Anti-inflammatory effects of muramyl dipeptide in experimental models of acute inflammation

Synthetic muramyl dipeptide,N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), has been tested for activity against acute inflammation. In all models employed (Bayol + Arlacel paw oedema in rats, carrageenan pleurisy in rats, and carrageenan of dextran paw oedema in mice), MDP given in admixture with the phlogistic agents significantly lowered the resulting inflammatory reaction by about 30–40%. 0.1–0.2 mg of MDP per animal was applied. The mechanism of anti-inflammatory activity of this substance remains unknown.     

Proinflammatory effects of interleukin 1 in the rat air pouch

We have utilized the 6-day air-pouch model in rats to study the local tissue response to interleukin-1 exposure. Injection of either recombinant human interleukin 1 alpha (rIL-1 alpha) or interleukin 1 beta (rIL-1 beta) directly into preformed air pouches caused a 10- to 100-fold increase in the number of white blood cells present within the pouch. On a weight basis, rIL-1 beta was more active than rIL-1 alpha. Polymorphonuclear neutrophils (PMN) represented the majority of cells entering the pouch following either a single injection or repeated daily injections of rIL-1 alpha or rIL-1 beta. Significant increases in the number of mononuclear cells present were observed only following repeated injections. Repeated injections of rIL-1 beta, but not rIL-1 alpha, also caused the accumulation of large amounts of fluid within preformed pouches and a grossly apparent thickening of the connective-tissue lining of the pouch. Microscopic examination of stained sections of pouch lining tissue indicated a proliferation of the connective-tissue elements of the lining and deposition of large quantities of extracellular collagen within the pouch wall. These findings are entirely consistent with a role for interleukin 1 in the development and perpetuation of inflammatory reactions.     

Elevation of serum interleukin 6 prior to acute phase proteins on the inflammation by surgical operation

Serum levels of interleukin 6 (IL-6) and acute phase proteins were measured in patients who underwent surgical operation. Elevation of IL-6 preceded that of acute phase proteins, indicating that the measurement of serum IL-6 may be helpful for the early detection of an inflammatory state.     

Pharmacological investigation of acute cellular accumulation in immunological air pouch inflammation

Immunologically-mediated cellular accumulation was measured 24 hours after antigenic challenge using a rat subcutaneous air pouch model. This response was inhibited by treatment with prednisolone, colchicine, anti-thymocyte serum and systemic antigen. In contrast, administration of a range of other pharmacological and clinically active agents had little effect. The profile of inhibitory activity suggested that this response was mainly due to delayed type hypersensitivity with little anaphylactic or Arthus-type component.     

Some characteristics of inflammation induced by muramyl dipeptide,endotoxin, and concanavalin A

Inflammatory reactions induced by muramyl dipeptide (MDP), endotoxin, and concanavalin A (Con A) were examined in the skin of rabbits. The neutrophil influx induced by MDP peaked at 2 h and declined to a low level by 4 h, thus resembling the response induced by endotoxin (2). MDP and endotoxin exhibited homologous desensitization when extant lesions were restimulated at 6 h. These agents did not, however, induce heterologous desensitization. Con A induced a biphasic influx of neutrophils into inflammatory lesions with peaks at 2 h and 12 h. Neither the first nor second peak exhibited desensitization to homologous restimulation; however, the cell influx in restimulated lesions assumed a monophasic character peaking at 3 h. Con A lesions were not desensitized to restimulation with MDP or endotoxin. The results suggest that these chemotaxinogens rely on different endogenous mediators to induce an inflammatory response. The protracted second period of neutrophil infiltration of Con A-induced lesions and the failure of desensitization of this response to develop suggest that the mediator of Con A-induced inflammation may play an important role in the sustained recruitment of neutrophils in some inflammatory diseases.     

Comparative effects of tetrandrine and berbamine on subcutaneous air pouch inflammation induced by interleukin-1, tumour necrosis factor and platelet-activating factor

The effects of the bisbenzylisoquinoline alkaloids tetrandrine and berbamine on the action of IL-1, TNF and PAF were investigated in the rat subcutaneous air pouch model of inflammation. Both compounds were equipotent in the suppression of leukocyte infiltration into air pouches induced by IL-1 and TNF, with ED₅₀ values in the range 20–30 mg/kg/3 days. Both were also equiptent in suppression of PMN infiltration induced by PAF with ED₅₀ values in the same range as that for IL-1 and TNF. However, tetrandrine was more potent than berbamine as a suppressant of PAF-induced MNC infiltration, but much less potent than berbamine in carageenen-induced PMN infiltration. These results suggest that these bisbenzylisoquinolines may have value in the therapy of chronic inflammatory diseases where IL-1, TNF and PAF have a role in pathogenesis.     

Increased expression of alkaline phosphatase activity in stimulated B lymphocytes by muramyl dipeptide

We have recently shown that the synthetic immunomodulator muramyl dipeptide (MDP) acts on murine B lymphocytes. It synergizes with interleukin 2 and interleukin 4 to stimulate, respectively, the differentiation and the proliferation of B cells. In the present study, MDP was shown to increase the proliferation of B cells stimulated by lipopolysaccharide (LPS). Moreover, the expression of alkaline phosphatase activity induced by LPS was markedly enhanced by MDP. These effects were time- and dose-dependent. The present report suggests that the biochemical mechanism by which MDP exerts its effects may involve protein phosphorylation-dephosphorylation pathways.     

T-cell-dependent immunobiological activity of a desmuramyl analog of muramyl dipeptide,adamantylamide dipeptide (AdDP), and D-isoglutamine

The present experiments demonstrate that, similar to immunomodulatory muramyl dipeptide, its desmuramyl analog adamantylamide dipeptide is able to induce mild and fully reversible paw edema in mice. This effect is an immune-related phenomenon depending on the activation of T-cell/macrophage interactions and on production of prostaglandins. Possible involvement of certain immunoregulatory/inflammatory cytokines (e.g. IL-1, IL-2) has been suggested. The most probable intrinsic moiety of the adamantylamide dipeptide molecule responsible for triggering the edema formation is obviously D-isoglutamine.     

Demonstration of muramyl dipeptide (MDP)-induced T suppressor cells responsible for MDP immunosuppressive activity

Muramyl dipeptide (MDP), a synthetic immunostimulant, has been previously shown to increase or decrease the humoral and cellular immune responses, depending upon the experimental conditions used. In the present study we have investigated the mechanisms of MDP-induced immunosuppression. After repeated injections of high dosages of MDP in vivo, both adherent and B cell-enriched cell populations from MDP-treated mice were able to collaborate with normal complementary populations. In contrast, T cell-enriched populations exhibited suppressive activity which could be removed by treatment with anti-Thy-1.2 antiserum and complement. These results clearly indicate that MDP-induced immunosuppression is mediated by T cells.     

Involvement of reactive oxygen metabolites in the candidacidal activity of human neutrophils stimulated by muramyl dipeptide or tumor necrosis factor

In the presence of the adjuvant glycopeptide muramyl dipeptide (MDP), purified human PMN exhibited an enhanced capacity to kill Candida albicans cells at various cell ratios. A significant effect was obtained at 100 ng/ml MDP, and the maximum was reached at 1 micrograms/ml MDP. Recombinant human tumor necrosis factor (rHuTNF), a monokine that enhances host resistance to bacterial and fungal infections, also stimulated the candidacidal potency of PMN with a maximal effect at 10(-2) ng/ml rHuTNF. When MDP- or rHuTNF-stimulated PMN were cultured with yeast cells, the intracellular production of oxygen metabolites was enhanced. Pretreatment with inhibitors of oxidative burst demonstrated that the yeast cell killing by MDP-stimulated PMN was not affected by SOD but was inhibited by sodium azide, indicating the involvement of myeloperoxidase (MPO)-halide system in fungicidal mechanisms induced by MDP. When PMN were stimulated with rHuTNF, the killing of yeast cells was neutralized by iodoacetamide, showing that the candidacidal potency of stimulated-PMN was due to oxygen derivatives. Inhibition by sodium azide and sodium benzoate indicated that these oxygen metabolites could be derived from the MPO-halide system but also from hydroxyl radical production. Moreover, SOD partially inhibited the fungicidal potency of rHuTNF-stimulated PMN, thus indicating a possible reutilization of the released O2- anion for intracellular killing. Cytochalasin B abrogated the PMN fungicidal potency in all cases.     

Agglutination of intravenous lipid emulsions by acute phase proteins of inflammation

Agglutination of intravenous fat emulsions (IVFE) by sera of acutely ill children was studied in vitro in 23 patients. C-Reactive protein (CRP) has been showed to agglutinate with IVFE; after discarding CRP, serum of acutely ill patients still agglutinate with IVFE. The agglutination score in generalized sepsis is significantly highest than in localized sepsis. We studied agglutination during three periods: first days of treatment by antibiotics (period 1), between the fourth day and the end of treatment (period 2), and after the end of treatment (period 3). In period 2, when orosomucoid concentrations; nevertheless, the observation of an agglutination in periods 1 and 3 where the mean orosomucoid level is within normal range, strongly suggests that one or more others acute phase proteins are also involved in occurrence of agglutination.     

Different pro-inflammatory profiles of interleukin 1 (IL 1) and tumor necrosis factor (TNF) in anin vivo model of inflammation

The pro-inflammatory properties of IL 1 and TNF were investigated in anin vivo model of inflammation. IL 1 induced PMN leukocyte accumulation that was slow in onset, reaching a peak rate at 3–4 h and that was inhibitable by Actinomycin D and Cycloheximide. PMN leukocyte emigration was not associated with any significant plasma leakage. In contrast, TNF induced PMN leukocyte accumulation and oedema formation, that were rapid in onset and very short of duration (t1/2 6–10 min). TNF-induced plasma leakage was PMN leukocyte-, but not protein biosynthesis-dependent. The differences in time course and biological profile suggest that IL1 and TNF exert their pro-inflammatory effectsin vivo via different mechanisms.MR is a Senior Research Assistant, Supported by the Belgian National Fund for Scientific Research (NFWO).     

Augmentation of immune responses by a muramyl dipeptide analog,MDP-Lys(L18)

The effects of N²-(N-acetyl-muramyl-l-alanyl-d-isoglutamyl)-N⁶-stearoyl-l-lysine (MDP-Lys(L18)), a muramyl dipeptide (MDP) analog, on the immune responses in mice were studied. MDP-Lys(L18) augmented the mitogenic responses of splenic lymphocytes to phytohemagglutinin (PHA) and lipopolysaccharide (LPS) at 0.1–10 g/ml, and antibody formation to sheep red blood cell (SRBC) in normal and immunosuppressed mice, and to dinitrophenyl (DNP)-Ficoll. In addition, MDP-Lys(L18) potentiated polyclonal B cell activation bothin vivo andin vitro. It was also found that MDP-Lys(L18) augmented the cellular immune responses, such as mixed lymphocyte reaction (MLR) and delayed type hypersensitivity (DTH). These effects of MDP-Lys(L18) were more potent than those of MDP. These findings may be attributed to the interleukin 1 (IL-1)-inducing activity of MDP-Lys(L18).     

A comparison of air pouch,sponge and pleurisy models of acute carrageenan inflammation in the rat

A comparative assessment of the time course of changes in three models of acute carrageenan-induced inflammation, the six-day air pouch, polyester sponge and pleurisy models, was obtained by measuring exudate volume, leucocyte numbers, PGE₂ concentrations and lactate dehydrogenase activity at 2, 6 and 24 h. The greatest increases in exudate volume, leucocyte numbers and PGE₂ concentration and the smallest rise in protein occurred in the air pouch model. Increases in lactate dehydrogenase were greatest in the sponge and least in the pleural exudate, indicating that the least cell damage occurred in the pleurisy model. PGE₂ was not detectable in most pleural exudate samples. The actions of two steroids, betamethasone and dexamethasome, at two dose levels, 80 and 160 g/kg, were assessed in each model. Overall, the six-day air pouch was found to be most satisfactory and most sensitive for assessing the actions of the steroids. The sponge model was either less sensitive or gave inconsistent responses, for the variables measured, than the cavity models of inflammation. Since the six-day air pouch has previously been shown to resemble synovium, our findings indicate that it is likely to be superior to the other two models as a model of joint inflammation.     

Fate of the synthetic immunoadjuvant, muramyl dipeptide (14C-labelled) in the mouse

Synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide or MDP) represents the smallest unit that can substitute for whole Mycobacteria in Freund's complete adjuvant. In this paper the fate of 14C-labelled (on the muramyl moiety) MDP is reported. Following intravenous or subcutaneous injection into mice, more than 50% of 14C-MDP was recovered in the urine after 30 min and more than 90% after 2 h. The labelled compound was found unchanged in the urine, as shown by detailed analyses. However, MDP was sequestered for a longer time at the site of injection when administered as a water-in-oil emulsion. Considering the relatively rapid elimination observed, it is suggested that the biological effects of MDP and related compounds, when administered in an aqueous medium, may be due to their activity at minute concentrations and/or an immediate action at the cellular level.