The adjuvant activity of synthetic N-acetylmuramyl-dipeptide: evidence of initial target cells for the adjuvant activity.

MurNAc-l-Ala-d-isoGln (N-acetylmuramyl-l-alanyl-d-isoglutamine, MDP), a synthetic compound, acts as an adjuvant on the humoral immune response and on the T cell-mediated immune response. In this report, we attempted to directly demonstrate the initial target cells of MDP for its adjuvant activity in vitro by using cell separation procedures.It was demonstrated that MDP enhanced the immune response following direct interaction with antigen-stimulated T and B lymphocytes, but nonstimulated lymphocytes, shortly after triggering by antigen, and that there was no macrophage requirement for MDP to elicite the adjuvant action in the primary anti-SRBC PFC response in vitro. It has also been demonstrated that the adjuvant activity of MDP is due to an enhancing effect which is different from the possible mitogenic activity to spleen cells and MDP replaces neither a function of macrophages, which is substituted by 2-mercaptoethanol nor a helper function of T cells.     

Components of mycobacteria and muramyl dipeptide with adjuvant activity induce lymphocyte activating factor

Several components of mycobacteria including a water-soluble extract (WSA) and an interphase material (IPM) as well as the synthetic cell wall analog muramyl dipeptide (MDP) all stimulated human mononuclear cells (MNL) to produce a factor which was mitogenic for murine thymocytes. The mediator induced by MDP is probably lymphocyte-activating factor (LAF) because it was produced by adherent but not nonadherent MNL and yields two characteristic peaks of activity in the 16,000–22,000 and 60,000–70,000 molecular weight range when eluted from Bio-Gel P-100 columns. The 6-O-stearoyl derivative of MDP was an active inducer of MNL LAF production, whereas, the d-alanine analog of MDP was somewhat less potent. Unfractionated as well as adherent, but not nonadherent, mouse peritoneal cells also produced LAF in response to WSA, IPM, and MDP. P388D₁ cell line macrophages, which are completely devoid of lymphocytes, could be stimulated by WSA, IPM, and MDP to produce LAF after prolonged incubation. These adjuvants did not stimulate nonadherent Balb/C or human blood cells to produce a mitogenic factor. However, when the P388D₁ macrophages were stimulated with these adjuvants in the presence of nonadherent murine or human peripheral blood cells, a mitogenic activity was produced in a shorter period of incubation suggesting that activated lymphocytes can facilitate the production of LAF by macrophages.     

Activation of the production of cytotoxic factors by mouse spleen cells exposed to the combined action of lipopolysaccharide and muramyl dipeptide in vitro

Lipopolysaccharide (LPS) and muramyl dipeptide (MDP) stimulated murine splenocytes in vitro to produce cytotoxic factors (CF) that killed target cells L-929. This effect was synergic at LPS dose of 10 ng/ml and MDP dose of 10 micrograms/ml. CF production started 2 hours after spleen cell activation and was maximum in 6 hours. CF were produced by macrophages as well as by lymphocytes stimulated by LPS, MDP or their combination. However, synergic effect of immunomodulators was registered only if nonfractionated spleen cells were stimulated during 24 hours. Lymphocytes depleted on T cells did not lack the ability to generate CF upon activation. In addition, LPS and MDP activated synergically the production of interleukin-I by spleen cells in vitro.     

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.     

Enhancement of B-cell stimulation by muramyl dipeptide through a mechanism not involving interleukin 1 or increased Ca2+ mobilization or protein kinase C activation

Muramyl dipeptide (MDP) enhanced mitogenic stimulation of mouse lymphocytes by polyclonal B cell activators (peptidoglycan, lipopolysaccharide, Staphylococcus aureus Cowan I cells, and pokeweed mitogen), but not by T-cell mitogens (phytohemagglutinin and concanavalin A). Only adjuvant-active MDP analogs were effective, whereas adjuvant-inactive MDP analogs, muramic acid, peptidoglycan pentapeptide, and low Mr digests of peptidoglycan were not. The half-maximal enhancement was seen at 5-10 microM MDP and occurred at both optimal and suboptimal concentrations of B cell mitogens. The enhancing effect of MDP was exerted on the B cells, since it was T cell- and macrophage-independent and was not mediated by IL-1. MDP was effective during the first 12 hrs of culture, and most strongly enhanced the mitogen-induced DNA synthesis, although significant enhancement of RNA synthesis and B cell differentiation into antibody-secreting cells was also observed. The enhancement of mitogenic response was not due to changed requirements for extracellular or intracellular Ca2+ or to increased activation of protein kinase C. These results demonstrate a novel immunoenhancing effect of MDP that should be useful in the studies on the mechanism of B cell activation.     

Inhibitory and stimulatory effects of a synthetic glycopeptide (MDP) on the in vitro PFC response: factors affecting the response.

A synthetic adjuvant active glycopeptide, N-acetyl-muramyl-l-alanyl-d-isoglutamine (MDP), has been previously shown to enhance the in vitro immune response of mouse spleen cells to T-dependent or independent antigens. Data presented here show that the net activity of MDP on the in vitro immune response is closely related to the cell culture conditions: Two distinct patterns of MDP activities could actually be detected. Marked stimulation of the PFC response was observed at “low density” cell cultures. In contrast, suppression could be seen at “high density” cell cultures. Moreover, the culture conditions which permitted characterization of either the enhancement or suppression of the immune response by MDP were strongly dependent on the strain of mouse used. However, these activities were not dependent on antigen concentration, on kinetics of responses, or on cytotoxic effects.     

In vitro spleen cell responsiveness to various analogs of MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine), a synthetic immunoadjuvant, in MDP high-responder mice

N-Acetylmuramyl-l-alanyl-d-isoglutamine (MDP), a synthetic immunoadjuvant, was incubated with spleen cells of DBA/2 or Balb/c mice and optimal responses were obtained after 4 or 5 days of culture in a serum-free medium supplemented with 2-mercaptoethanol. In contrast, lymphocytes of (C57B1/6 × AKR)F₁ hybrids responded weakly under the same conditions. The results reported here show that like in the case of DBA/2 and Balb/c strains, spleen cells of Swiss mice and of inbred AKR and CBA mice could be stimulated in vitro whereas C57B1/6 and LPS-refractory C3H/He mice did not respond. Fourteen synthetic MDP analogs (eight known to be adjuvant active and six devoid of activity) were tested in DBA/2 high-responder mice. A good correlation was observed between in vitro stimulation and the presence or absence of adjuvant activity in vivo of these compounds.     

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).     

The synergistic action of lipopolysaccharide and muramyl dipeptide in the immunotherapy of DBA/2 mice with mastocytoma P815

The intratumoral administration of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) in combination, but not separately, resulted in necrosis and rejection of subcutaneous P815 mastocytoma nodules in DBA/2 mice with 30 to 40% survival. Previous sensibilization of animals by LPS + MDP, treatment by indomethacin, cyclophosphamide or syngeneic lymphocytes did not augment the immunotherapeutic action of LPS + MDP combination. Reinoculation of P815 cells into cured DBA/2 mice 8 months after the disappearance of the primary tumor led to rejection of new nodules with 50% survival rate. In LPS + MDP immunotherapy of these tumors two stages may be distinguished by a thrombo-necrotic stage and that of development of immunity.     

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.     

On the mechanism of early recovery of specifically depleted lymphoid cell populations by nonspecific activation of T cells.

Specific depletion from normal CBA mouse spleen cells of those bound on pigeon erythrocyte (PRBC) immunoabsorbent columns before transfer of the depleted population into irradiated syngeneic recipients resulted in elimination of the anti-PRBC responsiveness as assessed by rosette (RFC) and hemolytic plaque (PFC) formation. The anti-sheep erythrocyte (SRBC) responses of cell populations treated in the same manner remained unimpaired. When, however, these populations were stimulated with both PRBC and muramyl dipeptide (MDP), an early recovery of specific anti-PRBC responsiveness was produced. PFC response in particular, suddenly increased between the fourth and fifth day after transfer and stimulation thus exhibiting a doubling time of only 4 to 6 hr. This effect of MDP was T-cell dependent since treatment of the depleted population with anti-θ antigen serum and complement hindered early recovery. Depleted populations stimulated with PRBC alone resumed their T-dependent RFC (but not PFC) responsiveness after the eighth day. In spite of the existence of these educated T cells, a second stimulation on the tenth day with PRBC was unable to elicit a specific PFC response. On the other hand stimulation with MDP alone on the day of cell transfer (Day 0) followed by stimulation with PRBC on Day 10 resulted in a specific PFC response on Day 15. Thus, MDP appeared to do more than simply promote education of T cells by antigen. In vitro cultures of depleted populations also recovered their specific reactivity when stimulated by antigen and MDP.     

Nonspecific activation of murine spleen cells in vitro by a synthetic immunoadjuvant (N-acetyl-muramyl-L-alanyl-D-isoglutamine)

We reported previously that synthetic N-acetyl-muramyl-l-alanyl-d-isoglutamine (MDP) displayed marked adjuvant activity but was devoid of mitogenicity in vitro. The data reported here establish that, under different cultural conditions, thymidine uptake and blast cells can be increased by MDP in spleen cells of DBA/2 and Balb/c mouse strains. Optimal responses were obtained on culture in a serum-free medium supplemented with 2-mercaptoethanol for 4 or 5 days. This effect was also obtained with spleen cells of Balb/c nude mice. When the synthetic MDP was compared to a natural water-soluble adjuvant (neo-WSA), extracted from Mycobacterium smegmatis cells, both were found to stimulate [³H] thymidine incorporation by mouse spleen cells. However, with the neo-WSA, the effect peaked on Day 2 and was weak or absent on Days 4 and 5. When the cells were cultured in a medium containing fetal calf serum, neo-WSA activation was completely abolished, while MDP-mediated stimulation was decreased.     

Macrophage Activation by Muramyl Dipeptide (MDP) without Lymphocyte Participation

An increase in the numbers of spread macrophages caused by macrophage stimulants was found to be a very sensitive measure for macrophage activation. Muramyl dipeptide (MDP), bacterial lipopolysaccharide (LPS), and lymphokines were found to activate macrophages dose-dependently as measured by this parameter. Macrophage activation by MDP was strictly dependent on its adjuvant-active stereochemically specific structures. Macrophage activation by MDP and LPS occurred without lymphocyte participation. It is suggested that LPS also activates macrophages via lymphocytes.     

Structural specificity of synthetic peptide adjuvant for induction of experimental allergic encephalomyelitis

The peptide N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), which has adjuvant activities, and 17 of its derivatives and analogs were synthesized and assayed to elucidate the structure necessary for adjuvant activity in induction of experimental allergic encephalomyelitis (EAE) in guinea pigs. The results revealed the importance of the d configuration and the α-carboxamide group of the isoglutaminyl residue of MDP for adjuvant activity. Replacement of the l-alanyl residue of MDP by d-alanine, but not by l-serine or glycine, resulted in a marked decrease in the activity. The β-methyl glycoside of MDP was found to be more active than the α-methyl derivative. 6-O-Stearoyl-N-acetylmuramyl-l-alanyl-d-isoglutamme showed activity.     

Study of the possibility to abolish the action of immunosuppressive factors of tumor cells]

We investigated the efficacy of IL-2, LPS, MDP, TRA, ionomycin and contrykal on proliferation of lymphocytes treated by tumor cell immunosuppressive factors (ISF). IL-2, LPS and/or MDP did not abolish the influence of P815 and B16 ISF on Con A or alloantigen-induced lymphocyte proliferation. TPA and in less extent ionomycin and combination of the above preparations totally abrogated the suppression of Con A-induced lymphocyte proliferation. In inverted experiments Con A abrogated ISF-mediated suppression of lymphocyte proliferation induced by TPA plus ionomycin.     

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.     

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.     

The acute phase response in Japanese quail (Coturnix coturnix japonica)

Experiments were conducted to determine the effect of injection of lipopolysaccharide (LPS, from S. typhimurium) or muramyl dipeptide (MDP, N-acetylmuramyl-L-ala-isoglutamine) in Japanese quail. Doses of MDP between 0.3 and 10 mg/kg body wt. had no effect on body temperature. In contrast, doses of 1.0-22.5 mg LPS/kg body wt. caused significant increases in body temperature. None of the doses of LPS or MDP resulted in mortality. The febrile response to LPS was diminished following a second injection 48 h after the first, and was absent following a third injection. Plasma zinc, an indicator of the acute phase response, was significantly reduced by either LPS or MDP after the first injection (P<0.001), but not after the second or third injection. Splenic interleukin 1-beta (IL-1beta) mRNA expression was increased after the first and last injection of LPS (P<0.001), but only after the first injection of MDP (P<0.005). Hepatic IL-1beta mRNA expression was increased after the first, but not the third injection of LPS (P<0.001), while MDP had no effect. These data indicate that Japanese quail are less sensitive to MDP than LPS, and that quail demonstrate tolerance to LPS following repeated injections.     

Role of the hepatic function in the development of the pyrogenic tolerance to muramyl dipeptide

We have demonstrated that the hepatic function may have an important role in the development of tolerance to the pyrogenic effect induced by endotoxin. To further investigate if the role of the hepatic function in the development of tolerance also extends to that induced by other pyrogenic stimuli, we investigated the effect of galactosamine, a specific inhibitor of the hepatic protein synthesis, on the development of tolerance to the pyrogenic effect induced by muramyl dipeptide (MDP) in rats. Pyrogenic tolerance was observed after the second intravenous or intraperitoneal injection of MDP (500 microgram/kg), 24 h after the first injection, similar to what was observed with endotoxin. Pyrogenic tolerance was abolished when galactosamine (300 mg/kg ip) was injected simultaneously with MDP (500 microgram/kg iv) on the first day. When uridine (600 mg/kg ip) was administered simultaneously with galactosamine (300 mg/kg ip) and the first injection of MDP (500 microgram/kg ip), pyrogenic tolerance was again observed after the second injection of the peptidoglycan. In conclusion, the hepatic function may not be important only for the development of tolerance to endotoxin, but also to a totally different pyrogenic stimulus such as MDP.