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Originally published online as doi:10.1189/jlb.0706451 on January 30, 2007

Published online before print January 30, 2007
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(Journal of Leukocyte Biology. 2007;81:925-933.)
© 2007 by Society for Leukocyte Biology

Peptidoglycan and mannose-based molecular patterns trigger the arachidonic acid cascade in human polymorphonuclear leukocytes

Isela Valera*, Ana González Vigo*, Sara Alonso*, Luz Barbolla{dagger}, Mariano Sánchez Crespo*,1 and Nieves Fernández*

* Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas, Valladolid, Spain; and
{dagger} Centro de Hemoterapia y Hemodonación de Castilla y León, Valladolid, Spain

1 Correspondence: Instituto de Biología y Genética Molecular, C/ Sanz y Forés s/n, 47003, Valladolid, Spain. E-mail: mscres{at}ibgm.uva.es

ABSTRACT

The release of arachidonic acid (AA) in response to microorganism-derived products acting on pattern recognition receptors (PRR) was assayed in human polymorphonuclear leukocytes (PMN). Peptidoglycan (PGN) and mannan were found to be strong inducers of AA metabolism, as they produced the release of AA at a similar extent to that produced by agonists of pathophysiological relevance such as complement-coated zymosan particles and IgG immune complexes. In sharp contrast, lipoteichoic acid, LPS, muramyldipeptide, and the bacterial lipoprotein mimic palmitoyl-3-cysteine-serine-lysine-4 failed to do so. Leukotriene B4 and PGE2 were synthesized in response to mannan and PGN, thus suggesting that the lipoxygenase and the cyclooxygenase routes are operative in human PMN in response to pathogen-associated molecular patterns (PAMP). Analysis of the lipid extracts of supernatants and cell pellets as well as pharmacological studies with the calpain inhibitor calpeptin and the cytosolic phospholipase A2 (PLA2) inhibitor pyrrolidine-1 showed the dependence of AA release on cytosolic PLA2-catalyzed reactions. The effect of PGN was not inhibited by previous treatment with anti-TLR2 mAb, thus suggesting a nonarchetypal involvement of the TLR2 signaling route and/or participation of other receptors. Because of the abundance of mannose-based and PGN-containing PAMP in fungi and bacteria and the wide array of PRR in human PMN, these finding disclose a role of prime importance for PAMP and PRR in AA metabolism in the inflammatory response mediated by PMN.

Key Words: phagocytosis • phospholipase • Toll-like receptors

INTRODUCTION

Polymorphonuclear leukocytes (PMN) are a central element of the innate immune system, as they are the first blood cells able to migrate into tissues following microbial invasion. PMN respond to a large list of stimuli, including inflammatory mediators and microbial products. This latter group of stimuli is most relevant, as microorganisms have unique pattern molecules, termed pathogen-associated molecular patterns (PAMP), which are recognized through pattern-recognition receptors (PRR) by the host innate immune system. The TLR family (for review, see refs. [1 , 2 ]) and nucleotide-binding oligomerization domain (NOD) family proteins (for review, see refs. [3 , 4 ]) are representative of what Janeway first called PRR [5 ]. In addition to the aforementioned families, lectin C-type receptors and the mannose receptor (MR) family represent other PRR able to interact with structural signatures expressed in microorganisms. The study of the response of PMN to PAMP via the TLR system has focused mainly on the induction of cytokines, the activation of oxidant production, and the release of granular components [6 7 8 ], but few data are available about the activation of the arachidonic acid (AA) cascade, neither in this context nor in response to the activation of the MR. Addressing this issue seems of relevance, as eicosanoids play an important role in connecting innate and adaptive immunity [9 , 10 ] and are pharmacological targets for human disease.

We have addressed the effect of a set of PAMP signatures on the release of AA and the production of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) in human PMN. Our findings have disclosed the occurrence of an active metabolism of AA in human PMN stimulated with mannan and peptidoglycan (PGN), which is comparable with the response elicited by other pathophysiologically relevant stimuli. In contrast, stimuli mimicking other bacterial PAMP, i.e., lipoteichoic acid, LPS, muramyldipeptide (MDP), and a bacterial lipoprotein analog, did not induce AA release. Taken collectively, these data indicate the existence in PMN of a set of responses to mannose- and PGN-containing PAMP similar to that already depicted for ß-glucan-containing particles in some cell types. This can be traced to the activation of the MR and to a more complex route, which might involve cooperation of TLR2 with other receptors, for instance, NOD2, which is involved in the recognition of MDP [11 ], and PGN recognizing proteins (PGRP), which interact with PGN [12 ].

MATERIALS AND METHODS

Reagents
Zymosan particles, laminarin from Laminaria digitata, mannan from Saccharomyces cerevisiae, MDP [N-acetylmuramyl (MurNAc),-L-alanyl-D-isoglutamine], Staphylococcus aureus PGN, Bacillus subtilis PGN, lipoteichoic acid, Escherichia coli LPS, lysostaphin, mutanolysin, porcin mucin-3, FITC-conjugated, mannosylated BSA (1.5–3 mol FITC per mol BSA), and FITC-conjugated BSA (7–12 mol FITC per mol albumin) were from Sigma Chemical Co. (St. Louis, MO, USA). The synthetic palmitoylated mimic of bacterial lipopeptides palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4) was from InvivoGen (San Diego, CA, USA). Antihuman TLR2 mAb (#16-9922) was from eBioscience (San Diego, CA, USA). Antihuman CD206/MR IgG1 mAb was from BD PharMingen (San Diego, CA, USA). Pyrrolidine-1 was from Calbiochem (San Diego, CA, USA). Enzymatic digestion of PGN with lysostaphin and mutanolysin was conducted at 37°C for 18 h under continuous stirring in potassium phosphate buffer according to protocols reported previously [13 , 14 ].

Cell culture, metabolic labeling, and assay of [3H]AA release
Human PMN were isolated from peripheral blood of healthy volunteer donors by centrifugation into Ficoll cushions and sedimentation in Dextran T500. Radioactive labeling with [3H]AA was performed by incubation for 2 h in the presence of 0.25 µCi [3H]AA in 0.25% essentially fatty acid-free BSA. After labeling, cells were washed and allowed to equilibrate at 37°C in medium containing 1% BSA before the addition of stimuli or vehicle solutions. The release of [3H]AA into the culture medium was measured by scintillation counting.

TLC analysis of [3H]AA-containing lipids
PMN and supernatants were subjected separately to extraction in chloroform/methanol (1:2, v/v), according to the Bligh and Dyer procedure. The lipids extracted into the chloroform layer were dried under N2 stream and developed by TLC on silica gel plates in the system n-hexane/diethyl ether/acetic acid (70:30:1, v/v). The radioactivity distributed in the different lipid fractions was quantitated using K/tritium imaging screens and a Molecular ImagerTM FX apparatus from BioRad Laboratories (Hercules, CA, USA).

Enzyme immunoassays for PGE2 and LTB4
Quantitation of PGE2 and LTB4 levels into cell culture supernatants was determined by using enzyme immunoassay kits (Amersham Biosciences, Little Chalfont, UK) according to the manufacturer’s instructions. These assays are based on competition between unlabeled PGE2 and LTB4 in the samples and a fixed amount of labeled antigen for specific antibody. The detection limit of these assays is 20 pg/ml for PGE2 and 6 pg/ml for LTB4.

RT-PCR assays for PGRP-S, dectin-1, Endo-180, TLR1, TLR2, TLR6, NOD1, NOD2, ß-actin, MR, and secreted phospholipase A2 (sPLA2) M-type receptor
Total cellular RNA was extracted by the TRIzol method (Life Technologies, Grand Island, NY, USA). The conditions for cDNA first-strand synthesis and PCR reactions were described previously [15 ]. PCR products were identified by automatic sequencing of the DNA eluted from the agarose gel by excision of the band under UV light, followed by purification using a QIAquick PCR purification kit (Qiagen Inc., Valencia, CA, USA). The characteristics of the oligonucleotide primers used for PCR reactions are shown in Table 1 . To address more exactly the expression of MR mRNA, real-time RT-PCR was carried out in RNA samples treated with DNase (Turbo-DNA freeTM, Ambion, Austin, TX, USA). The resulting cDNA was amplified in a PTC-200 apparatus equipped with a Chromo4 detector (BioRad Laboratories) using SYBR Green I mix containing HotStart polymerase (ABgene, UK). ß-actin was used as a housekeeping gene to assess the relative abundance of cyclooxygenase-2 (COX-2) mRNA, using the comparative cycle threshold (CT) method for relative expression. This method allows the relative expression for a given cDNA using the formula: 2–{Delta}CT, where {Delta}CT = {Delta}CTCOX-2 – {Delta}CTß-actin [25 ]. Therefore, 1 arbitrary unit (AU) corresponds to the expression of ß-actin.


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  		Table 1. Oligonucleotide Primers Used for the Detection of the mRNA Encoding for Receptors of the Innate Immune System

 
Flow cytometry
To assess the surface display of the MR, 5 x 106 PMN were incubated with 10 µg/ml antihuman CD206/MR IgG1 mAb. After washing with PBS, goat antimouse IgG-FITC conjugate was added and incubated for 30 min at 4°C. Subsequently, cells were washed and analyzed by immunofluorescence flow cytometry in a FACScan cytofluorometer (Becton Dickinson, San Jose, CA, USA). Positive cells were estimated using P3 x 63 myeloma supernatant as a negative control. To assay the endo/phagocytosis of FITC-conjugated mannosylated BSA, PMN were incubated at 37°C with FITC-conjugated mannosylated BSA and subsequently washed and resuspended in 500 µl PBS, supplemented with 1 mM EDTA for analysis by flow cytometry. Parallel controls were performed at 4°C to block endocytic uptake of the particles. The samples were analyzed using FACScan and CellQuest program (Becton Dickinson).

RESULTS

Human PMN express receptors for several PAMP
PMN display receptors of the TLR family, intracellular receptors of the NOD/Caterpiller family [11 ], and PGRP-S [12 , 26 ]. However, our knowledge about the full scope of signaling events coupled with the binding of ligands to these receptors is yet incomplete, and the expression of the MR family in this cell type has not been assessed, although two members of the MR family, the MR itself [15 , 27 , 28 ] and the M-type-secreted PLA2 receptor, can elicit inflammatory responses [29 ]. As shown in Figure 1A and 1B , human PMN show a high expression level of PGRP-S, the C-lectin-type dectin-1 displaying two predominant transcripts, TLR1, TLR2, and NOD2 mRNA. In contrast, they do not express Endo180, the type M-secreted PLA2 receptor, or NOD1 mRNA (data not shown), whereas a faint but significant expression of the mRNA encoding the MR could be detected. This was confirmed by real-time RT-PCR, which showed a value of 0.033 AU for the expression of MR mRNA, as compared with 1 AU for ß-actin mRNA. In experiments directed to assess the surface display of the MR (Fig. 1C) , PMN were found to express MR in resting conditions and after treatment with soluble mannan, thus indicating the constitutive expression of the MR and the absence of significant changes of its surface display upon challenge with its cognate ligand, what could suggest an efficient recycling of the MR to the cell surface. Endocytosis via the MR was assessed by incubation of PMN with FITC-conjugated mannosylated BSA at 37°C. Under these conditions, the fluorescence associated to the PMN increased significantly above the values detected in cells incubated at 4°C. Moreover, the uptake of FITC-conjugated BSA was lower than that observed with FITC-conjugated mannosylated BSA (Fig. 1D) , albeit the preparation of FITC-conjugated BSA was labeled with 7–12 mol FITC per mol BSA, as compared with 1.5–3 mol FITC per mol mannosylated BSA. Taken together, these findings suggest the occurrence of endocytic uptake of FITC-conjugated mannosylated BSA by a route most likely involving the MR.


Figure 1
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  		Figure 1. Expression of PRR in human PMN. Total RNA from human PMN was used for RT-PCR reactions with oligonucleotide primers designed from the encoding sequences of the receptors. RNA from other cell types was used for comparison. The left lanes show the position of DNA size markers obtained from digestion of phage {Phi}X174 with HaeIII (A and B). Resting PMN and PMN incubated with 10 mg/ml mannan for 1 h were used to assess the surface display of CD206/MR. Isotype-matched antibody was used as control (shown by the gray tracing on the left). These are representative recordings of three identical experiments (C). PMN were incubated with 200 µg/ml FITC-conjugated mannosylated BSA at 4°C and 37°C, and after 2 h, cells were used to assay particle uptake by fluorescence cytometry. The lowest panel shows an experiment carried out at 37°C, in which the uptake of FITC-conjugated mannosylated BSA is compared with the uptake of FITC-conjugated BSA. These data are typical recordings of three independent experiments (D).

 
PAMP signatures are strong inducers of [3H]AA release by human PMN
PGN from S. aureus and B. subtilis induced a robust release of [3H]AA (Fig. 2A ), which was comparable with that produced by the physiologically relevant stimulus C3bi-coated zymosan (Fig. 2B) . Mannan and mucin-3, a glycoprotein from the gastrointestinal tract, which is an endogenous ligand of the MR, also induced a significant release of [3H]AA at concentrations higher than 5 mg/ml, i.e., a dose dependence similar to that observed in response to the ß-glucan laminarin, a specific ligand of dectin-1 (Fig. 2B) . In sharp contrast, lipoteichoic acid and Pam3CSK4 did not elicit significant release, even at high concentrations (not shown). To determine if [3H]AA release could be the result of LPS contamination, we made use of the LPS-binding antibiotic polymyxin B. [3H]AA release produced by mannan and PGN was not reduced in the presence of 200 µg/ml polymyxin B. Moreover, incubation with PGN and LPS did not change the extent of [3H]AA release induced by PGN alone, thus ruling out contamination by LPS as well as a significant priming effect of LPS on PGN effect (not shown). [3H]AA release increased linearly up to 1 h (Fig. 3A ) and resembled the temporal pattern described for other stimuli of pathophysiological relevance [30 ].


Figure 2
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  		Figure 2. Effect of different stimuli on [3H]AA release. PMN, labeled with [3H]AA, were incubated for 1 h in the presence of different additions. The [3H]AA radioactivity released into the medium at the end of this period was assayed by scintillation counting. [3H]AA release is expressed as percentage of total [3H]AA incorporated. The PGN fraction of <30 kDa was obtained by ultrafiltration of S. aureus PGN solution with Ultracell Amicon® YM30 ultrafiltration discs (cut-off 30 kDa). Data represent mean ± SEM of four independent experiments with duplicate samples.

 

Figure 3
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  		Figure 3. Release of [3H]AA from human PMN: time-course. PMN were labeled with [3H]AA, and at the times indicated, the [3H]AA radioactivity released into the medium was assayed (A). The effect of different concentrations of S. aureus PGN, MDP, and a combination thereof on [3H]AA release is shown (B). The effect of the treatment with lysostaphin and mutanolysin on the [3H]AA-releasing activity of PGN is shown in the indicated columns. Data represent mean ± SEM of four experiments with duplicate samples.

 
The assignment of the biological effect of PGN to definite PRR has been a matter of close scrutiny [31 , 32 ], as TLR, PGRP, and NOD receptors have been involved, and at least PGN, MDP, and D-glutamic acid-meso diaminopimelic acid have been considered the moieties conveying the biological properties. The first approach to address this issue was to associate the [3H]AA-releasing activity with the fractions, where the compounds with the Mr of MDP and PGN are eluted [33 ]. Molecular weight fractionation of S. aureus PGN showed the association of [3H]AA-releasing activity with fractions of molecular weight >30 kDa, as judged from the retention of the activity after ultrafiltration of the samples in Ultracell Amicon® YM30 discs (Fig. 2A) , whereas no activity was detected in the <30-kDa ultrafiltrate (data not shown), which is consistent with the Mr of soluble PGN [14 ]. Sephadex G-25 desalting of S. aureus PGN did not show the association of the activity with the low molecular fractions that exhibit light absorbance at 206 nm, which is associated to MDP [31 , 34 ]. The L-isomer of MDP lacked [3H]AA-releasing activity, even at a concentration as high as 100 µg/ml (Fig. 3B) . Unlike treatment with lysostaphin and mutanolysin separately, the combined incubation suppressed the [3H]AA-releasing activity in soluble and insoluble fractions, thus indicating the requirement of the ß-1,4 glycosidic bond between MurNAc and N-acetylglucosamine (GlucNAc) for PGN to elicit this activity (Fig. 3B) . This result is in agreement with earlier reports using lysostaphin and mutanolysin or amidase, where the isolated treatment with these enzymes does not yield PGN monomers nor does it blunt the biological activity [13 , 35 ]. Preincubation of PMN with anti-TLR2 mAb at the concentration of 20 µg/ml prior to the addition of PGN did not inhibit [3H]AA release (Fig. 4A ), whereas this mAb did inhibit the induction of COX-2 protein in human PMN treated with the genuine TLR2 agonist Pam3CSK4 (Fig. 4B) . In contrast, anti-MR mAb showed a significant inhibition of [3H]AA release elicited by mannan, albeit this inhibition was not complete (Fig. 4A) .


Figure 4
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  		Figure 4. Effect of anti-TLR2 and anti-MR mAb on [3H]AA release. PMN were incubated with 20 µg/ml of the indicated mAb for 30 min prior to the addition of the indicated stimuli. The cell supernatants were collected for the assay of [3H]AA release 1 h after addition of the stimuli (A). The actual inhibitory effect of the anti-TLR2 mAb was confirmed in the PMN system after overnight incubation of cells with 1 µg/ml Pam3CSK4. At the end of this period, the cell lysates were used for the immunodetection of COX-2 and ß-actin to address the occurrence of similar protein loading across the gels (B). *, P < 0.05.

 
AA release is independent of microparticle formation and is blocked by inhibition of cytosolic PLA2
As the assay of [3H]AA release in the cell supernatants could not allow the distinction of unesterified arachidonate from the label incorporated into phospholipids, attempts were made to address unambiguously that the label assayed in supernatants was unesterified [3H]AA. Lipid extracts were analyzed by TLC in a system suitable for the separation of phospholipids and neutral lipids, which are the main pools in which AA is incorporated in human leukocytes [36 ]. [3H]AA in cell pellets appeared in two different areas, the retention factors, which are analogous to those of phospholipids and triglycerides (Fig. 5A , left-most lanes). In contrast, the analysis of cell supernatants showed a minor amount of the label in phospholipids and a major accumulation in the area where unesterified arachidonate migrates (Fig. 5A , right-most lanes). Of note, the label in the last area was absent in control cells, thus suggesting that the low amount of radioactivity detected under these conditions in cell supernatants could be accounted for by phospholipids rather than by unesterified [3H]AA. Stimulation of PMN in the presence of calpeptin, an inhibitor of the thiol-protease calpain involved in the formation of microparticles, only decreased [3H]AA release marginally (Fig. 5B) . In contrast, [3H]AA release was inhibited in a dose-dependent manner by treatment with the cytosolic PLA2 inhibitor pyrrolidine-1 [37 ] (Fig. 5B) , thus suggesting that the [3H]AA assayed in the supernatants is unesterified AA released by a cytosolic PLA2-dependent reaction.


Figure 5
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  		Figure 5. Distribution of [3H]AA label in the different lipid fractions in PMN and supernatants. [3H]AA-labeled PMN were stimulated for 1 h in the presence of 10 µg/ml PGN, 25 mg/ml mannan, and vehicle. At the end of this period, cell pellets were separated by centrifugation. The lipid extracts of cell pellets and supernatants were used for separation by TLC. The migration of the standards in TLC is indicated (A). Cells were incubated with calpeptin and pyrrolidine-1 prior to the addition of the stimuli and the [3H]AA released into the cell supernatants assayed. Data represent mean ± SEM of three experiments in duplicate. *, P < 0.05 (B).

 
Analysis of AA metabolites
Enzymes of the lipoxygenase and the COX routes are expressed in human PMN, which can allow the production of different types of eicosanoids, the most representative of which are LTB4 and PGE2. As shown in Figure 6A , PGN and C3bi-coated zymosan were the most robust stimuli for LTB4 production. In contrast, agonists unable to induce the release of [3H]AA, such as lipoteichoic acid and Pam3CSK4, did not elicit the production of LTB4. As to the production of PGE2 (Fig. 6B) , C3bi-coated zymosan and PGN elicited maximal production at 1 h, whereas the production elicited by mannan was somewhat lower.


Figure 6
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  		Figure 6. Effect of different stimuli on the release of LTB4 and PGE2. PMN were incubated with the stimuli for the times indicated, and then LTB4 (A) and PGE2 (B) were assayed in the supernatants. Data represent mean ± SEM of five experiments with duplicate samples.

 
DISCUSSION

The present results highlight the functional relevance of receptors for several PAMP in human PMN. This had been established previously for ligands of TLR2 and TLR heterodimers of TLR2 and TLR1/TLR6, but the description of productive binding by mannose-based stimuli and by PGN has not been reported in regard to the generation of lipid mediators. As these stimuli have been associated to the formation of microparticles [38 ], which in fact, contain phospholipids and have been involved in pathogenesis, we envisioned a possible connection between AA release and microparticle formation in response to microbial PAMP. However, we have found a vast predominance of [3H]AA in the unesterified form in cell supernatants; the independence of [3H]AA release from microparticle formation, as judged from the effect of calpeptin; and the dependence of [3H]AA release from cytosolic PLA2 activity, as judged from the inhibitory effect of pyrrolidine-1.

PGN and mannan elicited the production of LTB4 and PGE2 at a similar extent, thus indicating that they induce eicosanoid production and are relevant, functional stimuli. In contrast, our data do not support a similar effect of LPS, lipoteichoic acid, and MDP. Our results with LPS agree with earlier reports, where it was shown that LPS primes PMN for the response to other agonists, but it does not induce AA release on its own [39 40 41 ]. On this basis, AA release from PMN upon microbial invasion seems to be a hallmark of the host response to microorganisms containing PGN, mannose, and ß-glucans. PGN is found mainly in Gram-positive bacteria, where it accounts for approximately 90% (w/w) of the cell wall, thus explaining the high concentrations of PGN that can be reached in bacteria-infected tissues [32 ]. Mannose is found in Candida albicans, Leishmania donovani, and Pneumocystis carinni, whereas ß-glucan is found in C. albicans. The MR seems to be the most likely PRR for mannose-containing molecular patterns, as judged from the efficient uptake of mannosylated BSA, the effect of the endogenous ligand of the MR mucin-3, and the effect of anti-MR mAb. However, regarding PGN, at least three different types of receptors should be taken into account. We have confirmed the expression of the mRNA encoding TLR2, TLR1, TLR6, NOD2, and PGRP-S, all of which have some binding capacity for PGN or its elementary building blocks. As to the TLR2 route, PGN has been considered as an archetypal ligand, albeit this view has been a subject of controversy [31 , 32 ], as a recent study has reported that the ability of PGN to activate TLR2 might be lost after removal of lipoproteins and lipoteichoic acids [31 ]. It is therefore possible that the intracellular receptor NOD2 might be the actual PGN receptor, in view of its capacity to interact with MDP. Taking these data into account, we attempted to characterize the active moiety present in the PGN preparations and the possible signaling route. We found that PGN from S. aureus and B. subtilis elicits the same response, thus indicating that the effect is not restricted to diaminopimelic acid-type or L-alanyl-D-isoglutamine-type PGN. Moreover, the activity was not associated to lipoteichoic acid, LPS, MDP, and bacterial lipoproteins, as these compounds and the lipoprotein mimic Pam3CSK4 lacked any activity in the range of physiologically relevant concentrations tested. Molecular weight fractioning of PGN showed that the activity was retained in fractions containing eluates of molecular masses higher than 30 kDa, which is quite different from the 492.5-Da molecular weight of MDP. In addition, treatment with lysostaphin and mutanolysin suppressed the activity, which indicates that the integrity of the ß-1,4 glycosidic bonds between MurNAc and GlucNAc residues is necessary for the biological activity of PGN.

In regard to the type of receptor involved in the response, the absence of effect of other compounds acting on TLR2, i.e., Pam3CSK4 and lipoteichoic acid, and the lack of inhibitory effect of an anti-TLR2 mAb exhibiting strong blocking effects on TLR2-mediated cytokine production [42 ] and TLR2-dependent COX-2 induction do not point to an archetypal involvement of TLR2, thus suggesting that cooperation with other receptors could be necessary. So far, several examples of cooperation of TLR2 with NOD receptors have been reported in different cell types. This is the case of dendritic cells activated with bacillus-Calmette-Guerin-PGN, where activation of TLR2 and TLR4 is enhanced by the attendant recognition of MDP by NOD2 [43 ]. MDP also synergizes in THP-1 monocytes with archetypal TLR agonists mimicking bacterial components to induce IL-8 [44 ]. A further example of complexity has been shown in a recent study about the release of AA by RAW264.7 monocytes, where the response to ß-glucans was TLR2-dependent, whereas another TLR2 ligand, the macrophage-activating lipopeptide-2 (MALP-2) lipoprotein from Mycoplasma fermentans, was a weak agonist [41 ]. This seems to be a good example of the recently described cooperation of TLR with CD36, as CD36 is a selective and nonredundant sensor of microbial diacylglycerides, which signal via the TLR2/6 heterodimer, and senses the diacylated, bacterial lipopeptide MALP-2 but not triacylated lipopeptides or the TLR2-activating component of zymosan [45 ].

The possible involvement of PGRP-S is a challenging hypothesis within a rapidly moving field. Recent studies have disclosed the role of PGRP-S in defense against bacterial infection, as PGRP-S is present in PMN extracellular traps and binds to Gram-positive and Gram-negative PGN [28 , 46 ]. It is tempting to envision that notions already established in insect paradigms, i.e., the PGN catch-up receptor function of Drosophila PGRP-SA [16 ] and the essential role of PGRP-SC1a in Toll signaling and phagocytosis of S. aureus, disclosed by the study of the picky mutant of Drosophila [47 ], could be extended to the human immune system. Irrespective of the PRR involved, the present data disclose a central role for mannan- and PGN-based signatures in the induction of inflammatory responses in human PMN.

ACKNOWLEDGEMENTS

This work was supported by grants from Plan Nacional de Salud y Farmacia (grant SAF2004-01232), Junta de Castilla y León (Grupo de Excelencia), Red Brucella, Red Respira, and Red Temática de Investigación Cardiovascular from Instituto de Salud Carlos III. I. V. was the recipient of a grant from Banco de Santander-Central-Hispano. A. G. V. was the recipient of a grant from Instituto de Salud Carlos III. N. F. is under contract within the Ramón y Cajal Program of the Ministerio de Educación y Ciencia of Spain. We thank the staff of Centro de Hemoterapia y Hemodonación de Castilla y León for its help with the separation of leukocytes.

Received July 17, 2006; revised November 3, 2006; accepted November 30, 2006.

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I. Valera, N. Fernandez, A. G. Trinidad, S. Alonso, G. D. Brown, A. Alonso, and M. S. Crespo
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