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The Journal of Immunology, 2005, 174: 8154-8162.
Copyright © 2005 by The American Association of Immunologists

Mannose-Containing Molecular Patterns Are Strong Inducers of Cyclooxygenase-2 Expression and Prostaglandin E2 Production in Human Macrophages1

Nieves Fernández*, Sara Alonso*, Isela Valera*, Ana González Vigo*, Marta Renedo*, Luz Barbolla{dagger} and Mariano Sánchez Crespo2,*

* 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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The induction of cyclooxygenase-2 (COX-2) and the production of PGE2 in response to pathogen-associated molecular patterns decorated with mannose moieties were studied in human monocytes and monocyte-derived macrophages (MDM). Saccharomyces cerevisiae mannan was a robust agonist, suggesting the involvement of the mannose receptor (MR). MR expression increased along the macrophage differentiation route, as judged from both its surface display assessed by flow cytometry and the ability of MDM to ingest mannosylated BSA. Treatment with mannose-BSA, a weak agonist of the MR containing a lower ratio of attached sugar compared with pure polysaccharides, before the addition of mannan inhibited COX-2 expression, whereas this was not observed when agonists other than mannan and zymosan were used. HeLa cells, which were found to express MR mRNA, showed a significant induction of COX-2 expression upon mannan challenge. Conversely, mannan did not induce COX-2 expression in HEK293 cells, which express the mRNA encoding Endo180, a parent receptor pertaining to the MR family, but not the MR itself. These data indicate that mannan is a strong inducer of COX-2 expression in human MDM, most likely by acting through the MR route. Because COX-2 products can be both proinflammatory and immunomodulatory, these results disclose a signaling route triggered by mannose-decorated pathogen-associated molecular patterns, which can be involved in both the response to pathogens and the maintenance of homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Innate immunity is a mechanism of host defense widely conserved, the capacity of which to recognize pathogens depends on germline-encoded molecules called pattern recognition receptors (PRR).3 Unlike the receptors of the adaptive immune system, PRR do not undergo somatic mutation by recombinase-mediated rearrangement and recognize conserved microbial structures shared by large groups of pathogens, which are collectively called pathogen-associated molecular patterns (PAMP). LPS from Gram-negative bacteria and branched sugars with {beta}-glucan and {alpha}-mannose moieties from different microorganisms are archetypal examples of PAMP. Among PRR, the mannose receptor (MR), first described by Stahl et al. (1), has been the object of detailed scrutiny. This receptor recognizes glycosylated molecules with terminal mannose, fucose, or N-acetylglucosamine moieties and internalizes soluble and particulate ligands through the endocytic and phagocytic pathways. Its capacity for ligand recognition makes this receptor suitable to phagocytose Candida albicans, Leishmania donovani, and Pneumocytis carinni, among other microorganisms (2, 3, 4). The MR is the prototypic element of a homonymous family of C-type lectin receptors, which includes other members, such as the secreted phospholipase A2 M-type receptor, the dendritic cell receptor DEC-205, and Endo180/urokinase plasminogen-activated receptor-associated protein. These receptors contain carbohydrate recognition domains, although the chemical structure of the ligands interacting with those domains displays wide differences (for review, see Refs.5, 6, 7). It should be noted that the list of elements of this receptor family could be unexpectedly enlarged, because FcRY, the avian functional equivalent of the mammalian MHC-related FcR, is a homologue of the mammalian secreted phospholipase A2 (sPLA2) M-type receptor and shares a common domain architecture with other members of the MR family, although it binds IgY, the avian counterpart of IgG (8). The MR is mainly expressed in alveolar macrophages, peritoneal macrophages, and macrophages derived from blood monocytes (9) and seems to play a role in the early immune response against invading pathogens, but it has also been detected in other cell types, such as hepatic endothelium, kidney mesangial cells, tracheal smooth muscle cells, and retinal pigment epithelium (7).

There is some discrepancy regarding the full scope of molecules that may be bound by the MR. {beta}-Glucan- and mannan-containing particles, such as zymosan and C. albicans, have been proposed to be recognized by the MR (10), but other receptors involved in the innate immune response also bind mannosylated particles. Thereby, the mannose-specific, C-type lectin dendritic cell-specific ICAM-grabbing nonintegrin (DC-SIGN) recognizes lipoarabinomannan, a glycoconjugate component of the Mycobacteria cell wall (11, 12, 13). Some members of the MR family have been associated with signal transduction events, for instance, the secreted phospholipase A2 M-type receptor (14, 15), but few reports have addressed this functional capacity for the MR itself. In fact, the function of the MR has currently been related to the capture, internalization, and presentation of mannosylated Ags (16) bypassing the oxidative burst in human macrophages (17) as well as to the activation of an anti-inflammatory and immunosuppressive program in monocyte-derived dendritic cells (18). This agrees with the finding of normal defense against Candida and Pneumocystis infection in animals with targeted disruption of the MR gene (19, 20). Conversely, the synthesis of proinflammatory cytokines (21) and matrix metalloproteinase-9 (22) by engaging the MR has also been reported, and a recent study has described the ability of the MR to trigger activation of the transcription factor NF-{kappa}B and the induction of IL-8, thus suggesting a central role for the MR in the defense by human alveolar macrophages against Pneumocytis infection (23) in the context of HIV infection (4).

In a previous study we addressed the effect of both mannose- and {beta}-glucan-containing polysaccharides on the release of arachidonic acid by human monocytes (24), which might be of some relevance in the inflammatory response in view of the wide scope of physiological actions of eicosanoids. In the present study we addressed the effect of mannan-containing polysaccharide on the expression of cyclooxygenase-2 (COX-2), the inducible isoform of cyclooxygenase that converts arachidonic acid into the unstable PGG2. PGG2 is subsequently reduced to PGH2 and serves as a substrate for the production of other PGs, such as PGE2 and PGD2, through the action of isomerases and synthases. The effect of mannan is more potent than that elicited by similar concentrations of zymosan particles and seems to be mediated by the MR. The observed response cannot be accounted for by LPS contamination, is sensitive to blockade by mannosylated BSA, and is more remarkable in cells showing a robust expression of the MR, for instance, monocyte-derived macrophages (MDM). Because 15-deoxy-PGJ2, the final product of the metabolism of the PGD2 series, has been reported to modulate MR gene expression in macrophages (25), our findings disclose an autocrine/paracrine mechanism coupled to PG production that might influence the immune response and the defense against pathogens as well as the expression of their own MR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Zymosan, soluble {beta}-glucan from seaweed (laminarin, ~8 kDa), soluble {alpha}-mannan from Saccharomyces cerevisiae, and porcine mucin 3 (MUC-3), a mucin from the gastrointestinal tract, which is a natural ligand of the MR, were purchased from Sigma-Aldrich. 4-[5-(4-Chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (SC-236; a COX-2 inhibitor) and 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC-560; a COX-1 inhibitor) were obtained from Calbiochem. Mouse monoclonal anti-human CD206/mannose receptor and CD16/Fc{gamma}RIII were purchased from BD Pharmingen. IgG-OVA equivalence immune complexes (IC) were made according to classical procedures with optimal amounts of Ag using IgG Ab raised in rabbits. To obtain IC bound covalently to C3bi, IC were extensively washed with PBS and incubated with normal human serum as previously described (24). Coating of C3bi to zymosan was conducted by incubation with normal human serum, followed by extensive washing with PBS. The characteristics of the oligonucleotide primers used in PCRs for the detection of dectin-1 mRNA (26), DC-SIGN (27), DC-SIGN-related (DC-SIGNR) (28), Endo180 (29), TLR-2 (30), MR (31), and sPLA2 M-type receptor (32) are shown in Table I. Hemagglutinin-tagged cDNA of human TLR-2 and the mutation corresponding to the dominant negative TLR4 (P712H substitution) in C3H/HeJ-mice (33) cloned into the expression plasmid pRc/CMV (Invitrogen Life Technologies) were provided by Dr. M. Rehli (University of Regensburg, Regensburg, Germany).


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  		Table I. Oligonucleotide primers used for the detection of the mRNA encoding for receptors of the innate immune system

 
Cell culture

THP-1 cells were cultured in RPMI 1640 medium supplemented with 2 mM glutamine and 10% heat-inactivated FBS. Human monocytes were isolated from buffy coats of healthy volunteer donors by centrifugation onto Ficoll cushions and adherence to plastic dishes for 2 h. At the end of this period, nonadhered cells were removed by extensive washing. Differentiation of monocytes into macrophages was conducted by culture of adhered monocytes in the presence of 5% human serum for 2 wk in Primaria six-well dishes (BD Biosciences), in the absence of exogenous cytokine mixtures. HEK293 cells and HeLa cells were transiently transfected using the calcium phosphate method.

Assays for endo/phagocytosis and flow cytometry

Cells were incubated for different times at 37°C with FITC-labeled mannosylated BSA and subsequently washed and resuspended in 500 µl of PBS supplemented with 1 mM EDTA for analysis by flow cytometry in a FACScan cytofluorometer (BD Biosciences). Parallel controls were performed at 4°C to block endocytic uptake of the particles. The surface display of both CD16 and CD206/mannose receptor was determined by indirect immunofluorescence with mouse anti-human CD206 and CD16 IgG1 mAb, followed by washing with PBS and incubation with goatanti-mouse IgG-FITC conjugate Ab (Sigma-Aldrich; 1/100 dilution) for 30 min at 4°C. Isotype-matched irrelevant Ab was used as a control.

Confocal microscopy

Human monocytes were seeded in 35-mm Primaria culture dishes (BD Biosciences) to allow their differentiation into MDM as described above. Twenty-four hours before being used, cells were serum-starved. At different times after the addition of stimuli, MDM were extensively washed with HBSS to discard background extracellular fluorescence, and the dishes were observed in vivo by confocal microscopy using a Bio-Rad Laser scanning system Radiance 2100 coupled to a Nikon inverted microscope with a thermostatized chamber. The objective was a x20 and numerical aperture of 0.5. Green fluorescence (fluorescein) was monitored at 488 nm argon excitation using a HQ500 long-bandpass blocking filter. Images were merged using Adobe Photoshop 6.0 software.

Immunoblots of COX-2

The amount of protein in each cell lysate was assayed using the Bradford reagent, and 50 µg of protein from each sample was loaded on each lane of a 10% SDS-PAGE gel. Proteins were transferred to nitrocellulose membranes using a semidry transfer system. The membranes were blocked with dry milk and used for immunoblot using a goat polyclonal antiserum (SC-1745) and, in some experiments, rabbit anti-COX-1 Ab (SC-7950) from Santa Cruz Biotechnology. This was followed by incubation with donkey anti-goat IgG-HRP-conjugated Ab. Detection was performed using the Amersham Biosciences ECL system. {beta}-Actin immunodetection was used to address the occurrence of similar protein loading across the gels.

RT-PCR assays for dectin-1, Endo180, DC-SIGN, DC-SIGNR, TLR-2, MR, and sPLA2 M-type receptor

Total cellular RNA was extracted by the TRIzol method (Invitrogen Life Technologies). First-strand cDNA was synthesized from total RNA by RT reaction. The reaction mixture containing 0.2 mg/ml total RNA, 2.5 µl of H2O, 20 U of RNasin RNase inhibitor, 4 µl of 5x buffer, 2 µl of 0.1 M DTT, 4 µl of 2.5 mM dNTP, 1 µl of 0.1 mM hexanucleotide, and 200 U of Moloney murine leukemia virus reverse transcriptase. The reaction was conducted at 37°C for 60 min in a volume of 20 µl. The cDNA was amplified by PCR in a reaction mixture containing 2 µl of DNA template; 10 µl of H2O; 2.5 µl of 10x buffer; 0.75 µl of 50 mM MgCl2; 1.0 µl of 2.5 mM dNTP; 1.25 µl of each forward and reverse primer of dectin-1, DC-SIGN, DC-SIGNR, TLR-2, MR, and {beta}-actin; and 0.25 µl of 5 U/ml Taq DNA polymerase. The amplification profile for detection included one cycle of initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, primer annealing at 60°C for 30 s, and extension at 72°C for 30 s, and one cycle of final extension at 72°C for 7 min. The expression of {beta}-actin was used as a control for the assay of a constitutively expressed gene. 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).

ELISA for PGE2

Quantitation of cellular PGE2 levels was determined using an enzyme immunoassay kit (Amersham Biosciences) according to the manufacturer’s instructions. This assay is based on competition between unlabeled PGE2 in the sample and a fixed amount of labeled PGE2 for a PGE2-specific Ab. The detection limit of this assay is 20 pg/ml. The samples for the assay were collected 24 h after addition of the stimuli, because this allows the detection of PGE2 produced during a prolonged period by the action of COX enzymes on the arachidonate made available in the deacylation/reacylation cycle.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Induction of COX-2 protein and production of PGE2 by mononuclear phagocytes in response to different stimuli

Incubation of overnight-adhered monocytes with mannan induced a mild expression of COX-2 protein above basal levels, whereas treatment with laminarin, zymosan particles, complement-coated zymosan particles, and preformed IC did not noticeably increase COX-2 above the levels observed in resting adhered monocytes (Fig. 1A, left panel). Because mannan appeared as the most potent stimuli, additional experiments were conducted using different concentrations of this substance. However, a clear dose-dependency of the response was not observed for concentrations >5 mg/ml (Fig. 1A, right panel), most likely because of the possible induction of COX-2 expression by adherence-dependent signals (34), which could impede the correct appraisal of receptor-mediated induction. By contrast, in experiments conducted on monocytes cultured in the presence of human serum for 7 days, there was no expression of COX-2 in resting cells, suggesting that these conditions are most adequate for a fine-tuning assessment of ligand-induced COX-2 expression. In keeping with this view, both mannan and zymosan induced a net expression of COX-2 protein, with mannan clearly behaving as the most potent stimulus (Fig. 1B). MDM obtained after 2 wk of culture showed noticeable induction of COX-2 protein with concentrations of mannan as low as 0.1 mg/ml, 1 mg/ml zymosan, and 5 mg/ml soluble {beta}-glucan laminarin (Fig. 1C). Interestingly, the natural ligand of the mannose receptor, MUC-3, also induced COX-2 expression; this effect was not synergistic with that produced by the combination of MUC-3 and mannan, thereby agreeing with the hypothesis that both molecules act through the same receptor (Fig. 1C, lower panel). It is noteworthy that COX-1 expression was not influenced by mannan and zymosan treatment (Fig. 1D).



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  		FIGURE 1. Effects of mannan and {beta}-glucan particles on COX-2 expression. Monocytes adhered to plastic dishes by overnight culture at 37°C (A), monocytes cultured in the presence of 5% human serum for 7 days (B), and MDM (C and D) obtained after culture for 14 days in the presence of 5% human serum were incubated for 6 h in the presence of various stimuli, then the cell lysates were collected and used for the immunodetection of COX-2 and COX-1 (D). {beta}-Actin was immunodetected to address the occurrence of similar protein loading across the gels. These data are representative of three to five experiments with identical results. C3bi indicates particles coated with C3bi.

 
The time course of COX-2 induction was studied in MDM. Maximal induction occurred at 4–7 h with all agonists (Fig. 2A); the soluble {beta}-glucan laminarin was again the stimulus displaying the lowest potency, whereas mannan was the strongest stimulus. The production of PGE2 showed a good correlation with COX-2 induction, because in keeping with the noticeable basal expression of COX-2 protein, mannan slightly increased PGE2 production above that observed in resting adhered monocytes, whereas it was a potent stimulus in MDM (Fig. 2B). It should be noted that PGE2 production was most inhibited by 10 µM SC-236, a highly selective inhibitor of COX-2, the reported IC50 value of which for COX-1 inhibition is 17.8 µM. In contrast, a more limited inhibition was produced by 0.3 µM SC-560, i.e., a concentration significantly higher than its reported IC50 value of 9 nM for COX-1 inhibition (35). THP-1 cells are currently used as an archetypal model for human monocytes, because they display many different PRR even though they do not express CD14 (36) and do not adhere to plastic surfaces in the absence of phorbol ester treatment. On this basis, this cell line was used to obtain additional insight into the results observed in both monocytes and MDM. As shown in Fig. 2C, zymosan was a robust inductor of COX-2; this effect was enhanced by C3 bi-coating, which agrees with previous observations indicating a synergistic effect of complement coating on the proinflammatory effects of zymosan particles, most likely explained by the ability of zymosan-C3bi to concomitantly engage both dectin-1 (see below) and complement receptor 3 (CR3) (24). In contrast, the response to mannan was less remarkable, thus agreeing with the lower expression of MR in THP-1 cells (Fig. 2D) compared with MDM (see below). The possible autocrine effect of PGE2 on COX-2 induction (37) was addressed by assaying the effect of exogenous PGE2. However, concentrations of PGE2 as high as 15 ng/ml failed to induce COX-2 protein expression (data not shown), which most likely indicates that under these experimental conditions, E-prostanoid receptors engaged by PGE2 are not involved in autocrine COX-2 synthesis.



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  		FIGURE 2. Effects of mannan and zymosan particles on COX-2 expression and PGE2 production. MDM were incubated in the presence of the indicated additions for the times indicated, then the cell lysates were collected for immunodetection of COX-2 (A). Cell culture medium of both monocytes and MDM treated for 24 h in the presence of the indicated additions was used for the assay of PGE2 (B). These are typical experiments of three or four performed with identical results. In the case of the PGE2 assay, results express the mean ± SEM of three independent experiments (*, p < 0.05, SC-236-treated vs vehicle-treated MDM). THP-1 cells were incubated for 6 h in the presence of the various stimuli, then the cell lysates were collected for the immunodetection of COX-2 (C). {beta}-Actin was detected to address the occurrence of similar protein loading across the gels. These data are representative of three experiments with identical results. The surface display of both CD206/MR and CD16/Fc{gamma}RIII in THP-1 cells assayed by immunofluorescence flow cytometry is shown in D. Zymosan-C3bi indicates zymosan particles coated with C3bi.

 
Expression of PRR in monocytic cells

Because the effect of zymosan (a polymer of {beta}-glucan that can also contain mannose) on myeloid cells has been associated with the engagement of several receptors, namely, dectin-1 (38, 39), DC-SIGNR (40), MR (9, 17, 41), and CR3 (42), and the effect of mannose-containing particles has been associated to the engagement of MR, TLR-2 (43, 44, 45), and DC-SIGN (11, 12, 13), the expression of this set of receptors was assessed in different cell types.

As shown in Fig. 3A, the expression of dectin-1 mRNA was observed in all cell types studied at all stages of differentiation; however, semiquantitative PCRs showed the highest levels of expression in both polymorphonuclear leukocyte (PMN) and MDM. In keeping with the previously reported existence of two predominant and six minor transcripts obtained by alternative splicing and small insertions in the human dectin-1 gene (26), a main PCR product of ~558 bp was found in all cell types together with a lower Mr product and other minor products. The expression of Endo180 mRNA in monocytic cells was somewhat similar to that of dectin-1, inasmuch as it increased with monocyte differentiation into macrophages and was also detected in THP-1 cells. By contrast, Endo180 mRNA was not observed in PMN, and only a slight expression was observed in adhered monocytes (Fig. 3A). Interestingly, Endo180 mRNA showed a high expression level in both HEK293 cells and HUVEC, thus agreeing with the reported expression of this receptor in endothelial cells (29) (Fig. 3B). Of note, the mRNA of neither DC-SIGNR nor DC-SIGN could be detected in the same set of cell types (not shown). TLR-2 mRNA was detected in blood and THP-1 cells (Fig. 3A). It is noteworthy that THP-1 cells expressed high amounts of sPLA2 M-type receptor (Fig. 3C). As shown in Fig. 3D, maximal expression of MR was observed in MDM, although a weak, but significant, expression was also observed in nonadhered mononuclear cells, THP-1 monocytes, and HeLa cells (Fig. 3, C and D). This point was addressed in additional detail by indirect immunofluorescence flow cytometry. As shown in Fig. 4A, the surface display of MR was barely detectable in monocytes, but it blatantly increased after several days in culture, pari passu with the increasing size of the cells (Fig. 4B). The fluorescence intensity on day 1 was 3.6 ± 0.3 (mean ± SEM; n = 6; arbitrary units (AU)) and increased to 6.38 ± 0.4 and 39.2 ± 4 at 7 and 14 days, respectively. Moreover, FITC-conjugated, mannosylated BSA was readily uptaken by MDM, as assessed by flow cytometric assays (Fig. 5A). Confocal immunofluorescence microscopy showed that under these conditions, the uptake of FITC-conjugated, mannosylated BSA was very rapid, with focal images compatible with capping being detected as early as 2 min after addition of the stimulus, and label reaching the whole cytoplasm by 20 min. FITC-mannosylated BSA uptake was not observed at 4°C nor in the presence of 10 mg/ml mannose-BSA. Moreover, it was not observed in HEK293 cells after long periods of incubation (Fig. 5B).



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  		FIGURE 3. Expression of mRNA encoding for PRR in different cell types. Total mRNA extracted from blood leukocytes and cell lines was extracted and used for RT-PCR amplification with primers for dectin-1 (A), Endo180 (A and B), TLR-2 (A), MR (C and D), and type M sPLA2 receptor (C, lower panel). The expression of {beta}-actin mRNA is shown as an example of constitutive gene expression (A, lower panel). These data are representative of three experiments with similar results.

 


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  		FIGURE 4. Surface expression of CD206/MR and CD16/Fc{gamma}RIII in monocytes and MDM. Adhered monocytes were cultured in plastic dishes in the presence of 5% human serum for the times indicated. After these periods, cells were scraped and incubated with FITC-conjugated mouse anti-human CD206 and CD16 at a concentration of 1 µg/106 cells, then used for flow cytometry. Isotype-matched Ab was used as the control (shown by the thin tracing on the left). These data are representative recordings of six identical experiments (A). Lower panels, Phase-contrast transmission microscopic images of cells at various times of differentiation, indicating the blatant changes in cell size (B).

 


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  		FIGURE 5. Uptake of FITC-conjugated, mannosylated BSA by MDM. Adhered macrophages were incubated with various concentrations of FITC-conjugated, mannosylated BSA at both 4 and 37°C, as indicated, then cells were scraped and used for fluorescence flow cytometry. These data are typical recordings of three independent experiments (A). Typical patterns of merged images from phase-contrast transmission microscopy and green fluorescence of MDM are shown. Cells were incubated with 200 µg/ml FITC-conjugated, mannosylated BSA, and images were taken at the times indicated (B). The panel marked mannose-BSA indicates MDM incubated with 10 mg/ml mannose-BSA for 10 min at 4°C, followed by thermal equilibration at 37°C in the presence of FITC-conjugated mannose-BSA. Images were taken 30 min thereafter. The panel marked 60 min, 4°C, shows MDM incubated for 60 min at 4°C with FITC-conjugated, mannose-BSA. The panel marked HEK293 shows a typical image of HEK293 cells taken after 60 min of incubation with FITC-conjugated mannose-BSA.

 
The expression of CD16/Fc{gamma}RIII, a receptor involved in the adaptive humoral immune responses, also increased along the macrophage differentiation route (Fig. 4A), with mean fluorescence intensity values of 0.34 ± 0.05, 2 ± 0.03, and 4.77 ± 0.2 (mean ± SEM; n = 6; AU) on days 1, 7, and 14, respectively, thus indicating that this marker is a good indicator of macrophage differentiation, which is only detected in ~10% of peripheral blood monocytes (46).

COX-2 induction by mannan is not accounted for by TLR-2 activation nor LPS contamination

Because the most robust induction of COX-2 by mannan was observed in MDM after 14 days in culture, and the receptors capable of binding terminal mannose residues preferentially expressed in these cells are MR, Endo180, and TLR-2, these receptors were envisaged as being involved in conveying signals for COX-2 induction. Additional experiments were conducted in cell lines that have been used for similar purposes, namely, HEK293 (47) and HeLa cells (48). Unlike blood cells, these cell lines do not express type-M sPLA2 receptor (Fig. 3C) or TLR-2 mRNA (Fig. 6A), but readily express TLR-2 mRNA upon transfection with an expression vector encoding for human TLR-2 (Fig. 6A). However, upon stimulation with mannan, TLR-2-expressing HEK293 cells did not exhibit COX-2 protein expression (data not shown), whereas they produced an 8-fold increase in {kappa}B-driven transcriptional activity upon transfection with a firefly luciferase-linked 5x NF-{kappa}B reporter plasmid DNA and stimulation with 10 µg/ml peptidoglycan, i.e., a natural ligand for TLR-2 (49). In contrast, HeLa cells showed a noticeable basal expression of COX-2, which was enhanced by 10 mg/ml mannan (Fig. 6B). This response was not influenced by transfection of empty vector (pRc/CMC) and TLR-2-encoding vector (Fig. 6, C and D, upper panels), although transfection with TLR-2-encoding vector endowed HeLa cells with the ability to respond to the TLR-2 ligand peptidoglycan (Fig. 6, C and D, lower panels). Moreover, transfection of HeLa cells with the TLR-2 dominant negative mutant (P712H) did not abrogate mannan response, whereas these cells did not show COX-2 induction in response to peptidoglycan above the expression level observed in control cells (Fig. 6E). Taken together, these data indicate that a receptor other than TLR-2, most likely the MR, should be involved in mannan-induced COX-2 expression in HeLa cells. As shown in Fig. 7A, polymyxin B blocked the effect of Escherichia coli LPS on human MDM, whereas this was not observed after mannan treatment. This finding allows us to rule out a contamination of mannan by LPS. Conversely, mannose-BSA treatment inhibited in a dose-dependent manner the effect of mannan, whereas it did not influence LPS effect (Fig. 7, B and C). Of note, mannose-BSA partially inhibited the zymosan effect (Fig. 7C, right panel), whereas this was not observed on the response to the pure {beta}-glucan laminarin (Fig. 7C, left panel). These findings and the previously observed inhibition of zymosan uptake by mannan (24) are in keeping with previous reports indicating that the MR may be involved in a portion of the biological effects of zymosan, which are purportedly related to its content in mannose moieties (10, 17, 40).



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  		FIGURE 6. Effect of ectopic expression of TLR-2 on mannan-induced COX-2 expression. The expression of TLR-2 mRNA in THP-1 monocytes and HEK293 and HeLa cells in both the absence and the presence of transfection with a vector encoding human TLR-2 sequence is shown in A. The effect of various agonists on the expression of COX-2 in HeLa cells is shown in nontransfected cells (B) or after transfection of both a mock vector (pRc/CMV; C) and TLR-2 vector (D) 6 h after addition of stimuli. The effect of transfection of HeLa cells with both a TLR-2 vector and a dominant negative TLR-2 construct on the response to mannan and peptidoglycan is shown in E. These data are representative experiments of three with similar results. The expression of {beta}-actin is shown as an example of constitutive gene expression.

 


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  		FIGURE 7. Effects of polymyxin B and mannose-BSA on the induction of COX-2 produced by different stimuli. MDM were incubated with mannan and LPS in the presence and the absence of various concentrations of polymyxin B. After 6 h, cell lysates were collected and used for the immunodetection of COX-2 (A). The effect of the preincubation of MDM with mannosylated BSA on the induction of COX-2 by various stimuli is shown in B and C. These are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Molecular structures containing terminal mannose are unusual components of mammalian tissues; however, they are abundant in the walls of a variety of microorganisms, which is a prime condition for these structures to behave as PAMP. Moreover, host cells express different PRR able to recognize mannose-decorated components. The MR is the archetypal receptor, but DC-SIGN and Endo180 have the ability to recognize mannose moieties, thus making it difficult to ascertain the role displayed by each receptor in physiologically relevant cell systems. Inhibition of proinflammatory cytokines has been related to the engagement of the MR (44) and DC-SIGN (12, 13), whereas the proinflammatory effects of mycobacterial lipomannans depend on their ability to interact with TLR-2 (43, 44, 45), which is reminiscent of the ability of this receptor to interact with {beta}-glucans (50), although most recent studies have disclosed a more complex paradigm in which dectin-1 behaves as the recognition receptor (38, 39) and TLR-2 acts as a signal integrator targeting intracellular molecules into a signaling complex involving MyD88, IL-1R-associated kinase, and TNFR-associated factor-6 (51).

Our first approach to address this interplay of receptors was to analyze the pattern of receptors expressed in monocytic cells and its correlation with the cell response along the route to macrophage differentiation. We observed a reduced surface display of the MR on human monocytes, which agrees with previous reports (16), and an increased expression of this receptor along the macrophage differentiation route, pari passu with the expression of CD16/Fc{gamma}RIII, a receptor implicated in adaptive immunity that is a well-known marker of macrophage differentiation. In contrast, we have not observed the expression of DC-SIGN at any point in the differentiation process, which is in keeping with the reported pattern of expression of this receptor in cell microdomains during the development of human monocyte-derived dendritic cells (52). Furthermore, we have not detected the expression of DC-SIGNR mRNA at any time of macrophage differentiation. This finding and the unique activation of DC-SIGNR by {beta}-glucan make it unlikely that this receptor is involved in the response to {alpha}-mannan moieties. With regard to dectin-1, our data agree with the preferential involvement of this receptor in the response to {beta}-glucan particles, inasmuch as the response to zymosan is clearly observed in THP-1 cells, which definitely express dectin-1 mRNA, and is enhanced by C3bi coating, thus indicating a potentiation of the response by the simultaneous engagement of CR3. The involvement of MR in mediation of the COX-2 induction produced by mannan is suggested by several factors: 1) the correlation of the magnitude of the response with the extent of MR expression, as assessed by both RT-PCR and flow cytometry; 2) the inhibition by mannose-BSA, a ligand for the MR that is engulfed upon receptor binding and leads to noticeable endocytosis, but behaves as a weak agonist because it contains a lower ratio of attached sugar compared with pure polysaccharides (16); and 3) the similar effect of a natural ligand of the MR (MUC-3) together with the lack of synergism of mannan and MUC-3, thus pointing to an effect of both ligands on the same receptor. With regard to the possible involvement of Endo180, initial studies have stressed its preferential binding by N-acetylglucosamine, suggesting the existence of a different array of ligands for this receptor and the MR (29). Even though this idea has recently been modified by showing that under certain circumstances mannose oligosaccharides can bind to the receptor in a Ca2+-dependent manner (53), this has been observed for Ca2+ concentrations of ~10 mM, suggesting that this binding might only occur at concentrations well above physiological Ca2+ levels. Moreover, unlike HeLa cells, which express the MR and show COX-2 expression upon mannan challenge, HEK293 cells, which do express Endo180 mRNA, do not show COX-2 induction upon mannan challenge.

Regardless of the receptors involved in mannan effects, our data show that the soluble mannose polysaccharide mannan from S. cerevisiae as well as the natural ligand of the mannose receptor MUC-3 produce both COX-2 induction and PGE2 release in human macrophages, which, in combination with our previous description of the release of arachidonic acid by mannan, links this polysaccharide to the eicosanoid cascade (24). This finding raises several questions of functional relevance, because the MR might limit inflammation by counterbalancing signals from TLR and other PRR (54), and PGE2 down-regulates inflammation and dendritic cell migration via E-prostanoid receptor 2 (55). Interestingly, microarray analysis of gene expression in human dendritic cells stimulated with mannan has shown a significant enhancement of COX-2 expression in the context of an outstanding overlap of the set of genes elicited by C. albicans (56), thus attesting to the relevance of PRR in the recognition of mannose-decorated PAMP in antifungal responses (57). Moreover, tumor-associated macrophages show a functional polarization associated with MR expression, endocytic activity, and poor cytotoxicity, which can be explained by the action of tumor mucins on the MR (54, 58). That this could be due to PGE2 production elicited by mucins via the MR/COX-2 route is a tempting hypothesis. However, because several receptors can be activated by mannose-containing PAMP, it should be taken into account that the final response might be determined by a complex balance resulting from the concomitant activation of distinct receptors on host cells by microorganism- and tumor-derived products (59).

The characterization of the signaling route triggered by MR engagement is an important issue. In light of the mechanisms usually associated with the innate immune response, the NF-{kappa}B route would appear at first glance to be involved. This pathway has recently been explored in human alveolar macrophages and found to be involved in the defense against P. carinii infection (23), thus locating the MR with other PRR in the classical pathway of NF-{kappa}B activation leading to the increased transcription of genes encoding chemokines, adhesion molecules, and enzymes that synthesize inflammatory mediators, i.e., COX-2 (60). These data have recently been enlarged by showing the involvement of the GTPases, Cdc42 and Rho, in the nonopsonic phagocytosis of Pneumocystis by alveolar macrophages (61), and it has been suggested that reduced mannose receptor-mediated Cdc42 and Rho activation in alveolar macrophages in the context of HIV infection might be a mechanism that contributes to the pathogenesis of opportunistic pneumonia. In contrast, studies of monocyte-derived dendritic cells have revealed no activation of {kappa}B-driven transcription upon MR cross-linking, whereas blockade of the PI3K route showed marked inhibition of MR signaling (18). The possibility that cell-specific signaling pathways might be coupled to the MR, thus yielding different patterns of response, seems to be of pathophysiological relevance. In this connection, MR is to date the only innate PRR known to be significantly up-regulated in a typical chronic inflammatory condition such as nasal polyposis, thus suggesting a central role for MR in the pathophysiology of this disease (62).

In summary, our data have shown a strong capacity of ligands displaying terminal mannoses to induce COX-2 in human mononuclear phagocytes. This capacity increases along the differentiation of these cells into the macrophage route and best correlates with the expression of MR. These findings disclose the existence of a paracrine immunomodulatory route associated with PG production, which is associated with the endocytosis of mannose-decorated molecules and might also influence the expression of the MR (25).


   Acknowledgments
 
We thank Cristina Gómez for technical help, Dr. Padrón and the staff of Centro de Hemoterapia y Hemodonación de Castilla y León for their help with the separation and culture of blood monocytes, and Dr. Michael Rehli (University of Regensburg, Regensburg, Germany) for the generous gift of hemagglutinin-tagged cDNA of human TLR-2.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Plan Nacional de Salud y Farmacia (Grant SAF2001-0506), Red Brucella, Red Respira, and Red Temática de Investigación Cardiovascular from Ministerio de Sanidad y Consumo. N.F. is under contract with the Ramón y Cajal Program of Ministerio de Educación y Ciencia of Spain. 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. Back

2 Address correspondence and reprint requests to Dr. M. Sánchez Crespo, Instituto de Biología y Genética Molecular, Facultad de Medicina, 47005 Valladolid, Spain. E-mail address: mscres{at}ibgm.uva.es Back

3 Abbreviations used in this paper: PRR, pattern recognition receptor; AU, arbitrary unit; C3, the third component of the complement system; C3bi-IC, immune complex bound to C3bi; COX-2, cyclooxygenase-2; CR3, complement receptor 3; DC-SIGN, dendritic cell-specific ICAM-grabbing nonintegrin; DC-SIGNR, DC-SIGN-related; IC, immune complex; MDM, monocyte-derived macrophage; MR, mannose receptor; MUC-3, mucin 3; PAMP, pathogen-associated molecular pattern; PMN, polymorphonuclear leukocyte; sPLA2, secreted phospholipase A2. Back

Received for publication December 28, 2004. Accepted for publication April 12, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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S. K. Pathak, S. Basu, A. Bhattacharyya, S. Pathak, M. Kundu, and J. Basu
Mycobacterium tuberculosis Lipoarabinomannan-mediated IRAK-M Induction Negatively Regulates Toll-like Receptor-dependent Interleukin-12 p40 Production in Macrophages
J. Biol. Chem., December 30, 2005; 280(52): 42794 - 42800.
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