Effect of a synthetic adjuvant on the induction of primary immune responses in T cell-depleted spleen cultures.

N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide) stimulates in vitro primary immune responses to SRBC in T cell-depleted (nude) spleen cultures. The stimulation of immune responses by muramyl peptide was antigen dependent. A microculture system was used to compare the T cell-replacing activities of several structural analogues of muramyl dipeptide and to compare the activity of muramyl dipeptide to helper T cells. In a limiting dilution analysis with excess helper T cells or muramyl dipeptide, the frequency of B cell precursors that respond to SRBC was similar, ranging from 1.5 to 5 X 10(-5). Decreasing the cell density in microcultures did not affect the efficiency of B cell precursor responses in the presence of muramyl dipeptide. Muramyl dipeptide was examined for mitogenic activity in spleen cell cultures. In serum-free medium, muramyl dipeptide stimulates slight (3-fold) increases in DNA synthetic activity. In medium supplemented with 5 to 20% fetal calf serum, muramyl dipeptide showed no significant mitogenic activity. There are a number of possible explanations for the T cell-replacing activity of muramyl dipeptide. The most likely is that muramyl dipeptide interacts directly with B cells to mimic the helper T cell signal in the inductive stimulus.     

Muramyl peptides and serotonin interact at specific binding sites on macrophages and enhance superoxide release

Serotonin at low micromolar concentrations inhibited binding of two [125I]-labeled muramyl peptides to resident mouse peritoneal cells and to a macrophage-derived cell line, PU5-1.8-F7. Binding of [3H]serotonin was inhibited in parallel fashion. Overnight incubation with serotonin or muramyl peptide enhanced the release of superoxide by both types of cells when later stimulated with phorbol myristate acetate. Serotonin antagonists decreased binding of muramyl peptide and serotonin and diminished the subsequent enhancement of superoxide release. A cell line variant lacking detectable binding sites for muramyl peptide was far less responsive (superoxide release) than the parent line, to either drug. The data are consistent with sharing of a common set of receptors on the macrophage by muramyl peptide and serotonin and with involvement of these receptors in enhancing superoxide release.     

Immunopharmacology of muramyl peptides

In recent years the immunomodulatory activity of muramyl peptides has become of major interest because of their possible physiological and clinical importance. Many data suggest that this group of compounds has other pharmacological activities besides effects on the immune system. Some of these effects, such as pyrogenicity, sleep enhancement, and analgesic activity, are linked to the central nervous system (CNS). Other activities of muramyl peptides may involve CNS and peripheral mechanisms. These include antiinflammatory and hepatoprotective activities and the effect of muramyl peptides on blood pressure. The multiplicity of pharmacological actions of muramyl peptides suggests that these compounds might have a general modulatory role in physiological functions.     

Specific binding sites for muramyl peptides on murine macrophages

Two radiolabeled (125I) muramyl peptide derivatives of high specific activity were prepared: a tripeptide with an iodinated C-terminal tyrosine methyl ester (Ligand I), and a muramyl tripeptide with a C-terminal lysine derivatized with Bolton-Hunter reagent (Ligand II). These were used to characterize binding of muramyl peptides to monolayers of murine macrophages. Saturable high-affinity binding to resident, caseinate-elicited, and Listeria-activated peritoneal cells was observed with both radioligands. Binding affinities varied with the state of activation of the macrophages, and KD values ranged from 48 +/- 33 pM (for resident macrophages, Ligand I) to 1020 +/- 90 pM (for activated macrophages, Ligand II). Specific binding sites were also found on a macrophage-derived cell line. The ability of several unlabeled muramyl peptides to compete with Ligands I and II for their binding sites was tested. Competition was stereospecific and correlated with known biological activities of these compounds (i.e., immunoadjuvanticity, pyrogenicity, and somnogenicity). The sites identified here for Ligands I and II may mediate some of the effects that muramyl peptides have previously been demonstrated to have on macrophages.     

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.     

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.     

Muramyl dipeptide analogues as potentiators of the antitumor action of endotoxin

Summary The potentiation of endotoxin-induced necrosis and regression of solid syngeneic Meth A tumors in mice previously observed following administration of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) was investigated further by use of various muramyl peptide analogues and two unrelated synthetic adjuvants, viz. the pluronic polyol L121 and dimethyldioctadecylammonium bromide (DDA) instead of MDP. All agents were administered in aqueous solution by the IV route. None of the muramyl peptide analogues nor L121 or DDA had any strong antitumor action of their own. Two 6-O-acylated muramyl peptides (L2-MDP and B30-MDP) and muramyl dipeptide stearoyllysine [MDP-Lys (L18)] clearly potentiated endotoxin-induced necrosis and regression. In contrast, MDP with L- instead of D-isoglutamine was completely inactive. Optimal activity of B30-MDP and MDP-Lys (L18) was only achieved by adding of suitable amounts of a nonionic surfactant. L121 and DDA could not replace muramyl peptides as potentiating agent. The combination of endotoxin, MDP, and L121 caused complete tumor regression in all mice, but was highly toxic.On the basis of the data in the literature on the biological response-modifying activities of the agents used it is concluded that the potentiating activity of muramyl peptides cannot yet be related to their immunoadjuvant action or their capacity to activate macrophages or to enhance nonspecific bacterial resistance.The work described in this paper was supported by grant UUKC 82-15 from the Koningin Wilhelmina Fonds, The Netherlands Cancer Organization     

Muramyl dipeptide and its synthetic analog as possible inducers of interleukin-2 production

Muramyl dipeptide and its synthetic derivative N-acetyl-glucosaminyl-N-acetyl-muramyl-alanyl - D-isoglutamine induce mitogenic factor production for Con A activated blasts (Con A-blasts) in splenocytes. The maximal factor production was revealed 24 h after stimulation with muramyl dipeptide or its derivative. Addition of the inducers to the culture of Con A-blasts led but to the negligible proliferative response. Therefore, the mitogenic action of muramyl dipeptide on target cells is mediated by the growth factor, evidently by interleukin-2. It has been also shown that muramyl dipeptide and its derivative with Con A exerted a synergic action in interleukin-2 induction.     

hPepT1 selectively transports muramyl dipeptide but not Nod1-activating muramyl peptides

Muramyl peptides derived from bacterial peptidoglycan are detected intracellularly by Nod1 and Nod2, 2 members of the newly characterized nod-like receptor (NLR) family of pattern recognition molecules. In the absence of bacterial invasion into the host cytosolic compartment, it remains unclear whether muramyl peptides can cross the plasma membrane and localize into the cytosol. We have recently demonstrated that the plasma membrane transporter, hPepT1, was able to efficiently translocate muramyl dipeptide (MDP), a specific Nod2-activating molecule, into host cells. We aimed to characterize the transport properties of hPepT1 towards a spectrum of muramyl peptides, including Nod1-activating molecules. To do so, we designed an original procedure based on the ectopic expression of hPepT1 in oocytes from Xenopus laevis. Our results demonstrated that hPepT1 transports MDP but no other Nod2-activating molecule. Moreover, we observed that Nod1-stimulating muramyl peptides were not transported by hPepT1. Since hPepT1 expression is strongly associated with intestinal epithelial cells, where Nod1 and Nod2 have been shown to play a key role, these observations suggest a distinct contribution of Nod1 and Nod2 in mucosal homeostasis following the cellular uptake of muramyl peptides by hPepT1.     

Potential therapeutic value of muramyl peptides for modulating human immunologic responses

The clinical potential and the limitations of synthetic muramyl peptides have been suggested through extensive work describing their immunomodulating properties and toxicology. The intent of this paper is to offer the clinician a summary of these studies and to introduce the reader to the biological effects associated with administration of muramyl peptides.     

Inhibition of macrophage migration by synthetic muramyl dipeptide

N-acetylmuramyl-L-alanyl-D-isoglutamine, a synthetic compound which is known to have a minimal effective structure for an adjuvant activity of cell wall peptidoglycans, was found to inhibit the migration of normal macrophages. It was shown that the inhibition was neither due to cytotoxic or agglutinating effect of the muramyl dipeptide on macrophages nor due to lymphokine production uopn stimulation of lymphocytes by the muramyl dipeptide.     

PEPped up: induction of Candida albicans virulence by bacterial cell wall fragments

In this issue, Xu et al. (2008) report that Candida albicans, a prevalent fungal pathogen, expresses virulence-related attributes in response to bacterial peptidoglycan components, muramyl dipeptides, in human serum. Conserved elements in human and fungal detection of muramyl dipeptides highlight a common ability to perceive proximal bacterial populations.     

Modulation of natural killer activity by muramyl peptides: relationship with adjuvant and anti-infectious properties

Natural killer (NK) activity of spleen cells was studied in DBA/2 mice, 24 and 72 h after intravenous injection of various muramyl peptides: muramyl dipeptide (MDP) and derivatives which are both adjuvant-active and able to increase resistance against Klebsiella pneumoniae; derivatives which are adjuvant-active but devoid of anti-infectious properties; derivatives which are anti-infectious but devoid of adjuvant activity, and derivatives which are devoid of both activities such as the stereoisomer MDP[D-Ala]1. An early increase in NK activity was observed 24 h after injection of all nonadjuvant derivatives, whatever their effect on infection. A stimulation of natural cytotoxicity was always induced 72 h after injection of MDP and derivatives able to protect mice against Klebsiella pneumoniae infection. So, even if the reverse was not true, there seems to exist some correlation between the anti-infectious effect of muramyl peptides and the late increase in NK activity. The modulation of NK activity by muramyl peptides appeared to be independent of interferon production. Moreover, inhibition of the stimulatory effect by a cell cycle-specific drug, hydroxyurea, observed 72 h after MDP suggests a requirement for proliferation.     

Elucidating the molecular interaction of Zebrafish (Danio rerio) peptidoglycan recognition protein 2 with diaminopimelic acid and lysine type peptidoglycans using in silico approaches

Abstract

Peptidoglycan recognition proteins (PGRPs) belong to the family of pattern recognition receptor, represent the major constituent of innate immunity. Although PGRPs are structurally conserved through evolution, their involvement in innate immunity is different in vertebrates and invertebrates. They are highly specific towards recognition of ligands and can hydrolyze bacterial peptidoglycans (PGNs). Zebrafish PGRPs (zPGRPs) have both peptidoglycans lytic amidase activity and broad-spectrum bactericidal activity, but far less is known about how these receptors recognize these microbial ligands. Such studies are hindered due to lack of structural and functional configuration of zPGRPs. Therefore, in this study, we predicted the three-dimensional structure of zPGRP2 through theoretical modeling, investigated the conformational and dynamic properties through molecular dynamics simulations. Molecular docking study revealed the microbial ligands, that is, muramyl pentapeptide–DAP , muramyl pentapeptide–LYS, muramyl tripeptide–DAP, muramyl tripeptide–Lys, muramyl tetrapeptide–DAP, muramyl tetrapeptide–LYS and tracheal cytotoxin interacts with the conserved amino acids of the ligand recognition site comprised of β1, α2, α4, β4 and loops connecting β1 ? α2, α2 ? β2, β3 ? β4 and α4 ? α5. Conserved His31, His32, Ala34, Ile35, Pro36, Lys38, Asp60, Trp61, Trp63, Ala89, His90, Asp106, His143 and Arg144 are predicted to essential for binding and provides stability to these zPGRP–PGN complexes. Our study provides basic molecular information for further research on the immune mechanisms of PGRP’s in Zebrafish. The plasticity of the zPGRP’s binding site revealed by these microbial ligands suggests an intrinsic capacity of the innate immune system to rapidly evolve specificities to meet new microbial challenges in the future.

Communicated by Ramaswamy H. Sarma     

Enhancement in anti-Semliki Forest virus activity of ds RNA by a muramyl dipeptide

Antiviral activity of an interferon-inducing mycoviral ds RNA against Semliki Forest virus infection was considerably enhanced by N-palmitoylmuramyl-L-alanyl-D-isoglutamine (PMDP), a new muramyl dipeptide. This enhancement in activity was not due to increased production of interferon, but resulted probably from a PMDP-induced increase in nonspecific resistance to infection. These results indicate that a combined treatment with an interferon inducer and muramyl dipeptide may prove highly useful to control effectively viral infections.     

Selective recognition of synthetic lysine and meso-diaminopimelic acid-type peptidoglycan fragments by human peptidoglycan recognition proteins I{alpha} and S

The interactions of a range of synthetic peptidoglycan derivatives with PGRP-Ialpha and PGRP-S have been studied in real-time using surface plasmon resonance. A dissociation constant of K(D) = 62 mum was obtained for the interaction of peptidoglycan recognition protein (PGRP)-Ialpha with the lysine-containing muramyl pentapeptide (compound 6). The normalized data for the lysine-containing muramyl tetra- (compound 5) and pentapeptide (compound 6) showed that these compounds have similar affinities, whereas a much lower affinity for muramyl tripeptide (compound 3) was measured. Similar affinities were obtained when the lysine moiety of the muramyl peptides was replaced by meso-diaminopimelic acid (DAP). Furthermore, the compounds that contained only a stem peptide (pentapeptide, compound 1) and (DAP-PP, compound 2) as well as muramyldipeptide (compound 3) exhibited no binding indicating that the muramyltripeptide (compound 4) is the smallest peptidoglycan fragment that can be recognized by PGRP-Ialpha. Surprisingly, PGRP-S derived significantly higher affinities for the DAP-containing fragments to similar lysine-containing derivatives, and the following dissociation constants were measured: muramylpentapeptide-DAP, K(D) = 104 nm; muramyltetrapeptide-DAP, 92.4 nm; and muramyltripeptide-DAP, 326 nm. The binding profiles were rationalized by using a recently reported x-ray crystal structure of PGRP-Ialpha with the lysine-containing muramyltripeptide (4).     

Effects of nitric oxide synthase inhibitors on the febrile response to muramyl dipeptide and lipopolysaccharide in rats

We have administered aminoguanidine, a relatively specific inhibitor of inducible nitric oxide synthase, and N-nitro-L-arginine methyl ester (L-NAME), an unspecific nitric oxide synthase inhibitor, to rats made febrile with the gram-positive pyrogen, muramyl dipeptide and gram-negative pyrogen, lipopolysaccharide. Sprague-Dawley rats, housed individually at approximately 25 degrees C with a 12:12 h light:dark cycle (lights on 0700 hours), were injected (at 0900 hours) intraperitoneally with 50 mg/kg aminoguanidine, 25 mg/kg or 50 mg/kg L-NAME, and intramuscularly with 500 microg/kg muramyl dipeptide or 100 microg/kg lipopolysaccharide. Pyrogen injections were spaced at least 14 days apart. Body temperature was measured throughout the study in unrestrained animals using radio-telemetry. Neither muramyl dipeptide nor lipopolysaccharide-induced fevers were affected by aminoguanidine. However, L-NAME administration inhibited muramyl dipeptide and lipopolysaccharide-induced fevers, but only for the 1st 2-4 h of the fevers (two-way ANOVA, P<0.05). After the initial inhibition, lipopolysaccharide fevers developed normally. Therefore, constitutively expressed nitric oxide synthase appears to be involved in the initial phases of fever genesis of gram-negative and gram-positive fevers in rats. On the other hand, inducible nitric oxide synthase appears not to play a role in these fevers.     

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.     

Specific binding of glucosaminylmuramyl peptides to histones.

Intracellular N-acetylglucosaminylmuramyl peptide-binding proteins of murine macrophages and myelomonocytic WEHI-3 cells were characterized. SDS-PAGE and Western blotting revealed proteins with molecular masses of 18, 32 and 34 kDa retaining the ability to specifically bind glucosaminylmuramyl dipeptide. The inhibition analysis demonstrated that only biologically active muramyl peptides but not inactive analogs or fragments of glucosaminylmuramyl dipeptide could inhibit glucosaminylmuramyl dipeptide-binding to these proteins. Purification of these proteins and sequencing of peptides obtained after in-gel trypsin digestion enabled us to identify the above mentioned proteins as histones H1 and H3. These findings suggest that nuclear histones might be target molecules for muramyl peptides.     

Controlled reduction of cytochrome b in succinate-cytochrome c reductase complex by succinate in the presence of ascorbate and antimycin.

$\text{Bacillus subtilis}$ membranes can transfer either N-acetylmuramyl-pentapeptide phosphate or N-acetylglucosaminyl phosphate from UMP directly onto undecaprenyl phosphate. Tunicamycin blocks only the latter transfer and inhibits peptidoglycan synthesis by toluenized cells of $\text{Bacillus megaterium}$ utilizing added nucleotide sugar precursors or cell wall synthesis by intact cells of $\text{B. subtilis}$. Tunicamycin prevents formation of the cell wall disaccharide lipid intermediate by blocking transfer of N-acetylglucosamine onto undecaprenyl muramyl pentapeptidyl pyrophosphate.