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Incubation of rat liver macrophages in vitro with free or liposome-encapsulated muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine; MDP) resulted in a rapid but transient release of tumor necrosis factor-alpha (TNF-alpha), followed by a slow, steady release of nitric oxide (NO) and prostaglandin (PG) E. The secretion pattern induced in situ was determined by isolating the liver macrophages at 2, 12, 24, 48, and 72 h after injection of liposomal MDP and measuring the amounts of products secreted in a 24-h period following isolation. TNF-alpha secretion was detected only in macrophages isolated as early as 2 h after injection of liposomal MDP and not at later time points. Considerable heterogeneity was observed among macrophages of different size: For example, the large-sized cells were far more potent in TNF secretion than the smaller cells. Nitric oxide secretion, on the other hand, was maintained over a full 24-h period following MDP administration and was virtually independent of macrophage size. With regard to PGE release, similar to TNF-alpha secretion, considerable differences in secretory activity between cells of different size were observed. Also in this case the large cells were several times more active than the small cells. In contrast to TNF, however, PGE secretion could be detected up to 24 h after injection of liposomal MDP. These findings support the notion that the development and maintenance of the activated state of liver macrophages, induced by immunomodulators such as liposomal MDP, are under the control of a complex network of regulatory functions and that multiple secretory products play a role in the observed macrophage-mediated cytotoxicity.

In this study, we investigated different functional characteristics of the rat liver macrophage population after a single i.v. injection of liposome-encapsulated muramyl dipeptide (MDP) or its lipophilic derivative muramyl tripeptide-phosphatidylethanolamine (MTP-PE). The in situ induced tumoricidal activity of the liver macrophage population was determined in vitro against C26 colon adenocarcinoma cells. For investigating which cells are responsible for the observed cytotoxic effects, subfractions of the liver macrophage population, differing in cell size, were isolated at different intervals after injection of the liposomal muramyl peptides. From these subfractions, the number of cells, degree of cytotoxicity, and the response to an additional activation with free MDP in vitro were determined. Maximal induction of tumoricidal activity of the liver macrophage population was reached between 12 and 24 hours after injection of liposomal MDP, while no significant differences between the subfractions were observed. Heterogeneity of tumor cytolytic capacity was observed in subfractions of macrophages isolated at 2 and 48 hours after injection. At these time points, highest cytolytic activity was observed for the small to intermediate-size macrophages. No significant cytotoxicity was detectable in any subfraction 72 hours after injection of liposomal MDP. An identical pattern of macrophage tumoricidal activity was observed after injection of liposomal MTP-PE, although slightly lower cytotoxicity levels were found. When isolated during the first 12 hours after injection of liposomal MDP, the macrophage population was unable to respond to a subsequent in vitro exposure to MDP, with respect to tumor cytotoxicity. Twenty-four and 48 hours after injection, the smallest cells could be slightly reactivated, whereas the larger cells still remained unresponsive.(ABSTRACT TRUNCATED AT 250 WORDS)

The therapeutic effect of a combination of liposomal muramyl dipeptide (MDP) and 5-fluorouracil (5FU) was studied in a murine tumor model of hepatic metastases of the tumor cell line C26, a colon adenocarcinoma. Liposomal MDP (250 micrograms/kg body wt) and a low, nontoxic, dose of 5FU (10 mg/kg body wt) were mixed and administered during 6 consecutive days once daily, three times i.v. and three times i.p. Treatment was initiated 4 days after intrasplenic tumor cell injection. The combination of liposomal MDP and 5FU significantly reduced the number of liver metastases and the total tumor load in the liver. Liver weights of tumor-bearing mice treated with the combination were significantly lower than the liverweights of control mice (p < 0.005) and of mice treated with 5FU alone (p < 0.02) or liposomal MDP alone (p < 0.05). Liposomal MDP and 5FU, when given as single treatment modalities, had no significant effect on the number of metastases and liver weight. These results show that liposomal MDP can enhance the therapeutic effect of 5FU for the treatment of liver metastases.

The liver macrophage population was fractionated according to cell size into three subpopulations by means of elutriation centrifugation. The total liver macrophage population and the three subpopulations were cultured and exposed to the immunomodulators muramyl dipeptide (MDP), in a free or liposome-encapsulated form, and/or lipopolysaccharide (LPS). The tumor cytotoxic activity thus induced in the populations, the preservation of this activity and the response to a second stimulus were studied. The in vitro induced cytolytic activity was determined by a radioactivity release assay, using C26 colon adenocarcinoma cells, labeled with [methyl-3H]thymidine, as target cells. MDP or LPS readily activated the total macrophage population in maintenance culture to a tumor cytotoxic state during the first 2 days after isolation. Four days after isolation, the activation induced with both MDP and LPS was strongly reduced. The small to intermediate-size macrophages could be activated to tumor cytotoxic activity with MDP for up to 3 days and with LPS for up to 4 days in culture. The large-size macrophages could only be activated up to day 2 in culture with MDP or LPS or both. The combination of MDP and LPS, however, induced all cell populations in a synergistic way to become cytolytic for up to 4 days in culture. With free MDP as an activator, the activated state decayed within 1 day to almost zero levels, but less rapidly in the small cells than in the large cells. With liposome-encapsulated MDP, the activated state was preserved considerably longer, except in the largest cells.(ABSTRACT TRUNCATED AT 250 WORDS)