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.     

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).     

Inhibition of tumor growth in mice treated with synthetic muramyl dipeptide

Summary Treatment with synthetic MDP inhibited growth of transplantable, chemically induced tumors in syngeneic mice. The tumor-inhibitory effect was dependent on the schedule of MDP administration.Growth of SC transplants of a nonmetastasizing, MC-induced fibrosarcoma, MC11, was inhibited by local treatment with 200 g and 1,000 g MDP given SC 5–7 weeks before challenge. Treatment with lower (10 g and 100 g) doses of MDP and shorter (1–4 weeks) time intervals was not effective. Single doses of MDP (10–1,000 g) 1–3 weeks after challenge had no effect.Growth of IV-inoculated, metastasizing AAT-induced hepatoma A was inhibited by IV injections of 20 g MDP given 1 and 2 days prior to the challenge. Significant increases in the survival of hepatoma-bearing mice were observed only after injections of MDP incorporated in multilamellar liposomes.Abbreviations MDP n-acetylmuramyl-l-alanyl-d-isoglutamine - B10 C57BL/10ScSnPh mice - MC 3-methylcholanthrene - ATT o-amino-azotoluene - PBS phosphate-buffered saline     

Immunostimulating properties of synthetic derivatives of muramyl dipeptide and glucosaminyl muramyl dipeptide in vitro

Production of tumor necrosis factor (TNF) and interleukin-1 (IL-1) by macrophages of the spleen and peritoneal exudate of mice as well as cytotoxic factors (CFs) by murine splenocytes after in vitro activation was estimated. All the derivatives of muramyldipeptide (MDP) and glucosaminylmuramyldipeptide (GMDP) were able to induce production of TNF and CFs. In the presence of lipopolysaccharide (LPS), the effect was always higher. The response of the spleen macrophages to the effect of the preparations was higher than that of the peritoneal ones and ++non-fractionated splenocytes. GMDP and GMDP4 especially in the presence of LPS had the highest effect on induction of IL-1 by the murine peritoneal macrophages. On the contrary, MDP induced higher IL-1 synthesis by the spleen macrophages. The most active substances with respect to production of TNF, CFs and IL-1, i.e. MDP3 and GMDP4, might be recommended for immunotherapy of syngeneic tumors in animals.     

Immunostimulating effects of muramyl dipeptide, glucosaminyl muramyl dipeptide and their synthetic derivatives in vitro

The effect of muramyldipeptide (MDP), glucosaminylmuramyldipeptide (GMDP) and their six synthetic derivatives on production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-2) by murine spleen cells in vitro was studied. MDP induced insignificant TNF production and did not stimulate production of IL-1 by the murine splenocytes within a 24-hour cultivation period whereas in combination with lipopolysaccharide (LPS) it induced significant production of both the cytokins. GMDP induced marked production of TNF (54 per cent cytotoxic index) and IL-1 (stimulation index 8). Addition of LPS in an amount of 10 ng/ml increased production of TNF by the murine splenocytes under the effect of GMDP but had no effect on production of IL-1. Neither MDP nor GMDP even in combination with LPS induced production of IL-2 by splenocytes of mice DVA/2 and C57B1/6 at activation for 24 hours. All the synthetic derivatives of MDP and GMDP except the MDP polymer activated TNF production by the murine spleen cells. GMDP lysine had the highest effect: 67 per cent cytotoxic index. In combination with LPS its cytotoxic index amounted to 87 per cent. The TNF activity was always higher when LPS in an amount of 10 ng/ml was added to the glycopeptides.     

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.     

ImmTher, a lipophilic disaccharide derivative of muramyl dipeptide, up-regulates specific monocyte cytokine genes and activates monocyte-mediated tumoricidal activity

ImmTher, a liposome-encapsulated lipophilic disaccharide tripeptide derivative of muramyl dipeptide, previously showed activity against liver and lung colorectal metastases in a phase I trial. The purpose of the current studies was to investigate whether ImmTher could up-regulate specific cytokine gene expression and protein production, as well as activate the tumoricidal or cytostatic activity of human monocytes. ImmTher induced the expression and production of interleukin(IL)-1α IL-1β, IL-6, IL-8, IL-12, macrophage chemotactic and activating factor, and tumor necrosis factor α but not IL-2 or IL-10. Cytostatic or cytotoxic monocyte activity was stimulated against human Ewing's sarcoma, osteosarcoma, and melanoma cells but not breast cancer cells. Production and secretion of these cytokine proteins may play a role in the antitumor activity of ImmTher. Received: 15 December 1998 / Accepted: 18 March 1999     

Molecular mechanisms involved in the regulation of cytokine production by muramyl dipeptide

MDP (muramyl dipeptide), a component of peptidoglycan, interacts with NOD2 (nucleotide-binding oligomerization domain 2) stimulating the NOD2-RIP2 (receptor-interacting protein 2) complex to activate signalling pathways important for antibacterial defence. Here we demonstrate that the protein kinase activity of RIP2 has two functions, namely to limit the strength of downstream signalling and to stabilize the active enzyme. Thus pharmacological inhibition of RIP2 kinase with either SB 203580 [a p38 MAPK (mitogen-activated protein kinase) inhibitor] or the Src family kinase inhibitor PP2 induces a rapid and drastic decrease in the level of the RIP2 protein, which may explain why these RIP2 inhibitors block MDP-stimulated downstream signalling and the production of IL-1beta (interleukin-1beta) and TNFalpha (tumour necrosis factor-alpha). We also show that RIP2 induces the activation of the protein kinase TAK1 (transforming-growth-factor-beta-activated kinase-1), that a dominant-negative mutant of TAK1 inhibits RIP2-induced activation of JNK (c-Jun N-terminal kinase) and p38alpha MAPK, and that signalling downstream of NOD2 or RIP2 is reduced by the TAK1 inhibitor (5Z)-7-oxozeaenol or in TAK1-deficient cells. We also show that MDP activates ERK1 (extracellular-signal-regulated kinase 1)/ERK2 and p38alpha MAPK in human peripheral-blood mononuclear cells and that the activity of both MAPKs and TAK1 are required for MDP-induced signalling and production of IL-1beta and TNFalpha in these cells. Taken together, our results indicate that the MDP-NOD2/RIP2 and LPS (lipopolysaccharide)-TLR4 (Toll-like receptor 4) signalling pathways converge at the level of TAK1 and that many subsequent events that lead to the production of pro-inflammatory cytokines are common to both pathways.     

Macrophage activation by mycobacterial water soluble compounds and synthetic muramyl dipeptide.

The adjuvant effects of mycobacteria can be replaced by more chemically defined isolates of the cell walls including a water soluble fraction (WSA) and by the synthetic analog N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), which is the minimal structure required for adjuvanticity. These compounds can directly activate macrophages as determined by an increase in spreading and adherence and by an elevated synthesis of the enzyme collagenase. Moreover, this increase in collagenase production is modulated by enhanced production of prostaglandins that influences intracellular levels of cyclic AMP. In addition, both MDP and WSA induced macrophages to produce a biologically active mediator that triggers quiescent fibroblasts into active proliferation. It thus appears that a mechanism for mycobacterial adjuvant action as determined with MDP and WSA is via activation of macrophages, which may then precipitate a multiplicity of other reactions resulting in enhanced immune phenomena. Furthermore, the granulomatous and fibrotic reactions associated with mycobacterial infection may be a consequence of this direct activation of macrophages.     

Augmentation of the proliferative response of thymocytes to phytohemagglutinin by the muramyl dipeptide

A synthetic N-acetylmuramyl-l-alanyl-d-isoglutamine or muramyl dipeptide (MDP) and adjuvant-active analogs, but not lipopolysaccharide (LPS), exhibited the augmenting effect on the proliferative response of thymocytes to phytohemagglutinin (PHA). MDP also had a comitogenic effect on PHA-stimulated T lymphocytes. It was shown that the thymocyte-stimulating effect of MDP is not through the production of the monokines by MDP-stimulated macrophages and that MDP has a direct action on lymphocytes.     

Synthesis and antiproliferative activity of conjugates of adenosine with muramyl dipeptide and nor-muramyl dipeptide derivatives

We synthesized a series of MDP(D,D) and nor-MDP(D,D) derivatives conjugated with adenosine through a spacer as potential immunosuppressants. New conjugates 8a–k were evaluated on two leukemia cell lines (Jurkat and L1210) and PBMC from healthy donors. The conjugates 8a–k and MDP(D,D)/nor-MDP(D,D) derivatives 7e, f, i, j were active against L1210 cell line. Unconjugated nor-MDP(D,D) had better antiproliferative properties, but the conjugates 8b, f, g had the highest values of selectivity index. Both cell lines as well as PBMC were resistant to analogs 11a, b with the 6-aminohexanoic linker.     

Stimulation of an enhanced in vitro immune response by a synthetic adjuvant, muramyl dipeptide.

Various subcellular bacterial fractions are known to enhance immune responses and serve as potent adjuvants. Muramyl dipeptide (MDP), a synthetic adjuvant mimicking a component of mycobacterial cell walls, enhances humoral immunity to soluble antigens and can increase macrophage cytotoxicity toward mastocytoma cells in vitro. In the present study MDP was found to enhance the hemolytic antibody plaque response of normal mouse spleen cells in vitro to SRBC at a level equal to or greater than that induced by Escherichia coli lipopolysaccharide. Furthermore, MDP was found to enhance the antibody response to SRBC nonspecifically in unimmunized spleen cell cultures, suggesting that similar to LPS the synthetic dipeptide may induce a generalized clonal expansion of committed lymphocytes and thus serve as a "polyclonal activator." MDP also enhanced the immune responsiveness of normal splenocytes to suboptimum concentrations of SRBC, indicating that this material may be useful in enhancing immunity in situations where there would normally be a poor immune response.     

Augmentation of mouse natural killer cell activity by muramyl dipeptide and its analogs

The ability of muramyl dipeptide (MDP) and its structural analogs (des-MDP, abu-MDP, and des-abu-MDP) to influence mouse natural killer (NK) cells in two different strains of mice was examined. In CBA/J mice, administration of MDP by both intraperitoneal (ip) and intravenous (iv) routes enhanced splenic NK cell activity. Maximum augmentation of NK cell activity was observed 3 days after MDP treatment. NK cell activity was also stimulated upon in vitro culture of CBA/J mouse spleen cells with MDP. Only iv inoculation of MDP to C57BL/6 mice 7 days previously enhanced NK cell activity of spleen cells. Peritoneal NK cell activity was not affected in either strain of mice, regardless of the route of inoculation of MDP. Two structural analogs of MDP, abu-MDP and des-abu-MDP, enhanced peritoneal NK cell activity, whereas des-MDP had no effect when tested 3 days after ip treatment of CBA/J mice with these compounds. Peritoneal NK cell activity of C57BL/6 mice was not modulated by des-MDP, abu-MDP, or des-abu-MDP. A synergistic effect on peritoneal NK cell activity was observed in both CBA/J and C57BL/6 mice treated first with MDP and then with lipopolysaccharide (LPS) or Bacillus Calmette-Guerin (BCG).     

Conditions controlling tumor cytotoxicity of rat liver macrophages mediated by liposomal muramyl dipeptide

Activation of rat liver macrophages with free and liposome-encapsulated muramyl dipeptide (MDP) to a tumorcytotoxic state was characterized by employing various experimental conditions. Macrophage-mediated tumor cytotoxicity was determined using two standard assay systems: a [methyl-3H]thymidine release assay to measure the extent of tumor cell lysis and a [methyl-3H]thymidine incorporation assay to measure the combined effects of tumor cell lysis and stasis. The extent of cell lysis was not affected by the ratio of macrophages to tumor cells within the ratio range of 30:1 to 5:1, provided that the macrophages form a confluent monolayer. Tumor cell lysis, however, was significantly influenced by macrophage density; a low macrophage density for example resulted in a low percentage of tumor cell lysis. Tumor target cells used in this study, i.e., C26 adenocarcinoma, B16 melanoma and P815 mastocytoma, differed in their susceptibility towards macrophage-mediated cell lysis, whereas no differences were observed with respect to tumor cell stasis. Non-tumorigenic cell lines such as human fibroblastic cells and LLC monkey kidney cells were not lysed by activated macrophages, although proliferation of these cells was markedly inhibited. Additionally, the effects of liposomal lipid composition on macrophage activation were studied. With a basic composition of phospholipid/cholesterol/dicetylphosphate, we used either egg-yolk, dipalmitoyl-, distearoyl- or dihexadecylphosphatidylcholine as the bulk phospholipid constituent. Although these liposomes display a widely different susceptibility to lysosomal phospholipase activities, we could not detect any significant difference in either the extent or the duration of the tumoricidal activity induced by MDP encapsulated in these different types of liposomes.     

Activation of human NK cells by the bacterial pathogen-associated molecular pattern muramyl dipeptide

Muramyl dipeptide (MDP) is a bacterial pathogen associated molecular pattern derived from both Gram-positive and -negative bacteria. It is a specific ligand for nuclear oligomerization domain 2, a pattern recognition receptor best characterized for its role in immunosurveillance in the gut. In this study, we demonstrate that human peripheral blood NK cells express nuclear oligomerization domain 2 and respond to MDP. NK cells naturally internalize MDP leading to direct cell activation, including signaling through NFkappaB: characterized by p50/p65 heterodimers at early stimulations times and sustained activation of p50 homodimers. Moreover, MDP synergizes with IFN-alpha and IL-12 to activate NK cells and stimulate IFN-gamma secretion, suggesting a role for accessory cells in induction of an optimal NK cell response. Although IL-12 costimulation leads to a greater IFN-gamma response by NK cells, higher levels of CD69 in response to MDP are induced in the presence of IFN-alpha, suggesting that different pathogen-induced cytokine profiles will affect downstream NK cell responses. In contrast, MDP alone or in combination with either IFN-alpha or IL-12 only poorly increases NK cell cytotoxicity. In summary, this report identifies MDP as a bacterial pathogen associated molecular pattern that activates human NK cells.