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.     

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.     

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.     

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     

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.     

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.     

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.     

Immunological activities of muramyl peptides

Muramyl peptides are endowed with numerous modulatory effects on the immune and nervous systems. Studies with synthetic muramyl dipeptide (MDP), the smallest unit of bacterial cell walls that can replace Mycobacteria in Freund's complete adjuvant, revealed that this glycopeptide can regulate several functions of cells involved in the immune response. The adjuvanticity of MDP and the MDP-induced activation of macrophages against tumors were found to be potentiated in vitro and in vivo with monoclonal anti-MDP antibodies. When used on immunoadsorbent columns, the anti-MDP antibodies removed the somnogenic and pyrogenic activities contained in supernatants of stimulated rabbit peritoneal macrophages. Based on these data a hypothesis is put forward to explain the immuno- and neuro-modulatory effects of muramyl peptides.     

Induction, in vivo and in vitro, of macrophage membrane interleukin-1 by adjuvant-active synthetic muramyl peptides

Recent evidence has shown that a membrane form of interleukin-1 (IL-1) serves as a necessary signal for antigen presentation, leading to T-cell activation. The synthetic immunostimulant muramyl dipeptide (MDP) is known to induce secretion of IL-1 and its adjuvant effect was found to be mediated through enhancement of T-helper cells. We have investigated the ability of MDP and 19 other adjuvant-active or -inactive MDP analogs and derivatives to induce membrane IL-1 in mouse peritoneal macrophages. Enhancement in vitro of membrane expression and secretion of IL-1 in fresh or aged cultures of macrophages was observed after stimulation with MDP or with adjuvant-active but not with adjuvant-inactive muramyl peptides. Administration in vivo of adjuvant-active doses of MDP or of any of 12 other active analogs induced high levels of macrophage membrane IL-1 detected by the lymphocyte-activating factor assay. This effect was not observed when 7 other adjuvant-inactive derivatives were used. Moreover, under conditions where MDP did not exert an adjuvant effect, this immunomodulator was found to be incapable of inducing the expression of macrophage membrane IL-1. These results demonstrate a very high correlation between the ability to induce membrane IL-1 and the adjuvant activity of muramyl peptides. The correlation was observed irrespective of other biological effects of the synthetic adjuvants such as pyrogenicity and/or anti-infectious activity.     

A protein interaction network and cell signaling pathways activated by muramyl peptides

Review is devoted to studying the interaction muramyl peptides with protein components of immune system cells. Systems analysis of published results may be useful to select not only the strategy to further explore the function of this class of glycopeptides, but their use in clinical practice.     

Protein interaction network and signaling pathways activated by muramyl peptides

This review is devoted to studying the interaction of muramyl peptides with protein components of immune system cells. The systematic analysis of published results may be useful to select not only the strategy for the further exploration of the function of this class of glycopeptides, but also for their use in clinical practice.     

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).     

Peptide length requirement for experimental allergic encephalomyelitis in guinea pigs

Three peptides overlapping the tryptophan region of bovine CNS myelin basic protein were synthesized by the solid phase procedure of Merrifield. These were the nonapeptide H-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH, the octapeptide H-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH, and the heptapeptide H-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH. They were tested for encephalitogenic activity in guinea pigs with either Freund's complete adjuvant containing M. tuberculosis or muramyl dipeptide in incomplete Freund's adjuvant at doses of 10 microgram per animal. The results show that deletion of one or two residues from the amino-terminal end of the nonapeptide destroyed the ability of the shorter peptides to induce clinical but not histological signs of EAE.     

Influence of a muramyl dipeptide on human blood leukocyte functions and their membrane antigens.

Murabutide, which belongs to the immunomodulator family of muramyl peptides, was applied directly to fresh human blood to evaluate changes in leukocyte properties. After blood incubation with murabutide, lymphocytes presented a higher responsiveness to T-mitogens, and monocytes and polymorphonuclear cells exhibited an increase in their capacity to produce hydrogen peroxide. In addition, murabutide treatment enhanced phagocytic activity of neutrophils, whereas monocytes presented a decrease in this activity. Some surface markers were also investigated in the distinct leukocyte populations. After incubation with murabutide, a larger number of lymphocytes expressed Ta1 antigen (CD W26) and transferrin receptor (CD 71). In contrast, expression of interleukin-2 receptor (CD 25) was slightly decreased. Monocytes from treated blood displayed a larger number of receptors for C3bi (CD 11b), whereas the surface marker CD 14 and the class I receptor for the Fc portion of IgG were down-regulated. Activation of polymorphonuclear cells by murabutide was confirmed by the up-regulation of the C3bi receptor, Fc receptor, and CD 14 surface antigen. The effects of murabutide on leukocytes described in this paper may contribute to understanding mechanisms of the modulating activity of muramyl peptides on specific and nonspecific immunity.     

The frameshift mutation in Nod2 results in unresponsiveness not only to Nod2- but also Nod1-activating peptidoglycan agonists

NOD2/CARD15 is the first characterized susceptibility gene in Crohn disease. The Nod2 1007fs (Nod2fs) frameshift mutation is the most prevalent in Crohn disease patients. Muramyl dipeptide from bacterial peptidoglycan is the minimal motif detected by Nod2 but not by Nod2fs. Here we investigated the response of human peripheral blood mononuclear cells (PBMCs) from Crohn disease patients not only to muramyl dipeptide but also to several other muramyl peptides. Most unexpectedly, we observed that patients homozygous for the Nod2fs mutation were totally unresponsive to MurNAc-L-Ala-D-Glu-meso-diaminopimelic acid (DAP) (M-Tri(DAP)), the specific agonist of Nod1, and to Gram-negative bacterial peptidoglycan. In contrast, PBMCs from a patient homozygous for the Nod2 R702W mutation, also associated with Crohn disease, displayed normal response to Gram-negative bacterial peptidoglycan. In addition, the blockage of the Nod1/M-Tri(DAP) pathway could be partially overcome by co-stimulation with the Toll-like receptors agonists lipoteichoic acid or lipopolysaccharide. Investigation into the mechanism of this finding revealed that Nod2fs did not act as a dominant-negative molecule for the Nod1/M-Tri(DAP) pathway, implying that the blockage is dependent upon the expression or activity of other factors. We demonstrated that PBMCs from Nod2fs patients express high levels of the peptidoglycan recognition protein S, a secreted protein known to interact with muramyl peptides. We proposed that through a scavenger function, peptidoglycan recognition protein S may dampen M-Tri(DAP)-dependent responses in Nod2fs patients. Together, our results identified a cross-talk between the Nod1 and Nod2 pathways and suggested that down-regulation of Nod1/M-Tri(DAP) pathway may be associated with Crohn disease.     

Peptidoglycans as promoters of slow-wave sleep. II. Somnogenic and pyrogenic activities of some naturally occurring muramyl peptides; correlations with mass spectrometric structure determination

The structures of components of the sleep-promoting material purified from human urine were established by fast atom bombardment-mass spectrometry, as reported in the accompanying paper (Martin, S. A., Karnovsky, M. L., Krueger, J. M., Pappenheimer, J. R., and Biemann, K. (1984) J. Biol. Chem. 259, 12652-12658). We report here that two substances isolated from that preparation, viz. N-acetylglucosaminyl-1,6 -anhydro-N-acetylmuramyl-Ala-gamma-Glu-diaminopimelyl-Ala) and that compound lacking the terminal alanine, are active as somnogens. Cerebro-intraventricular administration of 1 pmol of the glycotetrapeptide was sufficient to induce prolonged excess sleep in rabbits. A similar substance obtained from Brevibacterium divaricatum in which the free carboxyls of the glutamic and diaminopimelic moieties, indicated above, were amidated (N-acetylglucosaminyl-1,6-anhydro-N-anhydromura-myl-Ala-iso- Gln- epsilon-amido-diaminopimelyl -Ala-Ala) was not active as a promoter of slow-wave sleep. Deamidation of this peptide to a mixture of the free dicarboxylic forms produced a somnogenic substance. Our findings show that in addition to the muramyl form of peptidoglycan monomers, the anhydro muramyl form, with no reducing end, is compatible with somnogenic activity. Furthermore, the data obtained with a natural product amplify our earlier observations with smaller synthetic molecules of the importance of amidation/deamidation in the structure-activity relationships of muramyl peptides.     

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.     

Tracheal cytotoxin structural requirements for respiratory epithelial damage in pertussis

The respiratory epithelial pathology of pertussis (whooping cough) can be reproduced by tracheal cyto-toxin (TCT), a disaccharide-tetrapeptide released by Bordetella pertussis. TCT is a muramyl peptide, a class of peptidoglycan-derived compounds which have many biological activities including adjuvanticity, somnogenicity, pyrogenicity, and cytotoxicity. The structural requirements for muramyl peptides to produce some of these biological effects have been partially characterized. Using in vitro assays with respiratory epithelial cells and tissue, we have previously determined that the disaccharide moiety of TCT is not involved in toxicity and that the side-chain functional groups of diaminopimelic acid (A₂pm) are crucial for toxicity. In this study, we determine the importance of every amino acid, functional group and chiral centre in the peptide portion of TCT. Although lactyl tetrapeptides are the most toxic of the TCT fragments, producing dose-response curves identical to TCT, the smallest analogues of TCT which are active in our assay are of the form X-γ-(d )-Glu-meso-A₂pm, where X may be an amino acid or a blocking group. Within this active substructure, main-chain chirality and all functional groups are essential for toxicity. This definition of the core region of TCT indicates that the TCT interaction site is unlike almost all other muramyl peptide interaction sites for which structure-activity data are available.     

Peptidoglycans as promoters of slow-wave sleep. I. Structure of the sleep-promoting factor isolated from human urine

Fast atom bombardment-mass spectrometry (FABMS) has been used to determine the structure of the urinary sleep-promoting factor (FSu), the nature of whose components had been reported earlier. Less than 1 nmol of the underivatized substance sufficed for the FABMS experiments. The major somnogenic constituent of the purified preparation was a peptidoglycan of Mr = 921 with the structure N-acetylglucosaminyl-N -acetylanhydromuramylalanylglutamyldiaminopimelylalanine. The anhydro linkage is between C-1 and C-6 of the muramyl entity. Two additional substances accompanied the above compound. These were the hydrated form (i.e. in which the muramyl entity had a free reducing end, and a free hydroxyl on C-6), and an anhydro analogue lacking the terminal alanine. The Mr values were 939 and 850, respectively. Methyl esters were prepared, and these were also acetylated. The mass spectra of the methyl ester of Mr = 921 displayed an increase in Mr of 42 (i.e. 3 X 14), indicating the presence, originally, of three free carboxyls. Acetylation increased Mr by a further 168 units (i.e. 4 X 42), indicating 4 hydroxyl or amino groups. These data are consistent with the structure cited above for the main entity of FSu. Similar confirmatory results were obtained for the two minor constituents described above. These operations were worked out on natural muramyl peptides of known structure, obtained from other sources, and the data are given for comparison.     

Muramyl Peptides as Possible Endogenous Immunopharmacological Mediators

Synthetic N-acetylmuramyl-L -alanyl-d-isoglutamine, also called MDP for muramyl dipeptide, is a copy of a fragment of bacterial peptidoglycan. Soon after the recognition of MDP as being the minimal subunit responsible for the activity of Freund's complete adjuvant, a great number of derivatives were synthesized. Because of their very low molecular weight it was hoped that they could retain selectively certain of the numerous effects produced by complex bacterial agents. Evidence was gathered showing MDP's direct effect on lymphocytes and on macrophages. The ensuing studies reviewed that MDP and several of its derivatives have marked immunopharmacological and neuropharmacological activities. Thus, besides being adjuvants, they are capable of producing hyperthermia by acting directly on thermoregulation centers or by inducing in vivo and in vitro endogenous pyrogens (EP). More recently, Krueger et al have shown that slow-wave sleep (SWS) factor was a muramyl peptide of a molecular weight close to 1,000 daltons. They have also shown that MDP and several of its synthetic analogs had a somnogenic activity. It has previously been hypothesized that several of the immunological activities of the muramyl peptides could be due to biological mimicry with endogenous products. Recent observations argue in favor of the presence of an MDP bacterial structure in mammalian mediators which increase slow-wave sleep and/or produce fever. The implications of these findings will be discussed.