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.     

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.     

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.     

Macrophage activation induced by a synthetic antioxidant

The effect of a synthetic antioxidant 2-tretbutyl-3-hydroxypyridine (TBHP) on the function of murine peritoneal macrophages (MP) has been studied. A direct contact of TBHP with MP in vitro increased the activity of a key enzyme of glucose monophosphate graft--glucose-6-phosphate dehydrogenase and the proportion of flattened MP. as compared to the control. Upon intraperitoneal MP injection the number of MP's in the abdominal cavity of mice increased. They differed from control MP's in enhanced flattening and phagocytosis. In mice with preinduced defect of abdominal clearance TBHP contributed to the recovery of the normal level of antibacterial protection. In all the in vitro and in vivo tests studying its activating effect on MP, the synthetic antioxidant was not inferior to the standard MP activator--bacterial lipopolysaccharide.     

Target cells for the activity of a synthetic adjuvant: muramyl dipeptide.

Muramyl dipeptide (MDP), a synthetic adjuvant, increased the primary response of CBA mice to sheep red blood cells (SRBC). In reconstituted irradiated recipients, cooperation between T and B lymphocytes was required for the expression of adjuvant activity and MDP increased the efficiency of SRBC-educated T cells. The role of T-derived lymphocytes in mediating the MDP adjuvant activity was also demonstrated in irradiated mice and in mice reconstituted with various splenic cellular types of donors which had received SRBC and MDP 24 hr earlier. In our experiments, the macrophage did not seem to be involved, since MDP did not increase the phagocytic capacity of peritoneal exudate cells and MDP- and SRBC-pretreated macrophages had no increased ability to induce an anti-SRBC immune response. These results demonstrate the importance of T lymphocytes as mediators of the adjuvant activity of MDP.     

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.     

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     

Effect of synthetic muramyl dipeptide derivatives on staphylococcal infection in mice

The work deals with the results of experimental evaluation of the influence of some new modified derivatives of muramyldipeptide (MDP) on the course of staphylococcal infection in mice. The preparations under study were found to produce rapid elimination of bacteria from kidneys and the increase of phagocytic activity of blood macrophages in animals. At the same time MDP and its derivatives stimulated natural killer cells whose activity was inhibited during infection. The dependence between the structure of these compounds and their protective action in staphylococcal infection, as well as the increase of the natural immunity characteristics of the body was followed.     

Comparison of murine B cell clonal expansions by synthetic lipid A and muramyl dipeptide analogs

Direct stimulations of murine B lymphocytes with synthetic lipid A analogs and synthetic muramyl dipeptide (MDP) derivatives were studied using a limiting dilution assay system. Synthetic lipid A analogs, GLA-27 and GLA-40, when conjugated with bovine serum albumin (BSA) had the ability to induce B cell clonal expansion of a single B cell from the spleen or bone-marrow. Their activities were almost the same as those of naturally obtained lipid A, but were lower than that of bacterial lipopolysaccharide (LPS). Addition of dextran sulfate (DXS) enhanced the effect of lipid A analogs. In contrast, synthetic MDP and its derivatives, although they had many biological and immunological activities in experimental animals, could not stimulate a single B cell to induce clonal expansion regardless of the presence or absence of DXS. These results suggested that lipid A analogs can directly cause the proliferation of B cells, but MDPs can not.     

Stereo-isomer specific induction of renal cell apoptosis by synthetic muramyl dipeptide lpar;N-acetylmuramyl-L-alanyl-D-isoglutamine)

In order to understand the modification of -adrenoceptor linked signal transduction by changes in the intracellular Ca²⁺, we examined the status of -adrenoceptors (-ARs), G-proteins and adenylyl cyclase (AC) in Ca²⁺-deficiency and Ca²⁺-overload by perfusing the isolated rat heart with Ca²⁺-free medium for 5 min and Ca²⁺-containing medium for 5 min following Ca²⁺-free perfusion, respectively. Ca²⁺-depletion caused not only an increase in basal, isoproterenol-, Gpp(NH)p-, NaF- and forskolin-stimulated AC activities but also produced an increase in the ₁-AR affinity and density as well as up-regulation of G_s-protein function and uncoupling of G_i-protein to AC. Ca²⁺-repletion for 5 min following 5 min Ca²⁺-free perfusion reversed the increased AC activities to varying degrees. The ₁-AR affinity was further increased upon Ca²⁺-repletion whereas its density was decreased. Ca²⁺-repletion also decreased protein content for AC and -AR kinase but augmented the changes in G_s- and G_i-protein functions. Although low Na⁺- medium perfusion during Ca²⁺-depletion prevented the changes in G-proteins during both Ca²⁺-depletion and Ca²⁺-repletion periods, the increased ₁-AR affinity and density as well as changes in AC activities due to Ca²⁺-depletion were not affected while alterations due to Ca²⁺-repletion were fully prevented. These results suggest that changes in Ca²⁺-homeostasis may represent a mechanism for alterations in the -adrenergic signal transduction pathway in the heart under pathological conditions.     

Synergistic activation by recombinant mouse interferon-gamma and muramyl dipeptide of tumoricidal properties in mouse macrophages

Mouse peritoneal macrophages were activated to become cytotoxic against B16-BL6 melanoma cells by the combination of subthreshold amounts of murine interferon-gamma (IFN-gamma; 0.1 to 10 U/ml) and N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP; 0.001 to 10 micrograms/ml), but not by the combination of pH 2-treated IFN-gamma and MDP, heat-treated IFN-gamma and MDP, or IFN-gamma and the inactive stereoisomer of MDP, N-acetyl-muramyl-D-alanyl-D-isoglutamine (MDP-D). The encapsulation of intact IFN-gamma and MDP within the same liposome preparation was synergistic for macrophage activation. In contrast, the presentation of identical concentrations of IFN-gamma and MDP in separate liposome preparations did not activate macrophages. These data allow us to conclude that the encapsulation of genetically engineered IFN-gamma and synthetically produced MDP within the same liposome is highly efficient in producing synergistic activation of tumoricidal properties in mouse macrophages.     

The activation of tumoricidal properties in human blood monocytes by muramyl dipeptide requires specific intracellular interaction

The purpose of this study was to identify the mechanism by which muramyl dipeptide (MDP) activates antitumor cytotoxic properties in normal and interferon-gamma (IFN-gamma)-primed human peripheral blood monocytes. The structurally and functionally active MDP analog, nor-muramyl dipeptide (nor-MDP), and [3H]nor-MDP were used as reference glycopeptides. Direct activation of normal, noncytotoxic monocytes by nor-MDP was enhanced by its encapsulation within multilamellar vesicles (MLV). Studies with [3H]nor-MDP revealed that the activation of monocytes by nor-MDP was not attributable to its interaction with a specific cell surface receptor, nor did it result merely from the internalization by monocytes of glycopeptide. Subthreshold concentrations of nor-MDP could activate tumor cytotoxic properties in IFN-gamma-primed monocytes. The intracellular interaction of [3H]nor-MDP with IFN-gamma-primed monocytes was specific in that intracellular levels of radiolabeled material could be displaced and recovered as intact molecules by unlabeled nor-MDP, but not by a biologically inactive MDP stereoisomer. Collectively, these results suggest that the activation of tumoricidal properties in human blood monocytes by MDP occurs subsequent to intracellular interaction with specific MDP receptors.     

Macrophage activation and immunomodulation by myeloperoxidase.

Myeloperoxidase (MyPo) is an enzyme found in neutrophils and monocytes that plays an important role in the microbicidal and cytocidal activities of these cells. The present studies show that this enzyme can also affect both capacities and functions of macrophages. When resident peritoneal macrophages from C57BL/6 mice were exposed to preparations of either human or canine enzyme in vitro, tumor necrosis factor (TNF) was released. The amount of TNF produced was dose dependent and could be neutralized with polyclonal anti-TNF. Low levels of interferon were also produced by these cells. In addition, exposure of murine macrophages in vitro to this enzyme resulted in increased ability to destroy 3T12 target cells. Intravenous injection of mice with myeloperoxidase induced the production of both TNF and interferon, which could be detected in the sera. Possible mechanisms of TNF induction include radical production by myeloperoxidase or ligand-receptor interaction by the binding of this enzyme to the mannosyl-fucosyl receptor. These results, when taken in their entirety, suggest that this enzyme can modulate the immune response through effects on macrophage function.     

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.     

Activation of mouse macrophages by muramyl dipeptide coupled with an anti-macrophage monoclonal antibody.

A rat IgG2a monoclonal antibody (mAb3A33) directed against the mouse Mac-1 antigen was conjugated with muramyl dipeptide (MDP) by using an intermediate polymer; under such conditions 75 MDP molecules were bound to one antibody molecule. A poly(L-lysine) polymer substituted with muramyl dipeptide and 3-(2-pyridyldithio)propionyl residues were prepared, the remaining lysine epsilon-amino groups were acylated with D-gluconolactone, leading to a neutral polymer; then a few polymer conjugates were coupled to mAb3A33 via a disulfide bridge. The binding capacity of the monoclonal antibody was preserved after conjugation with MDP-polymer molecules. Mouse peritoneal macrophages, incubated for 24 h with MDP-mAb3A33 conjugate became cytostatic against P815 mastocytoma cells, whereas unconjugated mAb3A33 and MDP-bound to a nonspecific rat IgG2a were ineffective. An enhancement of the cytostatic activity induced by MDP-mAb3A33 conjugate was obtained in the presence of gamma-IFN. These results show that several tens of MDP molecules can be linked to a macrophage-specific monoclonal antibody by using a neutral intermediate polymer without impairing the binding antibody capacity and that this type of MDP conjugate can efficiently activate macrophages and therefore could be the basis of the development of new antitumor therapy.