The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary Preinduction of potent haptenic muramyl dipeptide (MDP)-reactive helper T cell activity and subsequent immunization with MDP hapten-coupled syngeneic tumor cells resulted in enhanced induction of tumor-specific immunity through T-T cell collaboration between anti-MDP hapten helper T cells and tumor-specific effector T cells. The present study establishes two types of tumor-specific immunotherapy protocols utilizing helper T cells against MDP hapten cross-reactive with Bacillus Calmette Guérin (BCG). In the first model, naive normal C3H/He mice or mice in which MDP hapten-reactive helper T cells had been generated by BCG-sensitization were inoculated i.d. with syngeneic X5563 tumor cells. When both groups of mice were allowed to generate MDP hapten-modified tumor cells in the tumor mass in situ by intratumoral injection of MDP hapten, an appreciable number of growing tumors in the BCG-presensitized but not in the unsensitized group were observed to regress. In the second model, a growing X5563 tumor mass was removed by the surgical resection 9 days after the tumor implantation. Approximately 90% of C3H/He mice receiving such treatment died from tumor metastasis by about 30 days after the tumor resection. However, immunization of mice with MDP hapten-coupled X5563 tumor cells subsequent to the tumor resection resulted in an increased survival rate. Such protection from the tumor metastasis was appreciably stronger when compared to the protection obtained by immunization with MDP hapten-uncoupled tumor cells. The mice surviving in both models were also demonstrated to retain X5563 tumor-specific immunity. These results indicate that the presentation of MDP hapten-modified tumor cells to BCG-sensitized recipients results in potent tumor-specific immunity which contributes to the regression of the primary tumor or inhibition of metastatic tumor growth.This work was supported by a Grant-in-Aid for the Special Project Cancer Bioscience from the Ministry of Education, Science and Culture, Japan     

Dendritic cell maturation induced by muramyl dipeptide (MDP) derivatives: monoacylated MDP confers TLR2/TLR4 activation

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-Guerin (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.     

Calreticulin is a binding protein for muramyl dipeptide and peptidoglycan in RK13 cells

Calreticulin (CRT) was isolated and identified as a protein in rabbit kidney RK(13) cells that binds the apoptogenic bacterial cell wall (BCW) components, muramyl dipeptide (MDP) and peptidoglycan (PG). Mannan-agarose purified RK(13) cell CRT (rCRT) selectively bound sepharose-immobilized L,D-MDP and PG, but not L,L-MDP or D,D-MDP. Purified rCRT and bovine CRT (bCRT) also bound free PG and L,D-MDP demonstrated in bioassays of RK(13) cell apoptosis. The results suggest that, in RK(13) cells, (a) CRT is a specific binding protein for both L,D-MDP and PG and (b) CRT binding L,D-MDP or PG is dependent on the stereoisomeric configuration of the dipeptide (L-alanyl-D-isoglutamine) moiety. In addition, the results also suggest that, in RK(13) cells, the binding of L,D-MDP, L,L-MDP, D,D-MDP, or PG to CRT correlates with their capacities of inducing apoptosis.     

Marked enhancement of macrophage activation induced by synthetic muramyl dipeptide (MDP) conjugate using monoclonal anti-MDP antibodies

Activation of peritoneal exudate macrophages of mice to inhibit the in vitro proliferation of tumor target cells was achieved with low concentrations of N-acetyl-l-alanyl-d-isoglutamine (MDP for muramyl dipeptide) conjugated to a synthetic carrier. Addition to the cultures of monoclonal anti-MDP or anti-carrier antibodies renders a thousandfold-smaller concentration of the conjugate highly effective in activating macrophages. This synergistic effect was observed neither with a control monoclonal antibody of different specificity nor with an F(ab)₂ fragment of the monoclonal anti-MDP antibody. Other controls, such as addition to the cultures of the carrier alone with its specific monoclonal antibodies, also demonstrated that there exists a requirement for the presence of MDP in the conjugate. The possible uses of such a system as well as the underlying mechanisms are discussed.     

Lack of response of murine peritoneal macrophages to in vitro activation by muramyl dipeptide (MDP). I. Macrophage activation by MDP is species dependent

Peritoneal exudate macrophages from mice, rats, and guinea pigs were assessed using six different parameters of macrophage activation to determine whether the cells were stimulated under similar experimental conditions. Peritoneal exudate macrophages from mice, irrespective of strain, were far less responsive to a variety of in vitro stimulatory effects of N-acetylmuramyl-L-alanyl-D-isoglutamine than those from rats or guinea pigs, while no significant differences were noted with the reactivity to stimulation by endotoxic lipopolysaccharide. We conclude that macrophage activation by MDP in vitro is species dependent.     

Dissociation of immunostimulant activities of muramyl dipeptide (MDP) by linking amino-acids or peptides to the glutaminyl residue

N-acetyl-muramyl-L-alanyl-D-isoglutamine (muramyl dipeptide or MDP) is the minimal structure required for substituting mycobacteria in Freund's complete adjuvant. It is an adjuvant when injected in saline, protects mice non-specifically against infection, and enhances thymidine incorporation of lymphocytes. In this report it is shown that the linking of L-Lys, L-Ala-D-Ala, L-Lys-D-Ala or L-Lys-L-Ala to MDP permits the dissociation of anti-infectious activity from adjuvant activity. The optical configuration of the added residues plays the major role in this dissociation. It can be noted that the muramyl tetrapeptide MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala mimicking the natural structure is devoid of anti-infectious activity.     

New muramyl dipeptide (MDP) mimics without the carbohydrate moiety as potential adjuvant candidates for a therapeutic hepatitis B vaccine (HBV)

A series of new muramyl dipeptide (MDP) mimics were designed and synthesized via a solid-phase synthetic route. Their adjuvant activities were evaluated ex vivo for investigation of the synergism of the S_28-39 peptide, which is an MHC class I binding epitope of recombinant hepatitis B surface antigen (HBsAg) for both humans and mice. Several compounds without the carbohydrate moiety exerted better adjuvanticity than the MDP-C that has been reported by our laboratory previously. A primary screening test revealed that compounds 6, 14 and 16 exhibited stronger adjuvanticity compared with other MDP mimics.     

The origin of the synergistic effect of muramyl dipeptide with endotoxin and peptidoglycan

Although the basis for the high mortality rate for patients with mixed bacterial infections is likely to be multifactorial, there is evidence for a synergistic effect of muramyldipeptide (MDP) with lipopolysaccharide (LPS) on the synthesis of proinflammatory cytokines by mononuclear phagocytes. In this study, co-incubation of human Mono Mac 6 cells with MDP and either LPS or peptidoglycan (PGN) resulted in an apparent synergistic effect on tumor necrosis factor-alpha (TNF-alpha) secretion. Although incubation of cells with MDP alone produced minimal TNF-alpha, it caused significant expression of TNF-alpha mRNA. These findings suggest that the majority of TNF-alpha mRNA induced by MDP alone is not translated into protein. Furthermore, simultaneous incubation of cells with MDP and either LPS or PGN resulted in TNF-alpha mRNA expression that approximated the sum of the amounts expressed in response to MDP, LPS, and PGN individually. These findings indicate that the apparent synergistic effect of MDP on TNF-alpha production induced by either LPS or PGN is due to removal of a block in translation of the mRNA expressed in response to MDP. In subsequent studies, the effects of MDP alone and its effect on the production of TNF-alpha by LPS and PGN were determined to be independent of CD14, Toll-like receptor 2, and Toll-like receptor 4. These findings indicate that MDP acts through receptor(s) other than those primarily responsible for transducing the effects of LPS and PGN. Successful treatment of patients having mixed bacterial infections is likely to require interventions that address the mechanisms involved in responses induced by a variety of bacterial cell wall components.     

Differences between macrophage migration inhibitions by lymphokines and muramyl dipeptide (MDP) or lipopolysaccharide (LPS): migration enhancement by lymphokines

To investigate the repertoire of molecules which are associated with cytolytic T-lymphocyte (CTL)-mediated killing, function-blocking monoclonal antibodies (MAb) have been selected and characterized. Spleen cells from rats immunized with secondary mouse CTL were fused with mouse myeloma cells. Antibodies secreted by 2400 hybrid cultures were selected solely by their ability to block CTL-mediated killing in a mouse anti-rat xenogeneic system. Fifteen cultures with antibodies which blocked CTL-mediated killing were chosen for cloning and further characterized by immunoprecipitation and immunofluorescence flow cytometry. One group of five monoclonal antibodies recognized the Lyt-2,3 molecule of 35,000 M_r. The second group of six MAb recognized the LFA-1 antigen containing two subunits of 180,000 and 95,000 M_r. One MAb giving only partial inhibition of killing was an IgM anti-Thy-1. It strongly agglutinated CTL. The target antigens defined by three other MAb were not definitively identified. Competition in cell binding between anti-Lyt-2,3 and anti-LFA-1 MAb suggested that their blocking effect in cytolysis is due to binding to distinct and spatially separate molecules on effector cells. The results of direct screening for functional blockade support the important role of Lyt-2,3 and LFA-1 molecules in T-cell-mediated cytolysis.     

Restoration and stimulation of the in vitro immune response of B cells to sheep erythrocytes by interleukins and muramyl dipeptide (MDP)

MDP, a synthetic muramyl dipeptide, is capable of increasing the primary in vitro antibody response, to sheep erythrocytes, of purified B cells restored with a monokine and helper T cell factors, including Interleukin 2 and the late-acting T cell replacing factor (TRF). First, the possibility that the adjuvanticity of MDP could be due to the elaboration of Interleukin 1, caused by its effect on macrophages, was excluded. In addition, a kinetic study showed that the effect of MDP was greater when added later, concomitantly with or one day after the helper T cell factors. Therefore, it appears that MDP acts directly on B cells, in a late stage of their differentiation to antibody-producing cells.     

Pannexin-1-mediated intracellular delivery of muramyl dipeptide induces caspase-1 activation via cryopyrin/NLRP3 independently of Nod2

Muramyl dipeptide (MDP), the microbial activator of nucleotide-binding oligomerization domain 2 (Nod2), induces NF-kappaB and MAPK activation, leading to the production of multiple anti-bacterial and proinflammatory molecules. In addition, MDP has been implicated in IL-1beta secretion through the regulation of caspase-1. However, the mechanisms that mediate caspase-1 activation and IL-1beta secretion in response to MDP stimulation remain poorly understood. We show here that fluorescent MDP molecules are internalized in primary macrophages and accumulate in granular structures that colocalize with markers of acidified endosomal compartments. The uptake of MDP was Nod2-independent. Upon ATP stimulation, labeled MDP was rapidly released from acidified vesicles into the cytosol, a process that required functional pannexin-1. Caspase-1 activation induced by MDP and ATP required pannexin-1 and Cryopyrin but was independent of Nod2. Conversely, induction of pro-IL-1beta mRNA by MDP stimulation was abolished in Nod2-deficient macrophages but unimpaired in macrophages lacking Cryopyrin. These studies demonstrate a Nod2-independent mechanism mediated through pore-forming pannexin-1 that is required for intracellular delivery of MDP to the cytosol and caspase-1 activation. Furthermore, the work provides evidence for distinct roles of Nod2 and Cryopyrin in the regulation of MDP-induced caspase-1 activation and IL-1beta secretion.     

Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease

NOD2, a protein associated with susceptibility to Crohn's disease, confers responsiveness to bacterial preparations of lipopolysaccharide and peptidoglycan, but the precise moiety recognized remains elusive. Biochemical and functional analyses identified muramyl dipeptide (MurNAc-L-Ala-D-isoGln) derived from peptidoglycan as the essential structure in bacteria recognized by NOD2. Replacement of L-Ala for D-Ala or D-isoGln for L-isoGln eliminated the ability of muramyl dipeptide to stimulate NOD2, indicating stereoselective recognition. Muramyl dipeptide was recognized by NOD2 but not by TLR2 or co-expression of TLR2 with TLR1 or TLR6. NOD2 mutants associated with susceptibility to Crohn's disease were deficient in their recognition of muramyl dipeptide. Notably, peripheral blood mononuclear cells from individuals homozygous for the major disease-associated L1007fsinsC NOD2 mutation responded to lipopolysaccharide but not to synthetic muramyl dipeptide. Thus, NOD2 mediates the host response to bacterial muropeptides derived from peptidoglycan, an activity that is important for protection against Crohn's disease. Because muramyl dipeptide is the essential structure of peptidoglycan required for adjuvant activity, these results also have implications for understanding adjuvant function and effective vaccine development.     

Pathogen sensing by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is mediated by direct binding to muramyl dipeptide and ATP

Nucleotide binding and oligomerization domain-containing protein 2 (NOD2/Card15) is an intracellular protein that is involved in the recognition of bacterial cell wall-derived muramyl dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders, including Crohn disease and Blau syndrome. NOD2 is a member of the nucleotide-binding domain and leucine-rich repeat-containing protein gene (NLR) family. Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by these proteins remain largely unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl dipeptide in cellular assays. Using purified recombinant protein, we now demonstrate that NOD2 binds and hydrolyzes ATP. Additionally, we have found that the purified recombinant protein is able to bind directly to muramyl dipeptide and can associate with known NOD2-interacting proteins in vitro. Binding of NOD2 to muramyl dipeptide and homo-oligomerization of NOD2 are enhanced by ATP binding, suggesting a model of the molecular mechanism for signal transduction that involves binding of nucleotide followed by binding of muramyl dipeptide and oligomerization of NOD2 into a signaling complex. These findings set the stage for further studies into the molecular mechanisms that underlie detection of muramyl dipeptide and assembly of NOD2-containing signaling complexes.     

Identification of the critical residues involved in peptidoglycan detection by Nod1

Nod1 is an intracellular pattern recognition molecule activated following bacterial infection, which senses a specific muropeptide (l-Ala-d-Glu-meso-DAP (diaminopimelic acid); "Tri(DAP)") from peptidoglycan. Here we investigated the molecular basis of Tri(DAP) sensing by human (h) Nod1. Our results identified the domain responsible for Tri(DAP) detection in the center of the concave surface of hNod1 leucine-rich repeat domain. Amino acid residues critical for sensing define a contiguous surface patch that is largely conserved in Nod1 proteins from different species. Accordingly, the distinct specificities of human versus murine Nod1 toward muropeptide detection were also found to lie in this central cleft. Several splicing variants of Nod1 lacking repeats 7-9 have been characterized recently, the relative balance of which is thought to correlate with the onset of asthma or inflammatory bowel disease. We demonstrated that these isoforms failed to transduce NF-kappaB activation upon muropeptide stimulation. This study provided insights into the molecular mechanisms responsible for the detection of bacterial peptidoglycan by Nod1 and suggested that defects in Nod1-dependent peptidoglycan sensing may contribute to elicit certain inflammatory disorders.     

In vivo regulation of humoral and cellular immune responses of mice by a synthetic adjuvant, N-acetyl-muramyl-L-alanyl-D-isoglutamine, muramyl dipeptide for MDP.

In vitro immunizations by T-dependent or T-independent antigens can be modulated by muramyl dipeptide (MDP). Enhancement or suppression of the antibody responses was observed according to the spleen-cell concentrations. Data presented here show that MDP can also suppress the immune response in vivo if used at relatively high dosage and injected before the antigen (SRBC). In vitro generation of cytotoxic T-lymphocyte recovered from mice which had been treated by MDP under the same experimental conditions was also decreased whereas macrophage cytostatic activity was not affected. By MDP pretreatment, a significant increase of antibody-dependent cell-mediated cytotoxicity was observed.     

Regulatory regions and critical residues of NOD2 involved in muramyl dipeptide recognition

Multiple genetic variants of CARD15/NOD2 have been associated with susceptibility to Crohn's disease and Blau syndrome. NOD2 recognizes muramyl dipeptide (MDP) derived from bacterial peptidoglycan (PGN), but the molecular basis of recognition remains elusive. We performed systematic mutational analysis to gain insights into the function of NOD2 and molecular mechanisms of disease susceptibility. Using an archive of 519 mutations covering approximately 50% of the amino-acid residues of NOD2, the essential regulatory domains and specific residues of NOD2 involved in recognition of MDP were identified. The analysis revealed distinct roles for N-terminal and C-terminal leucine-rich repeats (LRRs) in the modulation of NOD2 activation and bacterial recognition. Within the C-terminal LRRs, variable residues predicted to form the beta-strand/betaturn structure were found to be essential for the response to MDP. In addition, we analyzed NOD1, a NOD2-related protein, revealing conserved and nonconserved amino-acid residues involved in PGN recognition. These results provide new insights into the molecular function and regulation of NOD2 and related NOD family proteins.     

Analysis of NOD2-mediated proteome response to muramyl dipeptide in HEK293 cells

NOD2, a cytosolic receptor for the bacterial proteoglycan fragment muramyl dipeptide (MDP), plays an important role in the recognition of intracellular pathogens. Variants in the bacterial sensor domain of NOD2 are genetically associated with an increased risk for the development of Crohn disease, a human chronic inflammatory bowel disease. In the present study, global protein expression changes after MDP stimulation were analyzed by two-dimensional PAGE of total protein extracts of human cultured cells stably transfected with expression constructs encoding for wild type NOD2 (NOD2(WT)) or the disease-associated NOD2 L1007fsinsC (NOD2(SNP13)) variant. Differentially regulated proteins were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) peptide mass fingerprinting and MALDI MS/MS. The limited overlap in the responses of the NOD2-overexpressing cell lines to MDP included a down-regulation of heat shock 70-kDa protein 4. A complex pro-inflammatory program regulated by NOD2(WT) that encompasses a regulation of key genes involved in protein folding, DNA repair, cellular redox homeostasis, and metabolism was observed both under normal growth conditions and after stimulation with MDP. By using the comparison of NOD2(WT) and disease-associated NOD2(SNP13) variant, we have identified a proteomic signature pattern that may further our understanding of the influence of genetic variations in the NOD2 gene in the pathophysiology of chronic inflammatory bowel disease.     

Inhibition of murine hepatic tumor growth by liposomes containing a lipophilic muramyl dipeptide

Summary We have investigated the ability of liposomes containing a lipophilic muramyl dipeptide, N-acetylmuramyl-l-alanyl-d-isoglutamine glycerol dipalmitate (MDP-GDP) to activate Kupffer cell tumoricidal activity in situ and to inhibit the growth of experimental hepatic micrometastases of tumor cell line H-59, a liver-homing variant of the Lewis lung carcinoma. Liposomes prepared from distearoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DSPC/DMPG) and containing MDP-GDP (1 mol and 2 g, respectively) were efficiently taken up by the liver after i.v. administration. A single i.v. injection of DSPC/DMPG liposomes containing MDP-GDP was capable of inducing Kupffer cell tumoricidal activity against H-59 tumor cells as measured in vitro. Control liposomes or 100 g free MDP were ineffective in inducing Kupffer cell tumoricidal activity in situ. Two treatment regimens were evaluated in vivo: firstly, C57BL/6 mice were injected with tumor cell line H-59 and subsequently treated with multiple injections of liposomal MDP-GDP. Secondly, treatment with liposomal MDP-GDP was initiated prior to tumor cell injection and continued after tumor cell injection. The ability of liposomes containing MDP-GDP to reduce the number of hepatic micrometastases using the first protocol was related to the tumor cell inoculum, significant inhibition being observed at lower liver tumor burdens (<25 tumor nodules). Pretreatment of the mice prior to tumor cell challenge followed by treatment afterwards greatly enhanced the efficacy of liposomal MDP-GDP and brought about a highly significant inhibition of the growth of experimental metastases even at high liver tumor burdens (>50 nodules).     

Innate immune recognition of microbes through Nod1 and Nod2: implications for disease

Nod1 and Nod2 are cytosolic proteins involved in intracellular recognition of microbes and their products. Recently, it was shown that these proteins recognize different moieties of bacterial peptidoglycan (PGN) mediating non-specific pathogen resistance and possibly generating signals for the adaptive immune response. Moreover, mutations in the gene encoding Nod2 are associated with increased susceptibility to chronic inflammatory disorders.