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The Journal of Immunology, 2005, 174: 7096-7103.
Copyright © 2005 by The American Association of Immunologists

Dendritic Cell Maturation Induced by Muramyl Dipeptide (MDP) Derivatives: Monoacylated MDP Confers TLR2/TLR4 Activation1

Junji Uehori{dagger},¶, Koichi Fukase§, Takashi Akazawa{dagger}, Satoshi Uematsu{ddagger}, Shizuo Akira{ddagger}, Kenji Funami*, Masashi Shingai*, Misako Matsumoto*,{dagger},¶, Ichiro Azuma||, Kumao Toyoshima{dagger}, Shoichi Kusumoto2,§ and Tsukasa Seya3,*,{dagger},¶

* Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo, Japan; {dagger} Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, {ddagger} Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, and § Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan; Department of Molecular Immunology, Nara Institute for Science and Technology, Nara, Japan; and || Hakodate National College of Technology, Hakodate, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
6-O-acyl-muramyldipeptides (MDP) with various lengths of fatty acid chains were examined for their dendritic cell (DC) maturation activity expressed through TLRs. Judging from anti-TLR mAb/inhibitor-blocking analysis, MDP derivatives with a single octanoyl or stearoyl fatty acid chain were found to activate TLR2 and TLR4 on human DCs, although intact and diacylated MDP expressed no ability to activate TLRs. Human DC activation profiles by the monoacylated MDP were essentially similar to those by Calmette-Guérin (BCG)-cell wall skeleton (CWS) and BCG-peptidoglycan (PGN) based on their ability to up-regulate costimulators, HLA-DR, {beta}2-microglobulin, and allostimulatory MLR. Monoacylated MDP induced cytokines with similar profiles to BCG-CWS or -PGN, although their potency for induction of TNF-{alpha}, IL-12p40, and IL-6 was less than that of BCG-CWS or -PGN. The MDP derivatives initiated similar activation in normal mouse macrophages, but exhibited no effect on TLR2/4-deficient or MyD88-deficient mouse macrophages. Mutation of D-isoGln to L-isoGln in monoacylated MDP did not result in loss of the DC maturation activity, suggesting marginal participation of nucleotide-binding oligomerization domain 2, if any, in monoacyl MDP-dependent DC maturation. These results define the adjuvant activity of 6-O-acyl MDP compounds at the molecular level. They target TLR2/TLR4 and act through the MyD88-dependent pathway in DCs and macrophages. Hence, the unusual combined activation of TLR2 and TLR4 observed with Mycobacterium tuberculosis is in part reflected in the functional properties of monoacylated MDP compounds. These findings infer that the essential minimal requirement for TLR2/4-mediated adjuvancy of BCG lies within a modified MDP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Microorganisms have unique pattern molecules that the host immune system selectively recognizes by their specific receptors. TLRs (1, 2) on cell membrane and nucleotide-binding oligomerization domain (NOD)4 family proteins (3) in cytoplasm are representatives of such microbial pattern recognition receptors. They trigger a defense response in the host immune-competent cells. The minimal structural motif of ligands for each pattern recognition receptor has been examined, mainly using synthetic compounds (4, 5, 6). They usually exhibit weak receptor-activating ability, but retain the receptor usages and inducible cytokine profiles compared with parent pattern molecules. We have elucidated that the peptidoglycan (PGN) structure of bacillus Calmette-Guérin (BCG) specifically activates both TLR2 and TLR4 (7), although most of the PGN of Gram-positive bacteria stimulates TLR2, but not TLR4 (8, 9). The minimal essential requirement for simultaneous activation of TLR2 and TLR4 by BCG components remains unidentified. The purpose of the present study was to identify the structural signature for TLR2/4 activation using synthetic compounds.

Muramyldipeptide (MurNAc-L-Ala-D-isoGln; MDP) is the minimal structural unit of the PGN in bacterial cell wall skeleton (CWS) that is required to sustain the induction of the PGN-mediated immune response in host cells (10). It is recognized by NOD2, but not by TLRs, to induce activation of NF-{kappa}B (11). MDP-mediated NF-{kappa}B activation, however, inefficiently promotes TNF-{alpha} protein production. Translation of NF-{kappa}B-dependent cytokines is enhanced when TLR signaling coincides with MDP-mediated NF-{kappa}B activation (12). Thus, MyD88-dependent TLR signaling is crucial even for NOD2-dependent cytokine production. These results also have far-reaching implications in understanding adjuvant function and consequent effective vaccine development.

Using specific TLR-blocking mAbs, we have shown that TLR2 and TLR4 are receptors for BCG-PGN in human monocyte-derived dendritic cells (DCs) (7). Experiments with macrophages from TLR knockout mice have largely supported the possible application to analysis of BCG-mediated human cells as well (7, 13). The receptor usage of BCG-PGN is atypical, because most bacterial PGNs are exclusively recognized by TLR2 (1, 2, 8, 9). MDP is a main constituent of BCG-PGN, but has no ability to activate TLR2 or TLR4 (14). Thus, the precise patterns and distinct motifs in BCG-PGN (portion other than MDP) that are required for recognition and ligand activity of TLR2 or TLR4 (1, 14, 15, 16) remain unclear. These observations along with the known role of NOD2 (3) suggest that the molecular base of BCG-PGN recognition by DCs is more complex.

The PGN of BCG-CWS (BCG-PGN) binds galactofranose chains via the linker region (L-Rham-D-Gal) and phosphoric acid (17). The putative binding site of this sugar chain is the 6-O portion of MDP (18). Using activation markers of human dendritic cells (DCs), we studied molecular bases for activation of DCs by synthetic MDP derivatives. We found that TLR2 and TLR4 are simultaneously activated by the MDP derivatives with 6-O-octanoyl or stearoyl acylation. The monoacylated MDP exerts DC maturation activity with a profile similar to that of BCG-PGN, but is generally less potent than BCG-PGN when judged by cytokine induction. The DC maturation activity of the monoacyl MDP is independent of the NOD2 recognition peptide sequence, L-Ala-D-isoGln. Thus, the structural signature of TLR2/4-mediated MyD88 activation should be present within the molecular structure of monoacylated MDP.


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

FCS was obtained from BioWhittaker; DMEM and RPMI 1640 were purchased from Invitrogen Life Technologies; GM-CSF and IL-4 were obtained from PeproTech EC; LPS (Escherichia coli O127:B8) was obtained from Difco; peptidoglycan from Staphylococcus aureus was obtained from Fluka-Chemie; MDP was purchased from Calbiochem; MDP derivatives produced in Dr. S. Kusumoto’s laboratory (Osaka University, Osaka, Japan); mouse Ig G2b (IgG2b) was obtained from Sigma-Aldrich; anti-CD83 mAb was purchased from Cosmo Bio; anti-CD80 mAb was obtained from Immunotech; anti-CD86 mAb was obtained from Ancell; anti-HLA-DR (Immu-357) and anti-{beta}2-microglobulin mAbs were purchased from Immunotech; anti-TLR4 (HTA125) was a gift from Dr. K. Miyake (University of Tokyo, Tokyo, Japan); anti-TLR2 (TLR2.45) was produced in our laboratory as described previously (7, 19); FITC-conjugated goat anti-mouse IgG F(ab')2 was obtained from American Qualex. A well-characterized antagonist of LPS for TLR4 stimulation (E5531) was a gift from Eisai (20, 21).

Synthetic MDP derivatives

The MDP derivatives used in this study were synthesized as reported previously (22, 23, 24). Briefly, MDP with 6-O-acylation and those with long fatty acid chains were synthesized by the method of Kusumoto et al. (22, 23, 24). Fig. 1 shows the chemical formulas of the compounds used. 6-O-acylated MDP with L-Ala-L-isoGln dipeptide (6Ste(LL)-MDP) was provided by Dr. S. Kusumoto (25).



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  		FIGURE 1. Schematic representation of the synthetic MDP derivatives used in this study. R1 and R2 of MDP were replaced as follows: 6Dec-MDP R1, decanoyl residue; R2, H; 4,6Dec-MDP R1,R2, decanoyl residue; 6Ste-MDP R1, stearoyl residue; R2, H; 4,6Ste-MDP R1, R2, stearoyl residue.

 
Preparation of human immature DCs (iDCs)

Human PBMC were prepared from 400 ml of citrate-phosphate-dextrose-supplemented blood by methylcellulose sedimentation and density gradient centrifugation with Ficoll-Hypaque (Amersham Biosciences). Monocytes were isolated from PBMC with a magnetic cell sorting system by using anti-CD14-coated microbeads (Miltenyi Biotec). The iDCs were generated from monocytes (5 x 105 cells/ml) by culturing them for 6 days in RPMI 1640 (10 ml) supplemented with 10% heat-inactivated FCS in the presence of human GM-CSF (500 IU/ml) and human IL-4 (100 IU/ml). Preparations of iDCs were checked for the surface markers CD14–, CD40+, CD83–, CD80low, CD86low, and CD1a+ before use (13).

DC maturation

The iDCs were cultured (5 x 105 cells/ml) for 24 h in RPMI 1640 containing10% heat-inactivated FCS with GM-CSF (500 IU/ml) and human IL-4 (100 IU/ml). Then they were incubated for 24 h with LPS (100 ng/ml), S. aureus-PGN (Staph-PGN; 15 µg/ml), MDP, or its derivatives (1–20 µg MDP eq/ml). The cells were collected at 4°C by gentle pipetting with PBS containing 10 mM EDTA. DC maturation was assessed by testing for the surface markers CD80high, CD86high, CD83+, and CD1a+ (13).

Preparation of TLR2-, TLR4-, or MyD88-deficient mouse macrophages

MyD88-deficient (26) and TLR2/TLR4-deficient mice (27) were generated as described previously. Mouse macrophages were prepared as described previously. Briefly, mice (F2 interbred from 129/Ola x C57BL/6) were i.p. injected with 2 ml of 4% thioglycolate medium (Difco). After 72 h, peritoneal invasive cells were isolated from the peritoneal cavity by washing with ice-cold HBSS. Adherent monolayer cells were used as peritoneal macrophages (26).

Flow cytometric analysis of cell surface Ags

Cells were suspended in PBS containing 0.1% sodium azide and 0.1% BSA and were incubated with 5 µg of mAb for 30 min at 4°C. The cells were washed and counterstained with FITC-conjugated goat anti-mouse IgG F (ab')2 for 30 min at 4°C. Fluorescence intensity and mean fluorescence shifts were then determined by flow cytometry (FACSCalibur; BD Biosciences) (13).

ELISA

Human iDCs (5 x 105 cells/well) were plated in duplicate in 96-well, flat-bottom plates in the presence or the absence of anti-TLR2 mAb (10 µg/ml), anti-TLR4 mAb (10 µg/ml), or E5531 (10 µM) (7). The iDCs were stimulated with polymyxin B-treated Staph-PGN, polymyxin B-free LPS (100 ng/ml), or MDP or its derivatives (15 µg MDP eq/ml) for 24 h at 37°C. Appropriate amounts of these TLR agonists were determined based on the levels of surface markers, indicating the degree of DC maturation. For TLR inhibition studies, cells were incubated with each TLR inhibitor for 30 min at 4°C before the stimuli were added. Levels of human cytokines (TNF-{alpha}, etc.) in culture supernatants were measured by ELISA (Amersham Biosciences) as described previously (13).

In mouse experiments, peritoneal macrophages (2.5 x 105 cells/ml) were prepared from wild-type (C57BL/6) mice and TLR2-, TLR4-, TLR2/4-, or MyD88-deficient mice as previously described (26, 27). Cells were cultured in duplicate in 24-well, flat-bottom plates with polymyxin B-treated Staph-PGN (15 µg/ml) or polymyxin B-free LPS (100 ng/ml), MDP, and their derivatives (15 µg MDP eq/ml) for 24 h at 37°C (11, 13). Concentrations of TNF-{alpha} in culture supernatants were determined in duplicate by ELISA (Amersham Biosciences) as described previously (13, 28).

MLR assay

For the MLR assay, iDCs were generated from human monocytes (5 x 105 cells/ml) that were cultured for 6 days with GM-CSF and IL-4 (13), followed by incubation for 24 h in medium alone or medium containing polymyxin B-treated Staph-PGN (15 µg/ml), or MDP and its derivatives (15 µg MDP eq/ml). The iDCs and stimulated DCs were irradiated (3000 rad; 137Cs source) and cultured in 100 µl of RPMI 1640 medium containing 10% heat-inactivated FCS for 3 days with 1 x 105 allogeneic PBMC in 96-well, flat-bottom plates. The levels of PBMC proliferation in culture medium were measured by CellTiter 96 nonradioactive cell proliferation assay kit (Promega).

RT-PCR

Cells were harvested by trypsin treatment, and total RNA was extracted using an RNeasy mini kit (29). Two micrograms of total RNA was incubated at 70°C for 5 min and kept on ice for 2 min, and RT was performed with Moloney murine leukemia virus reverse transcriptase at 37°C for 90 min. PCRs were performed with long and accuracy (LA)-Taq polymerase using the primers listed in Table I.


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  		Table I. PCR primers used in this studya

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Structures of MDP derivatives

The four derivatives of MDP are shown in Fig. 1, and other derivatives with different lengths of fatty acids were synthesized as described previously (22, 23, 24, 25). Diacylated and monoacylated MDPs with different lengths of chains were used to test their functions. The methods of synthesis and the properties of these derivatives were reported previously (22, 23, 24). 6-O-octanoyl and 6-O-stearoyl MDPs (6Dec-MDP and 6Ste-MDP) were mainly used for the following experiments.

DC maturation induced by MDP derivatives

Consistent with the earlier reports, MDP exhibited no ability to up-regulate CD80, CD86, or CD83 on human monocyte-derived DCs (13). Under similar doses and conditions, 6Dec-MDP and, to a lesser extent, 6Ste-MDP up-regulated CD83 and CD86 on DCs (Fig. 2). 6Dec-MDP effectively up-regulated CD83 and CD86. Twenty micrograms of 6Dec-MDP was comparable in its potency to 15 µg/ml Staph-PGN or 0.1 µg/ml LPS, the representative agonists for TLR2 and TLR4, respectively. CD80 was also subtly induced by these MDP derivatives, but less prominently than Staph-PGN or LPS at the same weight ratio. 6Dec-MDP and 6Ste-MDP also induced high levels of MHC class I and class II in human DCs (Fig. 3A). These functions were not observed with intact MDP or diacylated 4,6Dec-MDP and 4,6Ste-MDP. Thus, of the synthetic MDP derivatives, 6-O-monoacyl MDPs possess the ability to mature DCs.



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  		FIGURE 2. Up-regulation of DC maturation markers by 6Dec- or 6Ste-MDP. Monocyte-derived iDC (5 x 105 cells/ml) were stimulated with medium alone (none), Staph-PGN (15 µg/ml), LPS (100 ng/ml), MDP, 6Dec-MDP, 4,6Dec-MDP, 6Ste-MDP, or 4,6Ste-MDP (1–20 µg MDP eq/ml). After 24 h, the cells were harvested, and the levels of expression of CD80, CD83, and CD86 were estimated by flow cytometry. Data from one of the three similar experiments are shown.

 


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  		FIGURE 3. Human DC maturation induced by monoacyl MDP. A, Up-regulation of MHC class I and class II in DCs by monoacyl MDP. Monocyte-derived iDCs were stimulated with medium alone, MDP, or monoacyl MDP (6DecMDP in this figure) for 24 h. Cells were then washed, treated with isotype-matched IgG or mAb against MHC-DR, {beta}2-microglobulin, or CD83, then treated with FITC-labeled second Ab and analyzed by flow cytometry. Basal shifts by IgG control are shown to the left (overlapping two thin lines). The fluorescence intensities are shown to the right. Gray lines indicate iDCs with no stimulation; thick solid lines indicate iDCs after monoacyl MDP stimulation. MDP did not allow the up-regulation of these molecules (not shown). Samples from two donors are shown. B, Allogeneic MLR using DCs treated with 6Dec-MDP or 6Ste-MDP. The iDCs were cultured for 24 h in medium alone or medium containing Staph-PGN (15 µg/ml) and with MDP, 6Dec-MDP, or 6Ste-MDP (15 µg MDP eq/ml). After 24 h, these DCs were irradiated and cultured for 3 days with allogeneic PBMC, and PBMC proliferation was measured using a cell proliferation assay kit. Data from one of the three similar experiments are shown.

 
The function of MLR induction by DCs matured by MDP derivatives was tested next. Although MDP had no ability to activate DCs, consistent with previous reports (30), 6Dec-MDP and 6Ste-MDP exerted potent DC-mediated MLR induction. Consistently, the degree of lymphocyte proliferation was similar to that induced by Staph-PGN (Fig. 3B). Thus, MDP derivatives appear to confer maturation on monocyte-derived DCs by augmenting MLR and the expression of MHC and costimulators.

Production of cytokines in DCs stimulated with MDP derivatives

DCs stimulated with various maturation inducers induce inflammatory and instructive cytokines in a stimulator-specific manner. The cytokine-producing profiles in DCs stimulated with MDP derivatives were examined by ELISA. The results showed that TNF-{alpha} (>600 pg/ml) could be detected in the supernatant of DCs matured by monoacyl 6Ste- or 6Dec-MDP (Fig. 4A). However, the levels of TNF-{alpha} induced by 6Ste- or 6Dec-MDP were 10-fold less than those induced by Staph-PGN or LPS. Other cytokines, such as IL-1{beta}, IL-6, and IL12p40, were also produced by DCs in response to 6Ste-MDP (not shown) or 6Dec-MDP; their expression profiles were similar to that of BCG-CWS (Table II), but the amount produced was less than that by BCG-CWS-stimulated DCs. IL-12p40 and TNF-{alpha} productions were increased in DCs in a dose-dependent fashion (Fig. 4B). Although cytokine levels did not reach the levels induced by BCG-CWS at the doses tested, ligand density would be an important factor for monoacyl MDP-mediated cytokine induction in DCs. Together, the MDP derivatives induce cytokines in DCs in a similar profile as BCG-CWS. Both BCG-CWS and MDP derivatives failed to induce IFN-inducible protein 10 (IP10), which was produced by LPS in DCs (Table II and Fig. 4C); monoacyl MDP was distinct from LPS in the manner of activation of TLR4 signaling.



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  		FIGURE 4. Induction of cytokines in human DC treated with MDP derivatives. A, The iDC were cultured for 24 h in medium alone, medium containing Staph-PGN, or either MDP or MDP derivatives. After 24 h, the levels of TNF-{alpha} in the culture supernatants were measured by ELISA. The amounts of stimulators are: Staph-PGN, 15 µg/ml (left panel); and MDP, 6Dec-MDP, 4,6Dec-MDP, 6Ste-MDP, and 4,6Ste-MDP, 15 µg MDP eq/ml (right panel). ND, not detected. B, Production of TNF-{alpha} and IL-12p40 by iDC in response to MDP derivatives. The iDC were stimulated with the indicated concentrations of MDP, 6Dec-MDP, and 6Ste-MDP. TNF-{alpha} levels in the supernatants were measured by ELISA. One of the three independent experiments is shown. C, Cytokine profiling induced in ligand-stimulated DCs. The iDCs were cultured for 6 h in medium alone or in medium containing Staph-PGN, BCG-CWS, LPS, or the indicated MDP derivatives. Then the levels of cytokine messages in DCs were assessed by RT-PCR using specific primers. DCs were prepared from three donors. GAPDH and {beta}-actin were used as controls.

 

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  		Table II. Cytokine levels liberated from DCs stimulated with TLR ligandsa

 
Until 10 h, cytokine production by DCs stimulated with 6Dec-MDP vs LPS were not markedly different (<2-fold), except for IL-6 (Table II), suggesting that lacking late-induced events such as internalization (13) and/or activation of I{kappa}-B{zeta} (31) in monoacyl MDP causes a lesser cytokine production. Even in the late phase, IFN-{beta}- and IFN-inducible genes (represented by IP10) were barely induced by MDP derivatives or BCG-CWS (data not shown). Although donor-to-donor differences in the level of production of each cytokine by DCs were significant (Table II), the overall cytokine profiles induced in DCs by monoacyl MDP were similar among the donors. The MDP-mediated induction of these cytokines was not blocked by the addition of polymyxin B (not shown), which excludes the possibility of participation of contaminating LPS in the cytokine induction. Neither MDPs nor their derivatives with diacylation virtually promoted any TNF-{alpha} production.

mRNA levels of other cytokines were examined by RT-PCR (Fig. 4C). The results show that TNF-{alpha}, IL-12 p40, IL-23 p19, IL-6, and IL-1{beta}, but not IL-12 p35, IFN-{beta}, or IP10, were induced in DCs stimulated either with 6Dec- or 6Ste-MDP (Fig. 4C). The results were similar with 6Dec-MDP and BCG-PGN (not shown). Likewise, Staph-PGN induced these cytokine messages, but failed to induce IL-12p35, IFN-{beta}, and IP10 in DCs. LPS is an agonist of TLR4 and induces IL-12p35 and IP10. Again unlike LPS, 6Ste- or 6Dec-MDP barely induced IP10 or IL-12p35, although they are TLR4 ligands. These properties of monoacyl MDP are shared with those of BCG-PGN or BCG-CWS, suggesting that these BCG compounds commonly activate TLR2/4 via MyD88, but fail to activate the Toll-IL-1R homology domain (TIR)-containing adapter molecule-1 (TICAM-1)/TIR-domain-containing adapter-inducing IFN-{beta} (TRIF)-dependent pathway (29, 32).

Based on morphological changes and activation profiles (CD45RA vs RO), none of these compounds elicited activation of any lymphocyte population through direct interaction (data not shown). Furthermore, IL-18R or IL-12R complexes were not induced on lymphocytes in response to these compounds (data not shown). This suggests that DC maturation is an event specifically induced by these synthetic reagents.

MDP derivative-mediated DC maturation is TLR2/4 and MyD88 dependent

Using TNF-{alpha} production as a marker, we examined whether monoacylated MDPs activate DCs via TLRs. Although other TLR-blocking mAbs barely inhibited TNF-{alpha} production in human DCs, function-blocking mAbs against TLR2 or the TLR4 inhibitor E5531 partly (~50%) suppressed TNF-{alpha} production (Fig. 5A). Likewise, function-blocking mAbs against TLR4 partly inhibited TNF-{alpha} production, similar to E5531 (Fig. 5B). Combination usage of anti-TLR2 mAb and E5531 for TLR2/4 function blocking resulted in ~80% inhibition of TNF-{alpha} liberation (some data shown in Fig. 5C). Control studies using a TLR2 agonist (Staph-PGN) and a TLR4 agonist (LPS) showed that they inhibited TNF-{alpha}-inducing activity by >75 and >95%, respectively. Hence, we conclude that the monoacylated MDPs interact with TLR2 and TLR4 on the DC surface, resulting in function blockade by mAbs.



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  		FIGURE 5. Production of TNF-{alpha} by 6Dec-MDP or 6Ste-MDP in DCs is TLR2 and TLR4 dependent. A, The iDCs were precultured with medium as control (Cont), isotype-matched control IgG (10 µg/ml), anti-TLR2 mAb (10 µg/ml), or E5531 (10 µM) for 30 min at 4°C. Cells were then stimulated with Staph-PGN (15 µg/ml), LPS (100 ng/ml), MDP (15 µg MDP eq/ml), 6Dec-MDP (15 µg MDP eq/ml), or 6Ste-MDP (15 µg MDP eq/ml). Twenty-four hours after stimulation, TNF-{alpha} production was measured by ELISA. ND, not detected. Results from one of two similar experiments are shown. B, The iDCs were pretreated with medium only (None), anti-TLR4 mAb (10 µg of HTA125), or E5531 (10 µM) for 40 min at 4°C. Cells were then stimulated with the indicated TLR agonists (amounts described above). Twenty-four hours after stimulation, TNF-{alpha} production was measured by ELISA. ND, not detected. C, The iDCs were pretreated with medium only (None), isotype-matched control IgG (10 µg/ml), anti-TLR2 mAb (10 µg/ml), anti-TLR4 mAb (10 µg/ml), or anti-TLR2/anti-TLR4 mAbs (10 µg/ml each) for 40 min at 4°C. Cells were then stimulated for 24 h with 15 µg MDP eq/ml 6Ste(LL)-MDP (see the left scheme). TNF-{alpha} levels are shown as described in B. One representative result is shown. D, Overexpressed TLR2 plus TLR1, TLR2 plus TLR6, and TLR4 plus CD14 do not respond to monoacylated MDPs. HEK293 cells were transfected with the indicated combinations of plasmids (see the top of each panel). Twenty-four hours later, cells were stimulated with the indicated TLR stimulators (see upper right panel). Twenty-four hours after stimulation, NF-{kappa}B luciferase assay was performed with these transfectants. MALP-2 (a ligand for TLR2 plus TLR6), Pam3 (a ligand for TLR2 plus TLR1), and LPS (a ligand for TLR4) were used as positive controls. Although data are not shown, simultaneous expression of TLR2 and TLR4/CD14 on 293 cells barely responded to 6Ste-MDP (15 µg of MDP eq/ml). MDP was used as a negative control. Of note, TLR4 alone barely responded to LPS as in previous reports (21 ). Relative activation values are shown on the y-axis.

 
NOD2 was identified as a cytoplasmic sensor for the dipeptide portion of MDP (L-Ala-D-isoGln) (11). To test whether monoacyl MDPs activate NOD2 signaling, we synthesized a mock ligand to NOD2, 6Ste-MDP with L-Ala-L-isoGln dipeptide (6Ste(LL)-MDP). We determined its ability to induce TNF-{alpha} and the degree of inhibition by TLR2/4-blocking mAbs (Fig. 5C). D-IsoGln-containing and L-isoGln-containing 6Ste-MDPs induced almost identical levels of TNF-{alpha} in DCs. TLR2 and TLR4 blockers conferred similar inhibitory profiles on 6Ste(LL)-MDP-dependent TNF-{alpha} induction. Thus, NOD2 marginally, if at all, affects DC maturation in our system using monoacyl MDPs.

We tested whether TLR2 in conjunction with TLR1, TLR6, or TLR4 with CD14 could recognize the monoacylated MDP (Fig. 5D). In the overexpression studies, any of these combinations failed to activate NF-{kappa}B promoter above the level of TLR2 alone in HEK293 cells in response to monoacylated MDP (Fig. 5D). Thus, coupling of other TLRs with TLR2 on HEK293 cells did not simply reflect monoacylated MDP recognition in DCs. TLR4 recognized LPS only in the presence of MD-2 and CD14, and under these conditions, the TLR4 complex barely recognized monoacylated MDP (Fig. 5D). Simultaneous expression of TLR2 and TLR4 again failed to respond to MDP derivatives in HEK293 cells (data not shown). Thus, the MDP derivatives failed to activate reporters in the relevant TLR-expressing HEK293 cells, which is contrary to our expectation. These results are essentially consistent with previous unexpected results when we used BCG-CWS and PGN as TLR stimulators (7, 13). Possibly, an unidentified molecule expressed in DCs, but not HEK293 cells, plays an essential role in BCG constituent-mediated NF-{kappa}B activation. In fact, a catch-up receptor for PGN, such as peptidoglycan recognition protein (PGRP) or intelectin, is necessary for internalization of PGN and subsequent NF-{kappa}B activation in some cell systems (2, 33). We surmise that this is true in monoacyl MDP-mediated activation of NF-{kappa}B.

Finally, the TLR2/TLR4 receptor usage and signal selection via MyD88-dependent pathway for induction of TNF-{alpha} were confirmed using TLR2-, TLR4-, and MyD88-deficient macrophages prepared from gene-disrupted mice (Fig. 6). The results showed that 6Dec- or 6Ste-MDP allowed the production of TNF-{alpha} in wild-type mouse macrophages, but not MyD88–/– macrophages (Fig. 6A). Staph-PGN-mediated TNF-{alpha} production was also completely terminated in MyD88–/– DCs (Fig. 6A), but LPS-mediated TNF-{alpha} production remained in MyD88–/– macrophages to a minimal extent. 6Dec-MDP- and 6SteMDP-mediated TNF-{alpha} productions were partly impaired in mouse macrophages lacking TLR2 or TLR4 (Fig. 6B). More than 95% of monoacyl MDP-dependent TNF-{alpha} production was impaired in mouse macrophages lacking both TLR2 and TLR4 (Fig. 6B). Thus, the monoacylated MDP activates mouse macrophages with TLR2 and TLR4 via the MyD88-dependent pathway. Low induction of TNF-{alpha} in mouse macrophages in response to MDP derivatives compared with BCG-CWS was again observed. This tendency was prominent if the culture interval exceeded 6 h (data not shown).



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  		FIGURE 6. TNF-{alpha} induced by 6Dec-MDP or 6Ste-MDP in mouse macrophages. A, Mouse peritoneal macrophages were prepared as described previously (26 27 ). Peritoneal macrophages (2.5 x 105 cells/ml) from wild-type and MyD88-deficient mice were cultured for 24 h with medium alone (None) or medium containing Staph-PGN (15 µg/ml) or LPS (100 ng/ml; left panel). The same lots of macrophages were cultured for 24 h with 15 µg MDP eq/ml MDP, 6Dec-MDP, or 6Ste-MDP (right panel). The levels of mouse TNF-{alpha} in the culture supernatants were measured by ELISA. One of three similar experiments is shown. ND, not detected. B, Mouse macrophages (2.5 x 105 cells/ml) from wild-type and TLR2-, TLR4-, or TLR2/4 double-deficient mice were cultured for 24 h with medium only (None), Staph-PGN (15 µg/ml), or LPS (100 ng/ml; left panel). The same lots of macrophages were similarly cultured for 24 h with 15 µg of MDP eq/ml MDP, 6Dec-MDP, or 6Ste-MDP (right panel). The TNF-{alpha} levels are shown as described in A.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study we synthesized MDP derivatives with 6-O acylation, such as 4-N-acetylmuramyl-L-alanyl-D-isoglutaminyl-6-O-acylation (10), and demonstrated that monoacylated MDP derivatives 6Ste- and 6Dec-MDP activate monocyte-derived DCs similar to the BCG-PGN adjuvant. The agonistic activity of TLR2 and TLR4 of MDP derivatives was responsible for the DC maturation. MDP with no modification and diacylated MDP derivatives exhibited no activity for DC maturation even at high dosage. A NOD2 unstimulatory mutant of monoacylated MDP still reserved DC maturation activity. Hence, the minimal structural requirement for MyD88-dependent adjuvant activity of BCG-CWS (13) and BCG-PGN (7) is reflected in MDP with a single stretch of 6-O-acylation. This unique TLR activation profile in DCs may be a base for the potent in vivo adjuvancy of CFA and probably Mycobacterium tuberculosis (14).

The potency of cytokine production was relatively low in DCs stimulated with monoacylated MDP compared with BCG-CWS. At early time points, differences in cytokine levels between the two stimulators were not very marked. Probably, monoacyl MDP is a short-acting stimulator compared with BCG-CWS, which may explain the 100-fold lower induction of TNF-{alpha} by MDP derivatives than by BCG-CWS. A prominent structural difference is that MDP is a monomer, whereas BCG-CWS consists of multimeric MDPs with a sugar chain network (17). Additional structural moiety sustaining multimerization of MDP would be crucial for the robust immune response induced by BCG-CWS.

Unexpectedly, the TLR2/TLR4 usage by monoacyl MDP was not reproduced in the HEK293 expression system. In previous studies, BCG-CWS and BCG-PGN failed to activate TLR2/TLR4 in a similar overexpression system. Our interpretation was that an additional catch-up receptor for BCG-PGN, which exists in DCs, but not 293 cells, is required for effective NF-{kappa}B activation (13). However, transfection of plasmids for expression of PGRPs (peptidoglycan-recognition proteins) (34), DC-sign (35), or Dectin-1 (36) did not result in recovery of the function of monoacyl MDP in 293 cells (data not shown). The molecular bases required for recognition of monoacylated MDP need further investigation.

According to previous reports, TLR2 more efficiently responds to diacyl lipopeptide (MALP-2) with TLR6 or triacyl lipoprotein (Pam3) with TLR1 (37, 38, 39, 40). TLR5 can recognize flagellin in conjunction with TLR4 to induce type I IFN (41). The receptor complex that maximizes the response to monoacyl MDP has to be further elucidated. TLR2 and TLR4 appear to individually participate in recognition of the MDP derivatives, because blocking of either one only partially suppressed TNF-{alpha} production. TLR10 in conjunction with TLR2 is not a candidate for inducing an effective response to MDP derivatives (J. Uehori and T. Seya, unpublished observation). In conclusion, simple overexpression of TLR2 and/or TLR4/MD-2/CD14 on HEK293 cells did not result in MDP derivative-mediated NF-{kappa}B activation.

Recent reports suggested that NOD2 in the cytoplasm serves as an intracellular receptor for the L-Ala-D-isoGln peptide of MDP and NOD1 for the diaminopimelic acid-containing moiety of PGN (3, 10, 11, 42, 43). MDP of BCG-CWS possesses these structural signatures. However, we favor the interpretation that NOD2 activation does not virtually occur with monoacylated MDP. Firstly, blocking reagents of TLR2 and TLR4 suppress monoacyl MDP-mediated TNF-{alpha} production. Secondly, TNF-{alpha} production by monoacyl MDP is MyD88 dependent, supporting the participation of the TLR-MyD88 pathway in monoacyl MDP-dependent DC maturation. Thirdly, the cytokine profiles induced by 6Dec- and 6Ste-MDPs are almost identical with those of BCG-CWS and BCG-PGN. Intact MDP is recognized by NOD2 (11), but not by TLR2 or coexpressing TLR2 and TLR1 or TLR2 and TLR6. Finally, the NOD2-unstimulatory mutant 6Ste(LL)-MDP exhibits sufficient TNF-{alpha}-inducing activity. MDP-NOD2-mediated NF-{kappa}B activation appears distinct from the acyl MDP-induced TLR2/4-derived response.

A stearoyl-lysine MDP derivative serves as an adjuvant that exhibits biological activity, such as protection against a variety of bacterial, fungal, and viral infections in animals (44, 45). It appears to be a useful immunoadjuvant for vaccination against microbial agents. In addition, the stearoyl-lysine MDP derivative inhibits tumor metastasis by eliciting tumor immunity in mice. It also induces IL-1{beta}, IL-6, TNF-{alpha}, and GM-CSF (45). These functional profiles of the stearoyl-lysine MDP are reminiscent of the immunoadjuvant properties of BCG-CWS (46). However, it is unlikely that BCG-CWS and stearoyl-lysine MDP derivatives share the activation route in DCs, because stearoyl-lysine MDP does not activate any TLR (45) or adapters (44). Hence, we demonstrated that the synthetic monoacylated MDP is functionally distinct from stearoyl-lysine MDP.

Kusumoto et al. (22, 23, 24) have studied functional profiles of synthetic MDP derivatives since the 1970s. These compounds were active as adjuvants for eliciting cell-mediated immune responses in guinea pigs and mice, but were less active in the production of Abs than BCG-CWS (10). In particular, these compounds had virtually little ability to regress most of the implanted tumors (30, 47, 48). However, these earlier studies were performed when knowledge about the target cells for adjuvants and TLRs was not well established. Our studies are the first to characterize the adjuvant activities of these MDP derivatives using a TLR function-blocking system in ex vivo experiments and human monocyte-derived DC as a target of monoacyl MDP. This synthetic compound induces DC maturation, augmenting cytokine production and costimulator expression. Thus, the active center sustaining the BCG-CWS adjuvancy may contain a modified MDP.

Monoacylated MDP triggers a gene-inducing program similar to that of BCG-CWS in human monocyte-derived macrophages; 185 genes are BCG specific (K. Ishii and T. Seya, unpublished observations). The gene regulatory profile of the monoacylated MDP was similar to that of BCG-CWS (49). No liberation of IFN-inducible genes was noticed in either case (data not shown), suggesting no involvement of the TRIF/TICAM-1 pathway (29, 31). Because BCG-CWS has been applied in immunotherapy for cancer (46, 50), synthetic MDP derivatives with high DC maturation potential may serve as good candidates and clinically useful adjuvants that clear Good Manufacturing Practice criteria and promise high quality of life at low cost. Unfortunately, in previous reports monoacyl MDP could not exert sufficient adjuvancy in a tumor-implanting mouse model (30, 48). We now have knowledge about how BCG-CWS and its compounds function to regress tumor in the immune system (51). Designing new derivatives with high adjuvant activity will immensely help the advancement of adjuvant immunotherapy for cancer.


   Acknowledgments
 
We are grateful to Drs. K. Miyake (University of Tokyo, Tokyo, Japan) and R. Dziarski (Indiana University, Gary, IN) for providing function-blocking mAb against human TLR4 and PGRP cDNA plasmids, respectively. We thank Drs. H. Ishii, M. Tanabe, N. A. Begum, and N. O. Inoue (Osaka Medical Center for Cancer, Osaka, Japan) for invaluable discussions, and Dr. V. Kumar (St. Louis University, St. Louis, MO) for critical reading of the manuscript.


   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 in part by Core Research for Engineering, Science, and Technology; the Japanese Society for Technology; grants-in-aids from the Ministry of Education, Science, and Culture (Specified Project for Advanced Research); the Ministry of Health and Welfare of Japan; the Naito Memorial Foundation (to M.M.); and the Takamatsu Princess Cancer Foundation. Back

2 Current address: Suntory Institute for Bioorganic Research, Wakayamadai 1-1-1, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan. Back

3 Address correspondence and reprint requests to Dr. Tsukasa Seya, Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-8638, Japan. E-mail address: seya-tu{at}med.hokudai.ac.jp Back

4 Abbreviations used in this paper: NOD, nucleotide-binding oligomerization domain; BCG, bacillus Calmette-Guérin; CWS, cell wall skeleton; DC, dendritic cell; iDC, immature DC; IP10, IFN-inducible protein 10; MDP, muramyl dipeptide; PGN, peptidoglycan; PGRP, peptidoglycan recognition protein; Staph-PGN, Staphylococcus aureus-PGN; TIR, Toll/IL-1R hemology domain. Back

Received for publication October 28, 2004. Accepted for publication March 17, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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