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(Journal of Leukocyte Biology. 2001;70:723-729.)
© 2001 by Society for Leukocyte Biology

Muramyl dipeptide-Lys stimulates the function of human dendritic cells

Akihito Todate, Takafumi Suda, Hiroshi Kuwata, Kingo Chida and Hirotoshi Nakamura

Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan

Correspondence: Kingo Chida, M.D., Ph.D., 3600 Handa-cho, Hamamatsu, Shizuoka 431-3192 Japan. E-mail: chidak11{at}hama-med.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Muramyl dipeptide (MDP)-Lys (L18), a synthetic MDP analogue derived from bacterial cell walls, has been reported to be a potent immunoadjuvant that enhances protective immunity against pathogens and tumors by stimulating immune-competent cells, such as monocytes and macrophages. However, it is not known whether MDP-Lys modulates the function of dendritic cells (DCs), which are the most potent antigen-presenting cells and play a crucial role in initiating T cell-mediated immunity. Therefore, we examined the effects of MDP-Lys on the expression of surface molecules, cytokine production, and antigen-presenting function of human DCs generated from peripheral blood cells in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor. We found that MDP-Lys markedly up-regulated the expression of CD80, CD83, CD86, and CD40, but not human leukocyte antigen-DR, and stimulated the production of tumor necrosis factor-{alpha}, IL-6, IL-8, IL-10, and IL-12 (p40) by human DCs in a dose-dependent manner. Furthermore, MDP-Lys-treated DCs showed enhanced antigen-presenting function compared with untreated DCs, as assessed by an allogeneic mixed lymphocyte reaction. These results suggested that the immunoadjuvant activity of MDP-Lys in vivo is mediated, in part, by its stimulation of DC function.

Key Words: immunoadjuvant activity • antigen-presenting cells • T cell immunity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Muramyl dipeptide (MDP) is the smallest structural unit responsible for the immunoadjuvant activity of the peptidoglycan of bacterial cell walls [1 , 2 ]. MDP has been shown to exert diverse biological effects on immunocompetent cells in vitro [3 ]. MDP enhances phagocytic and microcidal activities of monocytes and macrophages [4 , 5 ]. It can also induce the mitogenic response of B cells [6 , 7 ] and augment the expression of immunostimulatory molecules, such as major histocompatibility complex (MHC) class II, CD40, and intercellular adhesion molecule-1 on monocytes and B cells [8 , 9 ]. To date, a number of its analogues and derivatives have been synthesized. Among them, MDP-Lys (L18) (N2-acetylmuramyl-L-alanyl-D-isoglutaminyl-N6-stearoyl-L-lysine), a stearoyl-MDP derivative, has been reported to be an adjuvant that exhibits more biological activity and less pyrogenenicity than other MDP derivatives [10 , 11 ]. MDP-Lys was shown to enhance the host defenses against a variety of bacterial, fungal, and viral infections in animals [12 13 14 ]. For example, in vivo administration of MDP-Lys restored the resistance to herpes simplex virus and Escherichia coli infections in immunosuppressed mice [15 , 16 ] and enhanced the host defense against hantaan virus infection in newborn mice [17 , 18 ]. MDP-Lys was also demonstrated to augment the immunogenicity of various vaccines, such as inactivated hantavirus and recombinant hepatitis B surface antigen, indicating its usefulness as an immunoadjuvant for vaccination against viral infections [18 , 19 ]. More recently, MDP-Lys was shown to inhibit tumor metastasis by amplifying the antitumor immunity in mice [20 ]. Furthermore, it was reported that MDP-Lys acts as a potent inducer of various cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor (TNF), and colony-stimulating factors (CSFs) in monocytes and macrophages [21 , 22 ]. Taken together, these data show the immunotherapeutic potential of MDP-Lys against infections and tumors in humans. Although the precise mechanisms by which MDP-Lys exerts its immunoadjuvant activity in vivo have not been fully elucidated, the above studies have suggested that its primary targets are monocytes and macrophages. However, it is not known whether MDP-Lys can stimulate the function of dendritic cells (DCs), which are the most potent antigen-presenting cells (APCs) and play a central role in the initiation of protective immunity against microorganisms and tumors.

DCs have a highly developed function in the immune system as specialized APCs for the primary immune response [23 , 24 ]. DCs act as sentinels and are widely distributed in virtually all organs [25 ]. They are strategically positioned to take up antigens, after which they migrate to lymphoid organs, where they present the antigens to naive T cells, leading to the initiation of T cell immunity [23 ]. The ability of DCs to act as potent APCs is largely attributable to their strong expression of MHC and costimulatory molecules as well as their capacity to produce various cytokines. Recently, factors present during the innate phase of the immune response, such as bacterial products and proinflammatory cytokines, have been reported to enhance the expression of MHC and costimulatory molecules and to induce cytokine production by DCs, leading to their activation [26 27 28 29 ]. Therefore, we considered it possible that MDP-Lys could directly augment DC function and that this activity might also be implicated in its immunostimulatory effect in vivo.

Thus, this study was conducted to explore the effects of MDP-Lys on DC function. For this purpose, using human DCs generated by culturing peripheral blood cells in the presence of IL-4 and granulocyte-macrophage (GM)-CSF, we examined their expression of surface molecules, production of cytokines, and allostimulatory capacity after treatment with MDP-Lys. We found that MDP-Lys markedly up-regulated the expression of CD80, CD83, CD86, and CD40 and stimulated the production of TNF-{alpha}, IL-6, IL-8, IL-10, and IL-12 (p40) by human DCs, resulting in enhancement of their antigen-presenting function. These results suggest that the activation of DCs by MDP-Lys is involved in its immunoadjuvant activities in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media and reagents
The culture medium consisted of RPMI 1640 (Gibco BRL, Tokyo, Japan) supplemented with 2 mM L-glutamine, 10 mM HEPES, 20 µg/mL of gentamycin (Gibco BRL), and 10% heat-inactivated fetal calf serum (Gibco BRL). Recombinant human IL-4 was purchased from R&D (Minneapolis, MN). Recombinant human GM-CSF was obtained from PeproTech (London, UK). Lipopolysaccharide (LPS) from E. coli was purchased from Sigma (St. Louis, MO). MDP-Lys (L18), which was synthesized according to the modified method of Kotani et al [30 ], was a gift from Dai-ichi Seiyaku Co. Ltd. (Tokyo, Japan). The compound was verified to be free of endotoxin by Limulus lysate assay using Limulus-Test Wako4 (Wako Pure Chemical Industries, Ltd., Osaka, Japan).

Cell preparations
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood of normal healthy donors by density gradient centrifugation with a Lymphoprep centrifuge (Nycomed, Oslo, Norway). PBMCs were harvested from the interface and washed twice in phosphate-buffered saline (PBS) supplemented with 5 mM EDTA and 0.5% bovine serum albumin (Sigma). Subsequently, CD14+ cells were separated by magnetic sorting with a MACS cell sorter (Miltenyi Biotec, Berglsh Gladbach, Germany) according to the manufacturer’s instructions. Briefly, PBMCs were incubated with saturating concentrations of anti-CD3, anti-CD19, and anti-CD56 monoclonal antibodies (mAbs) conjugated with superparamagnetic microbeads for 15 min on ice and then washed in PBS containing 5 mM EDTA and 0.5% human serum. Unlabeled cells were then isolated by elution from magnetic columns, routinely resulting in >98% purity of CD14+ cells as assessed by flow cytometric analysis.

Generation of DCs from CD3- CD19- CD56- cell culture
Isolated CD3- CD19- CD56- cells (2x106/mL) were cultured in 24-well tissue culture plates (Costar, Cambridge, MA) in 1 mL of the culture medium containing 1,000 U/mL of GM-CSF, 1,000 U/mL of IL-4, and 1% human plasma (complete medium) at 37°C in a 5% CO2 incubator. Every 2 days, half the medium was removed and an equivalent volume of fresh complete medium was added. For most experiments, the cells were collected routinely after 7–10 days of culture. The cells thus obtained contained >95% DCs as assessed by morphology analysis and flow cytometric analysis with anti-CD1a mAb. For stimulation with MDP-Lys, after 7 days of culture, nonadherent cells were harvested, washed, and subcultured in concentrations of 106 cells/mL in 24-well plates in 1 mL of complete medium with or without various doses of MDP-Lys for 72 h.

Flow-cytometric analysis
For immunophenotyping, cultured cells were analyzed by dual-color flow cytometry. The cells were washed in PBS and incubated with the appropriately diluted phycoerythrin (PE)-conjugated anti-CD1a [clone BL6, mouse immunoglobulin (Ig)G1] (Immunotech, Marseilles, France), anti-CD83 (clone HB15a, mouse IgG2b) (Immunotech), anti-CD40 (clone MAB89, mouse IgG1) (Immunotech), anti-CD80 (clone MAB104, mouse IgG1) (Immunotech), anti-CD86 (clone IT2.2, mouse IgG2b) (PharMingen, San Diego, CA), and fluorescein isothiocyanate (FITC)-conjugated anti-human leukocyte antigen-DR (HLA-DR) (clone Immu-357, mouse IgG1) (Immunotech) mAbs for 30 min on ice. Parallel incubations were also performed with FITC- or PE-conjugated irrelevant antibodies matched for the isotypes as controls. The cells were washed in PBS and then analyzed with an EPICS® Profile-II flow cytometer (Beckman Coulter, Fullerton, CA). The expression of cell surface markers was evaluated in terms of the percentage of positive cells and the mean fluorescence intensity (MFI). The cutoff level for the definition of positive cells was thus set so that <1% of irrelevant antibody-stained cells were positive.

Cytokine assays
The levels of the cytokines IL-6, IL-8, IL-10, IL-12 (p40), and TNF-{alpha} in the culture supernatants were measured using enzyme-linked immunosorbent assay kits from R&D Systems (Minneapolis, MN).

Allogeneic MLR
Stimulator cells from DC cultures that had been cultured for 72 h with MDP-Lys or LPS were harvested, washed, and irradiated (2,000 rad). Allogeneic T cells were prepared from PBMCs using a T cell Recovery Column (Hornby, Canada). Subsequently, CD45RA+ cells were purified from the allogeneic T cell populations by magnetic cell sorting with anti-CD45RA mAbs conjugated with super-paramagnetic microbeads (Miltenyi Biotec). Various numbers of DCs were then placed in 96-well flat-bottom tissue culture plates (Corning, Acton, MA) alone or with allogeneic CD45RA+ T cells (2x105 cells/well). The allogeneic mixed leukocyte reaction (MLR) was carried out in RPMI 1640 supplemented with 10% heat-inactivated normal human AB serum, 10 µg/mL of gentamycin, 25 mM HEPES, and 50 µM 2-mercaptoethanol (Sigma) and incubated at 37°C in an atmosphere with 5% CO2. On day 5, the cells were pulsed with [3H]thymidine (1 µCi/well) (Amersham Japan, Tokyo) for 16 h. The cultures were then harvested with a cell harvester, and the incorporated radioactivity was counted in a liquid scintillation counter (LSC-3100; Aloka Co. Ltd., Japan).

Statistics
For the statistical analysis, the Mann-Whitney test was used. A P value <0.05 was considered significant. All data are expressed as mean ± SE unless otherwise specified.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phenotypic analysis of cultured DCs by flow cytometry
Figure 1 shows representative profiles of surface molecule expression by cultured cells on day 7. On day 7 of culture, most cells (>99%) showed intense expression of both CD1a and HLA-DR, indicating that these cells were phenotypically DCs. More than 70% of cultured DCs were positive for costimulatory molecules CD80 and CD86. Moreover, the DCs expressed high levels of CD40, although they did not exhibit strong expression of CD83, a marker for mature DCs [28 , 31 ]. Approximately 20–30% of the cultured DCs were positive for CD83.



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  		Figure 1. Flow cytometric analysis of cultured human DCs. CD3- CD19- CD56- blood cells were cultured for 7 days with IL-4 (1,000 U/mL) and GM-CSF (1,000 U/mL). Cells were labeled with HLA-DR(FITC) and CD1a(PE), CD83(PE), CD80(PE), CD86(PE), or CD40(PE) mAbs. Data from one representative experiment out of 14 are shown.

 
MDP-Lys enhances the expression of costimulatory molecules on human DCs
On day 7, the cultured DCs were treated with various concentrations of MDP-Lys for an additional 72 h. As shown in Figure 2 , MDP-Lys further enhanced the expression of CD80, CD83, CD86, and CD40. However, it did not increase HLA-DR expression. In addition, MDP-Lys treatment resulted in a bimodal profile of CD83 expression, CD83low and CD83high populations. The MDP-Lys-induced up-regulation of CD80, CD83, CD86, and CD40 was dose dependent, and the expression of CD80, CD86, and CD40 was significantly elevated at concentrations of >100 ng/mL of MDP-Lys (Fig. 3 ). In DCs treated with 1,000 ng/mL of MDP-Lys, the MFIs of CD80, CD83, CD86, and CD40 were increased 2.5-fold, 2.0-fold, 2.2-fold, and 2.6-fold, respectively, compared with the MFIs of untreated DCs. No significant change in the MFIs of HLA-DR was found between untreated and MDP-Lys-treated DCs. In contrast, LPS increased the expression of HLA-DR, and the up-regulation of CD80, CD83, CD86, and CD40 induced by LPS (>=1.0 ng/mL) was greater than that stimulated by MDP (1,000 ng/mL) (Fig. 3) .



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  		Figure 2. Effects of MDP-Lys on the profiles of surface molecules expressed by human DCs. DCs incubated for 72 h in medium alone (line 2) or medium containing 1,000 ng/mL of MDP-Lys (line 3) were analyzed by flow cytometry for expression of HLA-DR, CD1a, CD83, CD80, CD86, and CD40. Line 1 indicates isotype controls. One representative experiment out of 14 is shown.

 


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  		Figure 3. Effects of MDP-Lys on the phenotype of human DCs. DCs were cultured for 72 h in medium alone, medium containing various concentrations of MDP-Lys, or LPS. DCs were then stained with the indicated mAbs, and their surface molecule expression was analyzed by flow cytometry. Results are expressed as MFI. Data are expressed as the mean ± SE of >10 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with MFI of untreated DCs.

 
MDP-Lys increases the production of cytokines by human DCs
To examine the effects of MDP-Lys on cytokine production by human DCs, we measured the levels of TNF-{alpha}, IL-6, IL-8, IL-10, and IL-12 (p40) in culture supernatants after stimulation with MDP-Lys. The production of cytokines was examined after 72 h of treatment with MDP-Lys because cytokine levels reached a plateau at this time (data not shown). We found that even unstimulated DCs produced low levels of these cytokines (Table 1 ). In response to the addition of various concentrations of MDP-Lys, the production of TNF-{alpha}, IL-6, IL-8, and IL-12 (p40) was significantly increased in a dose-dependent manner, with a significant effect already observed at a concentration of 1 ng/mL (Table 1) . MDP-Lys also tended to induce a dose-dependant release of IL-10, but the effect was not significant (control vs. 1,000 ng/mL; P=0.098) (Table 1) . Compared the cytokine producing capacity of MDP with that of LPS. TNF-{alpha}, and IL-10 production by 1,000 ng/mL of MDP were approximately comparable to those by 0.1 ng/mL of LPS. Treatment with 1,000 ng/mL of MDP induced the levels of IL-8 production similar to treatment with 1.0 ng/mL of LPS. However, the levels of IL-6 and IL-12 (p40) in the supernatants were much higher in LPS-treated DCs than in MDP-treated DCs.


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  		Table 1. Cytokine Production by DCs in Response to MDP-Lys or LPS

 
MDP-Lys enhances the allostimulatory capacity of human DCs
A hallmark of DCs among APCs is the capacity of DCs to stimulate naive T cells. We therefore evaluated the antigen-presenting function of cultured DCs, as measured by an allogeneic MLR using allogeneic CD45RA+ T cells, and examined whether MDP-Lys augmented the allostimulatory capacity of the DCs. Figure 4 shows that even untreated DCs cultivated in the presence of GM-CSF and IL-4 could strongly induce the proliferation of allogeneic T cells. When MDP-Lys-treated DCs were used as stimulators, the proliferation of the T cells was markedly increased. The enhancement of the allostimulatory capacity of the treated DCs was more marked at lower ratios of stimulators/responders. Compared the allostimulatory capacity of MDP-treated DCs with that of LPS-treated DCs, the stimulation with 1,000 ng/mL of MDP was similar to that of 0.1 ng/mL of LPS at the lowest DC/T cell ratio.



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  		Figure 4. MDP-Lys enhances the allostimulatory capacity of human DCs. DCs were incubated for 72 h in medium alone, 1,000 ng/mL of MDP-Lys, or 0.1 ng/mL of LPS. They were then washed, irradiated (2,000 rad), and added in various numbers to allogeneic CD45RA+ T cells (2x105/well) in 96-well flat-bottom microtiter plates. Thymidine incorporation was measured on day 4 by a 16-h pulse [3H]thymidine (1 µCi/well). Cultures were set up in triplicate. Data from one representative experiment out of four are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
MDP-Lys, a potent immunoadjuvant, has been demonstrated to enhance the protective immunity in vivo against pathogens and tumors. APCs are essential for triggering efficient T cell responses in the initiation of these types of immunity. Because DCs are the most potent APCs, the characterization of their response to MDP-Lys is crucial to understanding the mechanism by which MDP-Lys affects immunity in vivo. Therefore, we examined in this study the effects of MDP-Lys on the function of human DCs generated from peripheral blood cells in the presence of GM-CSF and IL-4. This study showed that MDP-Lys markedly stimulated the cytokine production and enhanced the expression of costimulatory molecules by human DCs along with the increased antigen-presenting function.

Human DCs generated from CD3- CD19- CD56- blood cells with GM-CSF/IL-4 were shown to exhibit high levels of expression of HLA-DR, CD1a, CD80, CD86, and potent antigen-presenting function, as assessed by an allogeneic MLR, indicating that they had the characteristic features of DCs. Upon stimulation with MDP-Lys, marked up-regulation of CD80, CD83, CD86, and CD40 was observed in a dose-dependent manner. Because costimulatory molecules such as CD80, CD86, and CD40 are particularly important for optimal activation of primed and unprimed T cells [32 , 33 ], DCs stimulated with MDP-Lys are likely to be more efficient for T cell stimulation. In addition, MDP-Lys might also augment their expression of CD83, which was recently shown to be a DC-specific marker expressed only by mature DCs [28 , 31 ]. Human DCs generated from peripheral blood with GM-CSF/IL-4 have been reported to be relatively immature in terms of their phenotype and antigen-presenting capacity [28 ]. In response to bacterial components (LPS and fixed Staphylococcus aureus), ligation via the CD40 ligand (CD40L), or inflammatory cytokines (TNF-{alpha} and IL-1), these immature DCs were shown to become fully mature DCs in which there were further increases in the expression of costimulatory molecules and CD83 [27 , 28 , 34 ]. Thus, the finding that MDP-Lys enhanced the expression of CD80, CD86, CD40, and CD83 on human DCs suggested that MDP-Lys promoted the phenotypic maturation of these DCs. It is interesting that we found two populations in MDP-Lys-treated DCs in terms of CD83 expression, CD83low and CD83high populations. It is possible that cultured DCs derived from CD14+ blood cells contain two different populations with regard to the induction of CD83 expression by MDP-Lys. In contrast to costimulatory molecules such as CD80, CD86, and CD40, MDP-Lys did not further increase HLA-DR expression on human DCs, although the DCs expressed high levels of HLA-DR even without stimulation. In monocytes and B cells, the data concerning the regulation of MHC class II expression by MDP have been controversial [8 , 9 , 35 ]. Several studies failed to demonstrate any MDP-induced enhancement of MHC class II expression in murine peritoneal macrophages and B cell lines [8 , 35 ]. In contrast, Heinzelmann and co-workers showed that MDP slightly increased HLA-DR expression on human monocytes [9 ]. Another study analyzed mRNA induction of HLA-DR by MDP and demonstrated only a minimal increase of HLA-DR mRNA in human monocytes [36 ]. These conflicting results may be attributable to the type of MDP analogues used, the nature of the responding cells, or the species employed. Taking our results together with the previous findings, MDP does not appear to be a potent inducer of MHC class II antigens.

Recently, it has been shown that DCs can secrete a large array of cytokines, including IL-6, IL-8, IL-10, IL-12, IL-18, and TNF-{alpha}, in response to a variety of stimuli such as bacterial products, phorbol myristate acetate/ionomycin, and CD40 ligation [27 , 37 ]. In addition to the T cell stimulatory property of DCs via direct cell-to-cell interaction, the cytokine production by DCs is also thought to play an important role in controlling immunity. In the present study, we observed that human DCs stimulated with MDP-Lys could secrete considerable amounts of TNF-{alpha}; IL-6; IL-8; IL-12 (p40); and, to a lesser degree, IL-10 in a dose-dependent manner. These cytokines are known to be crucial for the regulation of inflammatory and immunologic responses. TNF-{alpha} is an important proinflammatory cytokine that up-regulates adhesion molecules and primes T cells [38 ]. IL-6 induces proliferation and differentiation of B cells as well as stimulation of T cells [39 ]. IL-8 is a potent chemotactic factor for neutrophils and T cells [40 ]. IL-12 is an essential cytokine in promoting the T helper cell type 1 response [41 ]. Thus, it is suggested that MDP-Lys can induce the release of these cytokines by DCs in vivo, which, in turn, may augment protective immunity. It is interesting that MDP-Lys-treated DCs also secreted small amounts of IL-10, which is known to inhibit T cell proliferation and down-regulate expression of MHC class II and costimulatory molecules by monocytes and macrophages [42 , 43 ]. IL-10 was also shown to decrease IL-12 production by DCs and the antigen-presenting function of DCs [44 ]. In agreement with our results, recent studies using CD14+-derived human DCs showed their capacity to produce IL-10 in response to LPS and phorbol myristate acetate/ionomycin [27 , 37 ]. Considering the immunosuppressive role of IL-10, the production of IL-10 by DCs might be implicated in negatively controlling the level of T cell activation and DC function itself.

DCs are unique among APCs in their ability to stimulate naive T cells, as measured by an allogeneic MLR. The human DCs used in the present study were shown to induce strongly the proliferation of allogeneic naive T cells without stimulation. However, we found that MDP-Lys could further enhance the capacity of human DCs to stimulate allogeneic naive T cells. The increase in the allostimulatory potential of DCs by MDP-Lys was most noticeable when the cells were cultured using the smallest ratio of stimulators/responders. This suggests that MDP-Lys can augment the antigen-presenting function of DCs and that consequently even a small number of DCs might be able to efficiently provoke T cell-mediated immune responses.

To evaluate the potency of the ability of MDP-Lys to stimulate DC function in vitro, we compared the stimulatory activity of MDP-Lys with that of LPS, which is one of the most powerful DC activators [27 ]. In terms of the surface expression of immunostimulatory molecules and accessory cell function, stimulation with 0.1 ng/mL of LPS was shown to be comparable to stimulation with 1,000 ng/mL of MDP. It is interesting that LPS increased the expression of HLA-DR whereas MDP-Lys failed to do so. Although the reason for this discrepancy is not clear, the difference in signaling mechanism(s) between MDP-Lys and LPS might be responsible. As to cytokine production, LPS was shown to have a more potent capacity to produce cytokines than MDP-Lys. However, MDP-Lys could induce a significant increase in the production of TNF-{alpha}, IL-6, IL-8, and IL-12 (p40) in a dose-dependent manner. Collectively, although the ability of MDP-Lys to stimulate DCs in vitro was not as powerful as that of LPS, the present data clearly indicate that MDP-Lys did efficiently activate DC function. Considering the potential harm of in vivo administration of LPS and the low toxicity of MDP-Lys [45 ], MDP-Lys is thought to be a useful immunoadjuvant.

The mechanism by which MDP-Lys activates immune-competent cells remains unknown. There is growing evidence that pathogen-associated molecules such as LPS, peptidoglycan, and lipoteichoic acid are recognized by the specific receptors, including several toll-like receptors (TLRs) and CD14 [46 47 48 ]. Because MDP is a component of the peptidoglycan of bacterial cell walls, it is possible that these receptors are involved in the recognition of MDP. Recently, Yang et al. demonstrated that the activation of MDP on the human monocytic cell line THP-1 was not inhibited by anti-CD14 or anti-TLR4 mAbs [49 ]. Furthermore, MDP was shown to efficiently stimulate U937 cells differentiated by an analog of 1{alpha}, 25-dehydroxyvitamin D3, 22-oxyacalcitriol, which expressed almost no TLR2 [49 ]. These data suggest that MDP exerts its effects in a CD14-, TLR4-, and TLR-2-independent manner. However, there is the possibility that other TLRs act as receptors for MDP. It is interesting that MDP has been shown to interact with serotonin receptors on macrophages and to enhance superoxide production by them [50 , 51 ]. Further studies will be required to elucidate the recognition and signaling mechanisms of MDP by DCs.

In conclusion, our findings are the first to clearly indicate that MDP-Lys can activate human DCs to up-regulate the expression of costimulatory molecules and to produce various cytokines, resulting in enhancement of their antigen-presenting function. Thus, the activation of DCs by MDP-Lys is likely to be involved in its immunoadjuvant activities in vivo.


   ACKNOWLEDGEMENTS
 
T. S. was supported by a grant-in-aid for scientific research (11670572 and 13670595) from Japan Society for the Promotion of Science.

Received September 28, 2000; revised June 16, 2001; accepted June 18, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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