotriene B4, prostaglandin E2 and platelet-activating factor biosynthesis.


Key Words: PMN • lipid mediators


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
POLYMORPHONUCLEAR LEUKOCYTES (PMN) are the first immune cells to migrate at inflammatory sites and play a pivotal role in innate immunity. Like other mammalian immune cells, PMN detect the presence of bacteria, fungi, and viruses via the Toll-like receptors (TLR) that recognize pathogen-associated molecular patterns (1) . Human PMN express mRNA of all known TLRs except TLR3, whereas expression of TLR7 was found to be very low (2) . Viral single-stranded RNA (ssRNA) is the natural agonist of the intracellular (endosomal) TLR7 and TLR8; ssRNA viruses like influenza virus, vesicular stomatitis virus and the HIV have already been described as activators of TLR7/8 (3 4 5 6) . The imidazoquin-like molecule resiquimod R-848 is a potent synthetic ligand of TLR7/TLR8 (7) that possesses antiviral and antitumoral activities (8) . Treatment of human PMN with R-848 results in IL-8 production, increased phagocytosis of latex beads, and robust L-selectin shedding, and furthermore, it primes PMN for subsequent formyl-Met-Leu-Phe (fMLP)-induced superoxide generation (2) , demonstrating that R-848 stimulates functional responses in PMN.

So far, few studies have addressed the question of a putative link between TLR signaling and the lipid mediator network. The TLR2 activators, bacterial peptidoglycan, and yeast zymosan were shown to induce the generation of cysteinyl-leukotriene (LT) from human mast cells (9) . Moreover, recent studies demonstrated a clear connection between TLR4-signaling by bacterial lipopolysaccharides (lipopolysaccharide (LPS)) and cytosolic phospholipase A2{alpha} (cPLA2{alpha}) activation and arachidonic acid (AA) release in a mouse macrophage cell line (10) . This observation is important in view of the fact that the cPLA2{alpha} is a key enzyme in the release of AA and lyso-platelet-activating factor (PAF), the precursors of prostaglandins (PG), thromboxanes, leukotrienes (LT), lipoxins, and PAF, respectively (11) . Indeed, the stimulation of PMN with ligands such as C5a, fMLP, or PAF results in the concomitant release by the cPLA2{alpha} of AA and lyso-PAF from membrane phospholipids and their subsequent conversion into the lipid mediators of inflammation (12) . Among LT, leukotriene B4 (LTB4), a major product of AA metabolism in PMN and macrophages, is a potent chemoattractant and activator of phagocytes (13 , 14) .

Several recent studies strongly support an important role of LTB4 in host defense against infection. In addition, we previously demonstrated that exogenous LTB4 prevented cytomegalovirus (CMV) reactivation in a mouse model of infection (15) and that intravenous (i.v.) administration of LTB4 triggered the release of the potent antimicrobial peptides {alpha}-defensins in man (16) . A number of other studies also revealed the effect of viral infection or of viral proteins on the AA cascade in various leukocytes, although the specific issue of the involvement of TLR signaling in these responses of cells to viral infection was not addressed (17 18 19) . Therefore, it was of interest to determine whether activation of a TLR implicated in viral pathogen recognition and immune response would have an impact on LTB4 and other lipid mediator biosynthesis.

We report herein that the TLR7/8 ligand R-848 is a potent priming agent for the biosynthesis of three distinct classes of lipid mediators of inflammation in PMN, providing evidence that the TLR7/8 signaling pathway is coupled to the eicosanoid and PAF biosynthetic cascade. These data raise the possibility that the immune response to single-stranded RNA viruses via the TLR8 pathway may implicate the lipid mediators of inflammation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Resiquimod (R-848), imiquimod (R-837), and the thiazoloquinoline CL075 were purchased from PharmaTech (Shanghai, China) and Cedarlane Laboratories (Hornby, Canada), respectively; they were dissolved in sterile endotoxin-free water. Cytochalasin B (CB), dimethyl sulfoxide (DMSO), NS398, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), diisopropylfluorophosphate (DFP), lipopolysaccharides (LPS) (E. coli 0111:B4), the ionophore A23187, fMLP, and PAF were obtained from Sigma Chemical (St. Louis, MO, USA). Bafilomycin A1 was purchased from Cedarlane Laboratories and adenosine deaminase (ADA) from Roche (Laval, Canada). Pyrrophenone and MK-0591 were kindly provided by Shionogi and Co. (Osaka, Japan) and Merck-Frosst (Montreal, Canada), respectively. Stock solutions of bafilomycin A1, pyrrophenone, MK-0591, and A23187 were all prepared in DMSO and were directly added to cell suspensions; the final concentration of DMSO never exceeded 0.2% in PMN suspensions. Ficoll-Paque, HEPES, and Hanks’ Balanced Salt Solution (HBSS) were obtained from Wisent Laboratories (St. Bruno, Canada). GM-CSF, TNF-{alpha}, and IL-8 were purchased from Peprotech (Rocky Hill, NJ, USA). Anti-5-LO monoclonal antibody (mAb), anti-cPLA2 polyclonal antibody (pAb), and anti-COX-2 mAb were from Fitzgerald Industries International (Concord, MA, USA), Cell Signaling Technology (Beverly, MA, USA) and Cayman Chemical (Ann Arbor, MI, USA), respectively. Solid-phase extraction cartridges Oasis HLB (reverse phase, 60 mg) and Bond Elut Silica (silica, 100 mg) used for analysis of AA and PAF were purchased from Waters Corp. (Milford, MA, USA) and Varian (Mississauga, Canada), respectively. All eicosanoids and deuterium-labeled PAF were from Cayman Chemical.

Isolation of human PMN
Human PMN were isolated from citrated venous blood obtained from healthy donors by dextran sedimentation of erythrocytes, centrifugation over Ficoll-Paque cushions, and hypotonic lysis for elimination of contaminating erythrocytes, as described previously (20) . PMN were resuspended at 5 or 20 x 106/ml, as indicated in figure legends, in HBSS containing 10 mM HEPES and 1.6 mM CaCl2. The cell suspensions contained ≥95% neutrophils, with eosinophils as the major contaminant. Viability assessed by the trypan blue exclusion test was greater than 98%.

Cell stimulation
PMN suspensions were preincubated with 10 µM CB and either 15 µM R-848 or 700 pM GM-CSF or 1.5 nM TNF-{alpha} or 1 µg/ml LPS (in the presence of 5% heparinized plasma) or with combinations of these agents (as indicated in figure legends) for 30 min at 37°C before stimulation with 300 nM fMLP or 300 nM PAF for 5 min, or 100 nM IL-8 for 10 min. In addition, ADA was added (0.3 U/ml) during the last 15 min of this 30-min preincubation in order to eliminate the inhibitory effect of endogenous adenosine on LT biosynthesis (21) . When PMN were pretreated with LPS, cell suspensions were centrifuged and resuspended in fresh HBSS (containing HEPES, Ca2+ and ADA) to eliminate plasma before stimulation with the ligands. When A23187 was used as stimulus, PMN suspensions were subjected to a 30-min preincubation with R-848 without CB and ADA, and A23187 was added at 30 nM for 5 min. Where indicated, the cPLA2{alpha} inhibitor pyrrophenone and the 5-LO-activating protein (FLAP) inhibitor MK-0591 were added to the PMN suspensions 10 min either before the 30-min pretreatment with R-848, GM-CSF, TNF-{alpha} or LPS, or 10 min before stimulation with fMLP, as indicated in figure legends. Where indicated, the ATPase inhibitor bafilomycin A1 was added 20 min prior the 30-min pretreatment.

FACS analysis of TLR8 expression
Freshly isolated PMN were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Bioscience, San Diego, CA, USA). Cells were then stained with a phycoerythrin-tagged monoclonal anti-human TLR8 or an IgG1 isotype control (Cedarlane, Hornby, Canada). Expression was assessed on an EPICS XL Beckman-Coulter (Hialeah, FL, USA).

5-LO product analysis
For the determination of 5-LO product biosynthesis, PMN incubations were stopped by the addition of 0.5 volume of cold (4°C) methanol/acetronitrile (1/1, v/v) containing 25 ng/ml of each of PGB2 and 19-hydroxy-PGB2 as internal standards (stop solution-1). The denatured samples were centrifuged (600 g, 10 min at 22°C), and the supernatants were analyzed by reverse phase (RP)-HPLC using an on-line extraction procedure, as described previously (22) . LTB4, 20-carboxy-LTB4, 20-hydroxy-LTB4, 6(E)-LTB4, 6(E)-12-epi-LTB4, and 5(S)-hydroxy-eicosatetraenoic acid (5(S)-hydroxyeicosatetraenoic acid (HETE)) represent the major 5-LO metabolites of AA detectable by RP-HPLC and UV photometry in human PMN and are collectively referred to as 5-LO products. Quantitation of these metabolites was achieved by using the internal standard PGB2 for normalization and authentic standards of 20-hydroxy-LTB4, LTB4, and 5-HETE for calibration.

AA analysis
For the analysis of AA, incubations were stopped by the addition of 0.5 volume of cold (0°C) methanol containing 40 ng/ml of octadeutero (2H8)-AA (stop solution-2). The denatured incubation media were centrifuged (600 g, 10 min at 22°C), and the supernatants were subjected to solid-phase extraction. Briefly, 60-mg cartridges (Oasis HLB) were conditioned with 1.5 ml methanol and 3 ml of water containing 0.01% acetic acid. The samples were loaded on the cartridges, which were then successively washed with 1.5 ml of water containing 0.01% acetic acid, 1.5 ml of water, 1 ml of hexane, and 1.5 ml of methanol/water (65/35, v/v). AA was finally eluted with 1.5 ml of methanol. The AA-containing fractions were evaporated to dryness under reduced pressure using a Thermo Savant Speed-Vac concentrator model SPD121P (Thermo Electron Corp., Milford, MA) (drying rate set at "low") and the residues were dissolved in 50 µl ethanol/acetronitrile, (25/75, v/v) for analysis by liquid chromatography/mass spectrometry (LC/MS) using electrospray ionization in the negative ion mode, as described previously (23) .

PAF analysis
For the determination of PAF, PMN incubations were stopped by the addition of 1 volume of cold (4°C) ethanol containing 5 ng/ml of tetradeutero (2H4)-PAF as an internal standard (stop solution-3). The denatured samples were then centrifuged (600 g, 20 min at 22°C), and the supernatants were extracted as described previously (24) with minor modifications. Briefly, the samples were loaded on a conditioned (see paragraph above) 60-mg cartridge (Oasis HLB) and successively washed with 4 ml water and 2 ml ethanol/water (50/50, v/v). PAF was then eluted with 2 ml ethanol/water (98/2, v/v); this fraction was directly loaded onto an ethanol-conditioned 100 mg silica cartridge. The silica cartridge was then washed with 2 ml ethanol, and PAF was eluted with 1.1 ml acetronitrile/water (60/40, v/v). The fractions containing PAF were evaporated to dryness under reduced pressure in a Speed-Vac concentrator (drying rate set at "low") and the residues were dissolved in 50 µl of acetronitrile/10 mM aqueous ammonium acetate, 75/25 (v/v). Analysis of PAF was performed by LC-MS/MS using an API5000TM (MDS Sciex, Concord, Canada) by the measurement of the PAF/2H4-PAF ratio [(m/z 508/59)/(m/z 512/59)]. The HPLC separation was performed on a ZIC-HILIC, 5-µm particle, 4.6 x 100 mm column (Canadian Life Science, Peterborough, Canada) using acetonitrile/10 mM ammonium acetate, 72/28, v/v (isocratic) at a flow rate of 0.6 ml/min (no split). Quantitation was achieved using a standard curve generated by analysis (ratio determination) of solutions containing increasing amounts of PAF and a fixed amount of 2H4-PAF.

Analysis of cPLA2-phosphorylation and 5-LO translocation
For analysis of cPLA2-phosphorylation and 5-LO translocation, PMN incubations at 20 x 106/ml were stopped by the addition of 1 volume of cold HBSS (4°C). Cell suspensions were centrifuged (1200 g, 15 min, 4°C), supernatants were collected, and prepared for HPLC analysis as described above. Cell pellets were resuspended in 0.5 ml of sucrose buffer (10 mM HEPES, pH 7.4, 0.25 M sucrose, 1 mM EGTA) containing an antiprotease cocktail (1 mM PMSF, 1 mM DFP 10 µg/ml leupeptine, and 10 µg/ml aprotinin) and sonicated for 20 s (output setting at 1.5) using a Branson Sonifier 450 (Danbury, CT). Sonicated samples were then centrifuged (9,000 g, 15 min, 4°C) to remove intact cells and cell debris. Supernatants were subjected to ultracentrifugation (100,000 g, 45 min, 4°C). Pellets were resuspended in 150 µl of sucrose buffer and 37.5 µl of 5 times concentrated electrophoresis sample buffer (62.5 mM Tris, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 5% ß-mercapto-ethanol, 0.01% (w/v) bromphenol blue, 10 µg/ml aprotinin and leupeptin, 1 mM PMSF) and cytosols (320 µl) were mixed with 80 µl of 5 times concentrated electrophoresis sample buffer, and samples were heated to 100°C for 10 min. Twenty microliters of the cytosolic fractions and 30 µl of the membrane fractions were used for cPLA2 and 5-LO analysis by SDS-PAGE and Western blot analysis, respectively, as described previously (25) .

Analysis of type 2 cyclooxygenase (COX-2) expression
Total PMN RNA was isolated using Trizol (Life Technologies, Burlington, VT) according to the manufacturer’s protocol, with modifications (26) . First-strand cDNA synthesis was performed using 1 µg of total RNA with Superscript II (Invitrogen Lifetechnology, Carlsbad, CA) as recommended. Each sample for polymerase chain reaction (PCR) consisted of: 1 µg cDNA, 1.3 mM MgCl2, 0.2 mM dNTP, 500 nM of primers, 0.5 U of Taq polymerase (Amersham Biosciences, Buckinghamshire, UK), and SYBR Green dye (1:30 000 dilution; Molecular Probes, Eugene, OR) in a reaction volume of 20 µl. Amplification was performed in a Rotor-Gene 3000 (Corbett Research, Mortlake, Australia). Amplification conditions were as follows: 95°C (20 s), 58°C (20 s), 72°C (20 s) with 35 repetitions. Specificity of each reaction was ascertained by performing the Melt® procedure (58–99°C; 1°C/5 s), according to the manufacturer’s instructions. Primers used have been described previously (26) .

COX-2 protein expression was analyzed by Western blot analysis after separation of proteins by SDS-PAGE, as described previously (26) .

Analysis of PGE2 by immunoassay
PMN incubations were stopped by centrifugation (16,000 g, 10 s, 22°C); cell-free supernatants were collected and stored (–20°C) until their analysis by ELISA, according to the manufacturer’s instructions (Cayman Chemical).

Statistical analysis
Statistical analysis was performed by Student’s unpaired t test (two-tailed), and significance was considered to be attained when P was < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TLR8 expression in human PMN
Freshly prepared PMN were permeabilized and investigated for TLR8 expression by flow cytometry as described in Materials and Methods. As shown in Fig. 1 , the expression of TLR8 was clearly confirmed in the human PMN preparations used in the present study.


Figure 1
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  		Figure 1. TLR8 expression in human PMN. Freshly isolated human PMN (purity>95%) were permeabilized and analyzed for TLR8 expression by flow cytometry using an anti-human TLR8 antibody tagged with phycoerythrin or an isotype control (IgG1). The results shown are representative of 1 out of 3 different experiments.

R-848 primes human PMN for 5-LO product biosynthesis
Freshly isolated PMN were incubated with compound R-848 to investigate its effects on the AA cascade. R-848 was used at the concentrations of 10 and 15 µM, which is in the range of R-848 concentrations used by other investigators in experiments on PMN (27 , 28) . Figure 2 shows that R-848 triggered a slight but detectable release of AA and LT biosynthesis. These responses were first detectable at the 10-min time point and maximal at 10 and 15 min for AA release and 5-LO product formation, respectively. We next investigated whether R-848 might act as a PMN priming agent using fMLP as the second stimulus. In a first series of experiments, PMN were pre-exposed for various periods of time (up to 120 min) to 15 µM R-848, prior to stimulation with 300 nM fMLP. In control incubations, PMN were exposed for the same periods of time to the diluent (DMSO). Figure 3 A clearly shows that PMN exposed to DMSO for up to 120 min only very slightly responded (in terms of 5-LO product biosynthesis) to stimulation by the chemoattractant fMLP. In contrast, pretreatment of PMN with 15 µM R-848 enabled a very significant biosynthesis of 5-LO products in response to fMLP; the priming effect of R-848 was detectable after 15 min of pretreatment, maximal at 30 min, and then progressively declined. We next examined the concentration response curve of this priming effect of R-848. Figure 3B shows that the priming effect of R-848 on 5-LO products derived from leukotriene A4, that is, LTB4, the 6-trans isomers of LTB4, 20-hydroxy-LTB4 and 20-carboxy-LTB4, was detectable at 1 µM and maximal at 10 µM; the priming effect on 5-HETE biosynthesis closely paralleled that of the other 5-LO products (not shown).


Figure 2
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  		Figure 2. R-848 induces AA release and 5-LO product biosynthesis. Prewarmed (37°C) PMN (5x106/ml) were incubated for up to 60 min with 15 µM R-848. Incubations were stopped at the indicated time points by addition of 0.5 volume of ice-cold stop solutions-1 or -2 containing the internal standards for HPLC analysis of 5-LO products or LC-MS analysis of AA. The data shown (mean±SD) are from 1 representative experiment out of 3, each performed in duplicate. AA, arachidonic acid.


Figure 3
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  		Figure 3. R-848 primes for 5-LO product biosynthesis. Prewarmed (37°C) PMN were treated with 15 µM R-848 for the indicated times (A) and stimulated with 300 nM fMLP for 5 min, treated 30 min with different concentrations of R-848 and subsequently stimulated with 300 nM fMLP for 5 min (B), treated with different priming agents (15 µM R-848, 700 pM GM-CSF, 1.5 nM TNF-{alpha}, 1 µg/ml LPS with 5% plasma) for 30 min and stimulated with 300 nM fMLP for 5 min (C), treated 30 min with 15 µM R-848 and stimulated with either 300 nM fMLP or PAF for 5 min, 100 nM IL-8 for 10 min or 30 nM ionophore A23187 for 10 min (D). Incubations were stopped by addition of 0.5 volume of ice-cold stop solution-1, and 5-LO products were analyzed by HPLC. Data shown (mean±SD) are from 1 representative experiment out of 3, each performed in duplicate. *Significantly different from the corresponding control.

Compound R-848 has been shown to be a ligand for both TLR7 and TLR8. Although TLR7 expression is reported to be low in human PMN (2) , some experiments were carried out using the TLR7-specific ligand imiquimod (R-837) to assess whether the effects of R-848 described herein were the results of TLR8 activation. Figure 3B shows that R-837, at concentrations up to 300 µM did not show any priming effect on fMLP-stimulated 5-LO product synthesis. Finally, similar concentration-response experiments were performed with another TLR7/8 ligand, CL075 (also known as 3M002), a thiazoloquinoline compound structurally related to R-848 (29) . CL075 showed a priming effect similar to that of R-848 on fMLP-induced 5-LO product biosynthesis, however, with a three- to ten-fold enhanced potency (data not shown).

Several other agents, namely GM-CSF, TNF-{alpha}, and LPS have previously been shown to prime human PMN functional responses, including LT biosynthesis. A series of experiments was undertaken to compare the priming abilities of these agents to that of R-848 in fMLP-stimulated PMN. In these experiments, PMN were exposed to GM-CSF, TNF-{alpha}, and LPS under conditions (concentration and time) previously determined to be optimal for priming of LT biosynthesis. Figure 3C shows that the ability of R-848 to prime for 5-LO product biosynthesis exceeds that of GM-CSF and TNF-{alpha} and is also greater than that of LPS; Fig. 3C also shows that combination of R-848 with GM-CSF and TNF-{alpha} or R-848 with LPS did not afford a significantly enhanced priming effect.

Finally, we investigated the ability of R-848 to prime for LT biosynthesis with other stimuli. Figure 3D shows that 5-LO product biosynthesis induced by PAF was also strongly enhanced by pretreatment with R-848, whereas response to IL-8 was only slightly increased. Interestingly, LT biosynthesis induced by the ionophore A23187 was also significantly enhanced by R-848. It is noteworthy that the concentration of ionophore used in these experiments (30 nM) was submaximal and that R-848 did not enhance LT biosynthesis induced by optimal concentrations of ionophore (≥0.5 µM) (data not shown).

R-848 primes PMN for AA release and PAF biosynthesis
AA availability is a limiting step in LT biosynthesis. Thus, in the next experiments, we sought to determine whether enhanced AA release might account for increased 5-LO product biosynthesis in R-848-treated PMN. Figure 4 A shows that while fMLP alone caused a modest increase of AA release, its effect was greatly enhanced by pretreatment of PMN with R-848. The addition of the cPLA2{alpha} inhibitor pyrrophenone prior to PMN stimulation with fMLP strongly reduced AA release. Analysis of PAF biosynthesis in the same experimental setting (Fig. 4B ) demonstrated a similar result pattern. R-848 or fMLP alone triggered minimal PAF production, which was strongly increased when R-848-treated PMN were stimulated with fMLP. Pyrrophenone also strongly inhibited PAF biosynthesis in R-848-primed and fMLP-stimulated PMN. Figure 4C shows, for comparison purposes, the results of 5-LO product analysis in the same experiment, with a striking priming effect of R-848 on fMLP-induced 5-LO product biosynthesis and almost complete inhibition by pyrrophenone.


Figure 4
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  		Figure 4. R-848 primes PMN for AA release and PAF biosynthesis. Prewarmed (37°C) PMN (5x106/ml) were preincubated 30 min at 37°C with or without 15 µM R-848 and stimulated with 300 nM fMLP or treated with the vehicle (DMSO) for 5 min, as indicated under each bar. Where indicated, PMN were treated with 300 nM pyrrophenone 10 min before addition of fMLP (or DMSO), i.e., during the last 10 min of the 30-min pretreatment period. Reactions were stopped with cold (0°C) stop solutions-1, -2, or -3 containing the internal standards required for LC-MS analysis of AA (A) and PAF (B), and HPLC analysis of 5-LO products (C), as described in Materials and Methods. Data shown (mean±SD) are from 1 representative experiment out of 3, each performed in duplicate. N.D., not detectable. *Significantly different from control (no addition) value.

R-848 induces cPLA2{alpha} phosphorylation and 5-LO translocation
cPLA2{alpha} and 5-LO are key enzymes in lipid mediator production. Phosphorylation of Ser-505 of cPLA2{alpha} results in enhanced enzymatic activity, while translocation of cytosolic 5-LO to nuclear membranes is required for LT biosynthesis in intact cells. Given the observed effects of R-848 on AA release and LT and PAF biosynthesis, it appeared of interest to investigate whether R-848 affects these two important regulatory processes of lipid mediator biosynthesis. cPLA2{alpha} phosphorylation on Ser-505 was assessed by SDS-PAGE and Western blot analysis using an antibody reacting with both cPLA2{alpha} and phosphorylated (Ser-505) cPLA2{alpha}. Fig. 5 A shows that treatment of the PMN with R-848 resulted in phosphorylation of cPLA2{alpha}, as assessed by the band shift resulting from the decreased electrophoretic mobility of the phosphorylated enzyme (30) . Phosphorylation of the cPLA2{alpha} was detectable at 5 min and complete at 15 min. We next investigated whether priming with R-848 impacts on 5-LO translocation. Figure 5B shows that incubation of PMN with R-848 alone only slightly increased 5-LO level in the PMN nuclear fraction, while fMLP alone was even less efficient. In contrast, the further stimulation with fMLP of R-848-treated PMN, strongly induced 5-LO translocation to the cell membranes and 5-LO product biosynthesis, effects which were strongly inhibited when incubations were carried out in presence of pyrrophenone, or MK-0591, a potent inhibitor of FLAP. This latter observation supports that the 5-LO translocation promoted by R-848 is FLAP-dependent and relevant to 5-LO product biosynthesis rather than the consequence of an unspecific effect of R-848. Analysis of 5-LO in the cytosolic fraction was also performed but did not show significant changes between the different treatments due to the fact that only a very small fraction of 5-LO associates to membranes, even in R-848-treated and fMLP-stimulated PMN (data not shown).


Figure 5
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  		Figure 5. R-848 induces cPLA2{alpha} phosphorylation and 5-LO translocation. A) Prewarmed (37°C) PMN suspensions (20x106/ml) were incubated with 15 µM R-848 for the indicated times, and incubations were stopped by the addition of 1 volume of cold (4°C) HBSS. PMN suspensions were immediately centrifuged (1,200 g, 5 min, 4°C), and cell pellets were resuspended in sucrose buffer, sonicated, subjected to ultracentrifugation (100,000 g, 45 min at 4°C) and cPLA2{alpha} was analyzed in the cytosolic fraction as described in Materials and Methods. B) Prewarmed PMN suspensions (20x106/ml) were treated with 300 nM of the cPLA2{alpha} inhibitor pyrrophenone or 100 nM of the FLAP inhibitor MK-0591 (or DMSO) and then primed (or not) with 15 µM R-848 for 30 min at 37°C; PMN suspensions were then stimulated for 5 min with 300 nM fMLP (or DMSO), as indicated. Incubations were stopped as described above, and supernatants were processed for analysis of 5-LO products by HPLC. Cell pellets were fractionated as described above, and the cytosolic and cell membrane fractions were processed for analysis of 5-LO translocation as described in Materials and Methods. The immunoblots shown are representative of 1 out of three separate experiments. N.D., not detectable.

Priming with R-848 requires endosomal acidification
Recognition of ligand by TLR7/8 and signaling depend on endosomal acidification (4) . We have used bafilomycin A1, an efficient inhibitor of endosomal acidification, to confirm the involvement of TLR8 in the priming effects of R-848 on 5-LO product biosynthesis. The concentration-inhibition curve of bafilomycin A1 shown in Fig. 6 A demonstrates an 80% inhibition of 5-LO product biosynthesis induced by R-848 and fMLP at the concentration of 100 nM.


Figure 6
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  		Figure 6. A) Bafilomycin A1 selectively inhibits R-848 priming of 5-LO product biosynthesis. Prewarmed (37°C) PMN (5x106/ml) were incubated for 15 min at 37°C with various concentrations of bafilomycin A1. PMN were then primed with 15 µM R-848 for 30 min and stimulated with 300 nM fMLP for 5 min. PMN (5x106/ml) were also incubated with bafilomycin A1 and then stimulated with 100 nM ionophore A23187 for 5 min. B) PMN (5x106/ml) were treated with 100 nM bafilomycin A1 (or DMSO) during 15 min at 37°C and then treated with different priming agents (15 µM R-848, 700 pM GM-CSF/1.5 nM TNF-{alpha}, 1 µg/ml LPS with 5% plasma) for 30 min at 37°C and finally stimulated with 300 nM fMLP (or treated with DMSO) for 5 min as indicated. Incubations were stopped by addition of 0.5 volume of cold (4°C) stop solution-1 containing the internal standards, and samples were processed for HPLC analysis as described in Materials and Methods. Data shown (mean±SD) are from 1 representative experiment out of 3, each performed in duplicate. *, significantly different values.

In contrast, 5-LO product biosynthesis elicited by the ionophore A23187 was not affected by bafilomycin A1 (up to 1 µM), clearly demonstrating that the inhibitory effect of the drug is not the consequence of an unspecific effect on the enzymes involved in LT biosynthesis. Moreover, as shown in Fig. 6B , treatment of PMN with bafilomycin A1 prior to priming with agents that do not act through an endosomal TLR was not affected by the drug, while in the same experiment, R-848/fMLP-stimulated LT biosynthesis was inhibited by nearly 90%.

R-848 up-regulates COX-2 at the mRNA and protein levels and promotes PGE2 generation
Alongside LT, PMN also generate thromboxane A2 (TXA2) and prostaglandin E2 (PGE2) via the type-2 cyclooxygenase (COX-2) pathway. Therefore, we investigated the effect of TLR8 activation on the COX-2 metabolic pathway. PMN were incubated with R-848 for different times and analyzed for type-1 cyclooxygenase (COX-1) and COX-2 mRNA levels by real-time PCR. Figure 7 A shows that COX-2 mRNA levels were increased by up to 50-fold in PMN stimulated for 1 h with 15 µM R-848, which exceeded the stimulatory effect of combined GM-CSF and TNF-{alpha}. In the same experiment, R-848 and GM-CSF/TNF-{alpha} showed a very small effect only on COX-1 mRNA levels. When PMN were incubated with bafilomycin A1 prior to stimulation with R-848, the increase of COX-2 mRNA level was almost completely inhibited, but bafilomycin A1 only slightly inhibited the effect of GM-CSF/TNF-{alpha}. In the next step, PMN were incubated with R-848 for increasing lengths of time and processed for determination of COX-2 protein expression by SDS-PAGE and immunoblotting. COX-2 protein could be detected at the earliest 30 min after treatment with R-848, with maximum expression detected at 60 min and up to 180 min (Fig. 7B ); expression of COX-2 was comparable to that observed with PMN pretreated with GM-CSF/TNF-{alpha}, and was efficiently inhibited by pretreatment of the PMN with bafilomycin A1 prior to stimulation with R-848. In contrast, bafilomycin A1 treatment only slightly inhibited COX-2 expression in GM-CSF/TNF-{alpha}-stimulated PMN. Measurement of PGE2 biosynthesis was then carried out to assess the functional impact of enhanced COX-2 expression by R-848. PMN were preincubated with R-848 for 1 h and stimulated with 5 µM AA for 15 min. Cell-free supernatants were then analyzed for PGE2 production by ELISA. Figure 7C shows a strong concentration-dependent stimulatory effect of R-848 on PGE2 generation. PGE2 generation by R-848 was inhibited by ~70% by bafilomycin A1, while this compound had no effect on PMN treated with GM-CSF/TNF-{alpha}. In contrast, when PMN were treated with the specific COX-2 inhibitor NS398, PGE2 production was inhibited in both R-848 and GM-CSF/TNF-{alpha} -treated cells, confirming the involvement of COX-2 in the observed PGE2 biosynthesis.


Figure 7
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  		Figure 7. R-848 activates the COX-2 metabolic pathway in PMN. A) Prewarmed (37°C) PMN were incubated with R-848 or the combination of 700 pM GM-CSF and 1.5 nM TNF-{alpha} (GM/TNF) at 37°C for the indicated times. Inset: PMN were incubated 20 min at 37°C with or without 100 nM bafilomycin A1 and then treated for 60 min with R-848 or GM/TNF. Samples were processed for the determination of COX-1 and COX-2 mRNA levels by real-time PCR, as described in Materials and Methods. Results are the mean (±SEM) of 3 experiments. *Significantly different from nonstimulated cells. **Significantly higher inhibition than in GM/TNF-stimulated cells. B) Prewarmed PMN were incubated with R-848 at 37°C or GM/TNF for the indicated times (top). PMN were incubated with the indicated concentrations of R-848 or with GM/TNF for 60 min at 37°C, in the absence or presence of 100 nM bafilomycin (bottom). Samples were processed for the determination of COX-2 protein levels in PMN by Western immunoblotting, as described under Materials and Methods. Results presented are from 1 experiment, representative of 3 identical experiments. C) Prewarmed (37°C) PMN were incubated with the indicated concentrations of R-848 or with GM/TNF for 60 min at 37°C, then stimulated with 5 µM AA for 15 min. Inset: PMN were preincubated 20 min with or without 100 nM bafilomycin A1 or the specific COX-2 inhibitor, NS398, and then for 60 min with 15 µM R-848 or GM/TNF prior stimulation with 5 µM AA for 15 min. Incubations were stopped and cell-free supernatants were analyzed for their content in PGE2 by ELISA, as described under Materials and Methods. Maximal PGE2 biosynthesis was 3.7 ng ± 3.3/5 x 106 PMN. Results are the mean ± SEM of 3 identical experiments. **Significantly higher inhibition from GM/TNF-stimulated cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present investigation, we sought to assess the putative links between TLR8 and the lipid mediator network. The results clearly demonstrated that lipid mediators are components of the TLR8 signaling pathway in human PMN. Indeed, the TLR7/8 ligand R-848 elicited the release of AA and 5-LO product biosynthesis. However, the most striking effect of R-848 on the 5-LO pathway appeared to be a priming effect on subsequent activation of PMN by stimuli such as fMLP, PAF, and A23187. R-848 enhanced by up to 15-fold 5-LO product biosynthesis in fMLP-stimulated PMN. Priming was rapid and transient, being maximum after 30 min and not anymore observed at 90 min and above. The reported low expression of TLR7 in human PMN (2) and complete lack of effect of the selective TLR7 ligand imiquimod, support that the effects of the TLR7/8 ligand resiquimod reported herein are the results of TLR8 rather than TLR7 signaling. As further support to the involvement of TLR8 in the effect of R-848, the vacuolar membrane proton pump (ATPase) inhibitor bafilomycin A1, which inhibits endosomal acidification, a required condition for efficient endosomal TLR signaling (4) , inhibited R-848-induced responses, but not similar responses elicited by other agents (LPS, GM-CSF, and TNF-{alpha}), which do not act through endosomal TLR. It seems important to point out that while the experiments described above were carried out with PMN treated with CB to enhance PMN responsiveness to ligands (31) and 5-LO product biosynthesis (32) , the same experiments were also performed with PMN not exposed to CB. Both the kinetics and the concentration response curve to R-848 were qualitatively identical to those shown in Fig. 3A and 3B , however, with a 10- to 25-fold lower level of 5-LO product biosynthesis (data not shown). Thus, CB pretreatment of PMN was used throughout these studies to facilitate 5-LO product quantitation by RP-HPLC. The mechanism by which CB enhances 5-LO product biosynthesis in PMN is still incompletely understood. Cytochalasins are inhibitors of actin polymerization (33) and therefore hamper the formation of actin microfilaments. Cytoskeleton remodeling is essential to leukocyte function (34) , and proteins of the AA cascade, such as the cPLA2 and the 5-LO, have been shown to interact with cytoskeletal components (35 36 37 38 39) . It is therefore possible that alteration of the dynamics of the actin skeleton by cluster of differentiation (CD) in activated PMN has an impact on 5-LO product biosynthesis. Interestingly, it was recently shown that inhibitors of actin polymerization promote LT biosynthesis in PMN by strongly increasing AA release through enhanced Ca2+ mobilization involving Src kinase/PLC signaling (40) ; it was proposed that actin polymerization may represent a down-regulatory process of Ca2+-dependent PMN functions (such as 5-LO product biosynthesis).

The effects of R-848 reported herein are reminiscent of similar priming effects of GM-CSF, TNF-{alpha}, and LPS, which alone do not elicit (or to a very small extent only) 5-LO product formation in human PMN but strongly enhance responses to PMN ligands (41 42 43) . When the priming effects of R-848 and other agents were compared within the same experiments and using optimal conditions (as determined in previous studies), R-848 was found to be more potent than LPS or the combination of GM-CSF and TNF-{alpha}. Interestingly, combinations of R-848 and GM-CSF/TNF-{alpha} or R-848 and LPS did not result in significant additive effects, suggesting the involvement of the same priming mechanism(s). These experiments demonstrate that TLR8 activation in human PMN represents an efficient route for the potentiation of LTB4 biosynthesis at sites of infection/inflammation.

Significant insights on the priming mechanism were obtained through investigation of the effects of R-848 on AA release (a limiting step in LT biosynthesis in PMN) PAF biosynthesis and 5-LO translocation and cPLA2 phosphorylation, two critical processes in the regulation of these key enzymes in LT biosynthesis (30 , 44) . We observed that R-848 promoted AA release and LT and PAF biosynthesis in response to fMLP and that the cPLA2{alpha} inhibitor pyrrophenone blocked all of these effects of R-848, indicating a key role of cPLA2{alpha} in LT and PAF biosynthesis in PMN exposed to the TLR7/8 agonist. Accordingly, immunoblot analysis demonstrated that R-848 did cause the phosphorylation of cPLA2{alpha} (on Ser-505, as shown by a decreased electrophoretic mobility of the protein (30) . Moreover, immunoblot analysis of 5-LO in the PMN membrane fraction revealed a striking increase of fMLP-induced 5-LO translocation by R-848. This enhanced 5-LO translocation by R-848 was strongly inhibited by pyrrophenone, indicating that this effect of R-848 was related to the enhanced AA release, in accord with our recent observation that AA regulates 5-LO translocation in human PMN (45) . These data also revealed some similarities with the mechanism of priming by LPS (a TLR4 ligand) and other agents. Indeed, cPLA2{alpha} phosphorylation by MAP kinases and enhanced AA release are common features of the effects of LPS and cytokines on 5-LO product biosynthesis (41 42 43) , and it has been suggested that cPLA2 phosphorylation by GM-CSF prior to stimulation with the second stimulus is an important event in the priming effect of the growth factor (41) . We have also shown that LPS priming of PMN strongly enhances fMLP-induced 5-LO translocation. Interestingly, a recent study revealed that cPLA2{alpha} activation and AA release by LPS in the mouse macrophage cell line RAW264.7 is mediated by TLR4 and its adaptor protein (MyD88 and TRIF)-dependent MAP kinase (p38 and ERK) pathways (10) . Because TLR8 signaling also involves MyD88, it seems likely that p38 and ERK 1/2 MAP kinases may be implicated in the phosphorylation of cPLA2{alpha} by R-848. Additional studies are needed to define the signaling events in TLR8-mediated priming for LT and PAF biosynthesis in leukocytes.

Besides LT and PAF, PMN exposed to inflammatory stimuli also generate PGE2 (and TXA2) through the inducible form of cyclooxygenase (COX-2) (46) . PGE2 is a lipid mediator of inflammation with a complex profile of action. It is a potent vasodilator and mediates hyperalgesia and the pyretic effects of endotoxins and IL-1. On the other hand, PGE2, acting through EP2 receptors positively coupled to the adenylate cyclase efficiently suppresses functional responses in phagocytes and lymphocytes, thus acting as an endogenous antiinflammatory agent and immunosupressor (47 , 48) . In the present study, we found that R-848 was a potent stimulus of COX-2 (but not COX-1) mRNA and protein expression, strongly enhancing the ability of PMN to produce PGE2 from exogenous AA. This effect of R-848 on COX-2 expression is not unique to TLR8 activation and has been reported following TLR2, TLR4, and TLR9 signaling (through NF{kappa}B) in various cell types (49 50 51 52 53) . The observation that TLR8 signaling impacts on AA pathways leading to the formation of both pro- and antiinflammatory lipid mediators is intriguing. Given the dual nature of PGE2, it is distinctly possible that its enhanced formation by cells exposed to TLR8 ligands serves the purpose of either promoting the inflammatory response or on the contrary, supporting its resolution. The time course of release of various mediators is likely critical to the evolution and resolution of inflammation. Our data might suggest that the early release of LT (and PAF) following TLR8 activation support the development of the immediate innate immune response to viral infection, while the enhanced capacity for PGE2 synthesis at a later time may be part of the down-regulatory mechanisms leading ultimately to resolution of inflammation.

In conclusion, we have shown herein that activation of TLR8 has a strong impact on the ability of human PMN to produce three different classes of inflammatory mediators, namely LT, PAF, and PG. Although they were not specifically investigated in the present study, it is likely that lipoxin and TX formation is also up-regulated by TLR8 ligands, since their biosynthesis implicates the 5-LO and COX-2 pathways, respectively (46 , 54) . Our data emphasize that TLR8-dependent stimulation of functional responses in immune cells likely implicates these lipid mediators, in addition to inflammatory cytokines. While some studies have already established that TLR7 and TLR8 recognize ssRNA from viruses such as vesicular stomatitis virus, influenza virus, and HIV, further studies are needed to assess the importance of TLR8 in host defense against these and other ssRNA viruses and the role of lipid mediators in TLR8-induced immune processes.


   ACKNOWLEDGMENTS
 
This work was supported by the German Academy of Scientists Leopoldina with resources from the Bundesministerium for Education and Research (BMBF-LPD 9901/8–133) and by the Canadian Institutes for Health Research (CIHR).

Received for publication October 2, 2006. Accepted for publication December 14, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Takeda, K., Akira, S. (2005) Toll-like receptors in innate immunity. Int. Immunol. 17,1-14[Abstract/Free Full Text]
  2. Hayashi, F., Means, T. K., Luster, A. D. (2003) Toll-like receptors stimulate human neutrophil function. Blood 102,2660-2669[Abstract/Free Full Text]
  3. Diebold, S. S., Kaisho, T., Hemmi, H., Akira, S., Reis e Sousa, C. (2004) Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303,1529-1531[Abstract/Free Full Text]
  4. Lund, J. M., Alexopoulou, L., Sato, A., Karow, M., Adams, N. C., Gale, N. W., Iwasaki, A., Flavell, R. A. (2004) Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc. Natl. Acad. Sci. U. S. A. 101,5598-5603[Abstract/Free Full Text]
  5. Heil, F., Hemmi, H., Hochrein, H., Ampenberger, F., Kirschning, C., Akira, S., Lipford, G., Wagner, H., Bauer, S. (2004) Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303,1526-1529[Abstract/Free Full Text]
  6. Schlaepfer, E., Audige, A., Joller, H., Speck, R. F. (2006) TLR7/8 triggering exerts opposing effects in acute versus latent HIV infection. J. Immunol. 176,2888-2895[Abstract/Free Full Text]
  7. Jurk, M., Heil, F., Vollmer, J., Schetter, C., Krieg, A. M., Wagner, H., Lipford, G., Bauer, S. (2002) Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat. Immunol. 3,499[CrossRef][Medline]
  8. Dockrell, D. H., Kinghorn, G. R. (2001) Imiquimod and resiquimod as novel immunomodulators. J. Antimicrob. Chemother. 48,751-755[Abstract/Free Full Text]
  9. McCurdy, J. D., Olynych, T. J., Maher, L. H., Marshall, J. S. (2003) Cutting edge: distinct Toll-like receptor 2 activators selectively induce different classes of mediator production from human mast cells. J. Immunol. 170,1625-1629[Abstract/Free Full Text]
  10. Qi, H. Y., Shelhamer, J. H. (2005) Toll-like receptor 4 signaling regulates cytosolic phospholipase A2 activation and lipid generation in lipopolysaccharide-stimulated macrophages. J. Biol. Chem. 280,38969-38975[Abstract/Free Full Text]
  11. Chilton, F. H., Ellis, J. M., Olson, S. C., Wykle, R. L. (1984) 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine. A common source of platelet-activating factor and arachidonate in human polymorphonuclear leukocytes. J. Biol. Chem. 259,12014-12019[Abstract/Free Full Text]
  12. Rubin, B. B., Downey, G. P., Koh, A., Degousee, N., Ghomashchi, F., Nallan, L., Stefanski, E., Harkin, D. W., Sun, C., Smart, B. P., et al (2005) Cytosolic phospholipase A2-alpha is necessary for platelet-activating factor biosynthesis, efficient neutrophil-mediated bacterial killing, and the innate immune response to pulmonary infection: cPLA2-alpha does not regulate neutrophil NADPH oxidase activity. J. Biol. Chem. 280,7519-7529[Abstract/Free Full Text]
  13. Ford-Hutchinson, A. W., Bray, M. A., Doig, M. V., Shipley, M. E., Smith, M. J. (1980) Leukotriene B, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 286,264-265[CrossRef][Medline]
  14. Palmblad, J., Malmsten, C. L., Uden, A. M., Radmark, O., Engstedt, L., Samuelsson, B. (1981) Leukotriene B4 is a potent and stereospecific stimulator of neutrophil chemotaxis and adherence. Blood 58,658-661[Abstract/Free Full Text]
  15. Gosselin, J., Borgeat, P., Flamand, L. (2005) Leukotriene B4 protects latently infected mice against murine cytomegalovirus reactivation following allogeneic transplantation. J. Immunol. 174,1587-1593[Abstract/Free Full Text]
  16. Flamand, L., Borgeat, P., Lalonde, R., Gosselin, J. (2004) Release of anti-HIV mediators after administration of leukotriene B4 to humans. J. Infect. Dis. 189,2001-2009[CrossRef][Medline]
  17. Behera, A. K., Kumar, M., Matsuse, H., Lockey, R. F., Mohapatra, S. S. (1998) Respiratory syncytial virus induces the expression of 5-lipoxygenase and endothelin-1 in bronchial epithelial cells. Biochem. Biophys. Res. Commun. 251,704-709[CrossRef][Medline]
  18. Gosselin, J., Borgeat, P. (1997) Epstein-Barr virus modulates 5-lipoxygenase product synthesis in human peripheral blood mononuclear cells. Blood 89,2122-2130[Abstract/Free Full Text]
  19. Nokta, M. A., Hassan, M. I., Loesch, K. A., Pollard, R. B. (1995) HIV-induced TNF-alpha regulates arachidonic acid and PGE2 release from HIV-infected mononuclear phagocytes. Virology 208,590-600[CrossRef][Medline]
  20. Boyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand. J. Clin. Lab. Invest. Suppl. 97,77-89[Medline]
  21. Krump, E., Picard, S., Mancini, J., Borgeat, P. (1997) Suppression of leukotriene b-4 biosynthesis by endogenous adenosine in ligand-activated human neutrophils. J. Exp. Med. 186,1401-1406[Abstract/Free Full Text]
  22. Borgeat, P., Picard, S., Vallerand, P., Bourgoin, S., Odeimat, A., Sirois, P., Poubelle, P. E. (1990) Automated on-line extraction and profiling of lipoxygenase products of arachidonic acid by high-performance liquid chromatography. Methods Enzymol. 187,98-116[Medline]
  23. Surette, M. E., Palmantier, R., Gosselin, J., Borgeat, P. (1993) Lipopolysaccharides prime whole human blood and isolated neutrophils for the increased synthesis of 5-lipoxygenase products by enhancing arachidonic acid availability: involvement of the CD14 antigen. J. Exp. Med. 178,1347-1355[Abstract/Free Full Text]
  24. Harrison, K. A., Clay, K. L., Murphy, R. C. (1999) Negative ion electrospray and tandem mass spectrometric analysis of platelet activating factor (PAF) (1-hexadecyl-2-acetyl-glycerophosphocholine). J. Mass. Spectrom. 34,330-335[CrossRef][Medline]
  25. Pouliot, M., Mcdonald, P. P., Krump, E., Mancini, J. A., Mccoll, S. R., Weech, P. K., Borgeat, P. (1996) Colocalization of cytosolic phospholipase a(2), 5-lipoxygenase, and 5-lipoxygenase-activating protein at the nuclear membrane of a23187-stimulated human neutrophils. Eur. J. Biochem. 238,250-258[Medline]
  26. Cadieux, J. S., Leclerc, P., St-Onge, M., Dussault, A. A., Laflamme, C., Picard, S., Ledent, C., Borgeat, P., Pouliot, M. (2005) Potentiation of neutrophil cyclooxygenase-2 by adenosine: an early anti-inflammatory signal. J. Cell Sci. 118,1437-1447[Abstract/Free Full Text]
  27. Nagase, H., Okugawa, S., Ota, Y., Yamaguchi, M., Tomizawa, H., Matsushima, K., Ohta, K., Yamamoto, K., Hirai, K. (2003) Expression and function of Toll-like receptors in eosinophils: activation by Toll-like receptor 7 ligand. J. Immunol. 171,3977-3982[Abstract/Free Full Text]
  28. Radsak, M. P., Salih, H. R., Rammensee, H. G., Schild, H. (2004) Triggering receptor expressed on myeloid cells-1 in neutrophil inflammatory responses: differential regulation of activation and survival. J. Immunol. 172,4956-4963[Abstract/Free Full Text]
  29. Gorden, K. B., Gorski, K. S., Gibson, S. J., Kedl, R. M., Kieper, W. C., Qiu, X., Tomai, M. A., Alkan, S. S., Vasilakos, J. P. (2005) Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8. J. Immunol. 174,1259-1268[Abstract/Free Full Text]
  30. Lin, L. L., Wartmann, M., Lin, A. Y., Knopf, J. L., Seth, A., Davis, R. J. (1993) cPLA2 is phosphorylated and activated by MAP kinase. Cell 72,269-278[CrossRef][Medline]
  31. Bengtsson, T., Dahlgren, C., Stendahl, O., Andersson, T. (1991) Actin assembly and regulation of neutrophil function: effects of cytochalasin B and tetracaine on chemotactic peptide-induced O2- production and degranulation. J. Leukoc. Biol. 49,236-244[Abstract]
  32. Salari, H., Braquet, P., Naccache, P., Borgeat, P. (1985) Characterization of effect of N-formyl-methionyl-leucyl-phenylalanine on leukotriene synthesis in human polymorphonuclear leukocytes. Inflammation 9,127-138[CrossRef][Medline]
  33. Cooper, J. A. (1987) Effects of cytochalasin and phalloidin on actin. J. Cell Biol. 105,1473-1478[Free Full Text]
  34. Fenteany, G., Glogauer, M. (2004) Cytoskeletal remodeling in leukocyte function. Curr. Opin. Hematol. 11,15-24[CrossRef][Medline]
  35. Nakatani, Y., Tanioka, T., Sunaga, S., Murakami, M., Kudo, I. (2000) Identification of a cellular protein that functionally interacts with the C2 domain of cytosolic phospholipase A(2)alpha. J. Biol. Chem. 275,1161-1168[Abstract/Free Full Text]
  36. Rakonjac, M., Fischer, L., Provost, P., Werz, O., Steinhilber, D., Samuelsson, B., Radmark, O. (2006) Coactosin-like protein supports 5-lipoxygenase enzyme activity and up-regulates leukotriene A4 production. Proc. Natl. Acad. Sci. U. S. A. 103,13150-13155[Abstract/Free Full Text]
  37. Lepley, R. A., Fitzpatrick, F. A. (1994) 5-Lipoxygenase contains a functional Src homology 3-binding motif that interacts with the Src homology 3 domain of Grb2 and cytoskeletal proteins. J. Biol. Chem. 269,24163-24168[Abstract/Free Full Text]
  38. Provost, P., Doucet, J., Hammarberg, T., Gerisch, G., Samuelsson, B., Radmark, O. (2001) 5-Lipoxygenase interacts with coactosin-like protein. J. Biol. Chem. 276,16520-16527[Abstract/Free Full Text]
  39. Provost, P., Samuelsson, B., Radmark, O. (1999) Interaction of 5-lipoxygenase with cellular proteins. Proc. Natl. Acad. Sci. U. S. A. 96,1881-1885[Abstract/Free Full Text]
  40. Fischer, L., Poeckel, D., Buerkert, E., Steinhilber, D., Werz, O. (2005) Inhibitors of actin polymerisation stimulate arachidonic acid release and 5-lipoxygenase activation by upregulation of Ca2+ mobilisation in polymorphonuclear leukocytes involving Src family kinases. Biochim. Biophys. Acta 1736,109-119[Medline]
  41. DiPersio, J. F., Billing, P., Williams, R., Gasson, J. C. (1988) Human granulocyte-macrophage colony-stimulating factor and other cytokines prime human neutrophils for enhanced arachidonic acid release and leukotriene B4 synthesis. J. Immunol. 140,4315-4322[Abstract]
  42. Durstin, M., Durstin, S., Molski, T. F. P., Becker, E. L., Shaafi, R. I. (1994) Cytoplasmic phospholipase-a(2) translocates to membrane fraction in human neutrophils activated by stimuli that phosphorylate mitogen-activated protein kinase. Proc. Natl. Acad. Sci. U. S. A. 91,3142-3146[Abstract/Free Full Text]
  43. Surette, M. E., Dallaire, N., Jean, N., Picard, S., Borgeat, P. (1998) Mechanisms of the priming effect of lipopolysaccharides on the biosynthesis of leukotriene B4 in chemotactic peptide-stimulated human neutrophils. FASEB J. 12,1521-1531[Abstract/Free Full Text]
  44. Radmark, O., Samuelsson, B. (2005) Regulation of 5-lipoxygenase enzyme activity. Biochem. Biophys. Res. Commun. 338,102-110[CrossRef][Medline]
  45. Flamand, N., Lefebvre, J., Surette, M. E., Picard, S., Borgeat, P. (2006) Arachidonic acid regulates the translocation of 5-lipoxygenase to the nuclear membranes in human neutrophils. J. Biol. Chem. 281,129-136[Abstract/Free Full Text]
  46. Pouliot, M., Gilbert, C., Borgeat, P., Poubelle, P. E., Bourgoin, S., Creminon, C., Maclouf, J., McColl, S. R., Naccache, P. H. (1998) Expression and activity of prostaglandin endoperoxide synthase-2 in agonist-activated human neutrophils. FASEB J. 12,1109-1123[Abstract/Free Full Text]
  47. Christman, B. W., Christman, J. W., Dworski, R., Blair, I. A., Prakash, C. (1993) Prostaglandin-e(2) limits arachidonic acid availability and inhibits leukotriene-b(4) synthesis in rat alveolar macrophages by a nonphospholipase-a(2) mechanism. J. Immunol. 151,2096-2104[Abstract]
  48. Nataraj, C., Thomas, D. W., Tilley, S. L., Nguyen, M. T., Mannon, R., Koller, B. H., Coffman, T. M. (2001) Receptors for prostaglandin E(2) that regulate cellular immune responses in the mouse. J. Clin. Invest. 108,1229-1235[CrossRef][Medline]
  49. Ghosh, D. K., Misukonis, M. A., Reich, C., Pisetsky, D. S., Weinberg, J. B. (2001) Host response to infection: the role of CpG DNA in induction of cyclooxygenase 2 and nitric oxide synthase 2 in murine macrophages. Infect. Immun. 69,7703-7710[Abstract/Free Full Text]
  50. Chen, Y., Zhang, J., Moore, S. A., Ballas, Z. K., Portanova, J. P., Krieg, A. M., Berg, D. J. (2001) CpG DNA induces cyclooxygenase-2 expression and prostaglandin production. Int. Immunol. 13,1013-1020[Abstract/Free Full Text]
  51. Rhee, S. H., Hwang, D. (2000) Murine TOLL-like receptor 4 confers lipopolysaccharide responsiveness as determined by activation of NF kappa B and expression of the inducible cyclooxygenase. J. Biol. Chem. 275,34035-34040[Abstract/Free Full Text]
  52. Chang, Y. J., Wu, M. S., Lin, J. T., Chen, C. C. (2005) Helicobacter pylori-induced invasion and angiogenesis of gastric cells is mediated by cyclooxygenase-2 induction through TLR2/TLR9 and promoter regulation. J. Immunol. 175,8242-8252[Abstract/Free Full Text]
  53. Suram, S., Brown, G. D., Ghosh, M., Gordon, S., Loper, R., Taylor, P. R., Akira, S., Uematsu, S., Williams, D. L., Leslie, C. C. (2006) Regulation of cytosolic phospholipase A2 activation and cyclooxygenase 2 expression in macrophages by the beta-glucan receptor. J. Biol. Chem. 281,5506-5514[Abstract/Free Full Text]
  54. Serhan, C. N., Levy, B. (2003) Novel pathways and endogenous mediators in anti-inflammation and resolution. Chem. Immunol. Allergy 83,115-145[Medline]




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