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In this study we determined the in vivo localization of recombinant proteins expressed by intraperitoneally (i.p.) injected recombinant Semliki Forest virus (SFV) particles. Subsequently, we investigated the influence of i.p. administered SFV particles encoding recombinant murine granulocytemacrophage colony-stimulating factor (rmGM-CSF) on intraperitoneal recruitment and activation of cells. Finally, the therapeutic effect of SFV-GM-CSF treatment on an i.p. growing ovarian tumor was determined. Intraperitoneal injections of recombinant SFV particles encoding the reporter protein luciferase resulted in a high level of luciferase activity in cells of the peritoneal lining and tumor cells in the peritoneal cavity. Low levels of luciferase activity were found in liver, spleen and lungs. Injection of SFV-GM-CSF particles resulted in a slight increase in the number of peritoneal macrophages and in a significant increase in the number of neutrophils. In contrast to multiple i.p. injections with commercially available recombinant GM-CSF, i.p. injected SFVGM-CSF particles activated the macrophages to tumor cytotoxicity. Although treatment of tumor-bearing mice with SFVGM-CSF particles did not result in prolonged survival, tumor growth was inhibited for 2 weeks. Our findings indicate that macrophage-activating cytokines expressed by the efficient and safe recombinant SFV system when administered i.p. may provide an immunotherapeutic treatment modality additional to current chemotherapeutic treatment of intraperitoneally growing cancers. [ABSTRACT FROM AUTHOR]

We studied the effect of recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) on the cytotoxic potential of murine peritoneal cells. Mice received rmGM-CSF intraperitoneally using different dosages and injection schemes. At different time points after the last injection, mice were sacrificed, peritoneal cells isolated and their tumour cytotoxicity was determined by a cytotoxicity assay using syngeneic [methyl-[SUP3]H]thymidine-labelled colon carcinoma cells. Also, the cytotoxic response to a subsequent in vitro stimulation with lipopolysaccharide was determined. Upon daily injection of 6000-54 000 U rmGM-CSF over a 6-day period, the number of peritoneal cells increased over ten fold with the highest rmGM-CSF dose. Increases in cell numbers was mainly due to increases in macrophage numbers. Upon injection of three doses of 3000 U rmGM-CSF per day for 3 consecutive days, the number of macrophages remained elevated for minimally 6 days. Although the peritoneal cells from rmGM-CSF-treated mice were not activated to a tumoricidal state, they could be activated to high levels of cytotoxicity with an additional in vitro stimulation of lipopolysaccharide. Resident cells isolated from control mice could be activated only to low levels of tumour cytotoxicity with lipopolysaccharide. Tumour cytotoxicity strongly correlated with nitric oxide secretion. When inhibiting nitric oxide synthase, tumour cell lysis decreased. Thus, the expanded peritoneal cell population induced by multiple injections of rmGM-CSF has a strong tumour cytotoxic potential and might provide a favourable condition for immunotherapeutic treatment of peritoneal neoplasms. [ABSTRACT FROM AUTHOR]

One of the functions of macrophages is to provide a defense mechanism against tumor cells. In the last decades the mechanism of tumor cell killing by macrophages have been studied extensively. The tumor cytotoxic function of macrophages requires stimulation either with bacterial cell wall products such as lipopolysaccharide (LPS) or muramyldipeptide (MDP) or with cytokines such as interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Activated macrophages secrete several substances that are directly involved in tumor cell killing i.e. tumor necrosis factor (TNF) and nitric oxide (NO). On the other hand, substances are secreted that are able to stimulate tumor cell growth, depending on the stage and the nature of the tumor. Several clinical trials have been performed aiming at the activation of macrophages or dendritic cells, a subpopulation of the macrophages. In this review we will summarize and discuss experimental studies and clinical trials based on the activation of macrophages.

Liposomes of 400 nm in diameter can cross the 100-nm fenestrations in the endothelium of the hepatic sinusoid, provided they contain phosphatidylserine (PS) but not phosphatidylglycerol (PG) [Daemen et al. (1997) Hepatology 26, 416]. We present evidence indicating that (i) the PS effect does not involve a pharmacological action of this lipid on the size of the fenestrations, (ii) fluid-type but not solid-type PS liposomes have access to the hepatocytes and (iii) the lack of uptake of PG liposomes by hepatocytes is not due to a lack of affinity of the hepatocytes for PG surfaces. We conclude that the mechanism responsible for the uptake of large PS-containing liposomes by hepatocytes in vivo involves a mechanical deformation of these liposomes during their passage across the endothelial fenestrations.

Human serum albumin (HSA) derivatized with cis-aconitic anhydride was covalently coupled to liposomes with a size of approximately 100 nm [polyaconitylated HSA (Aco-HSA) liposomes]. Within 30 min after injection into a rat, Aco-HSA liposomes were completely cleared from the blood and almost exclusively taken up by the liver, whereas in control liposomes 80% was still present in the blood at that time. Endothelial cells were shown to account for almost two-thirds of the hepatic uptake of the Aco-HSA liposomes, the remainder being recovered mainly in the liver macrophages (Kupffer cells). With fluorescently labeled liposomes it was shown that the Aco-HSA liposomes target a vast majority (>85%) of the cells in the endothelial cell population. Control liposomes were not taken up to a significant extent by the endothelial cells. Uptake of Aco-HSA liposomes by both endothelial and Kupffer cells was inhibited by preinjection with polyinosinic acid, indicating the involvement of scavenger receptors in the uptake process. The uptake of Aco-HSA liposomes by liver endothelial cells was dependent on liposome size; with increasing liposome diameter endothelial cell uptake decreased in favor of Kupffer cell uptake. We have demonstrated that massive in vivo targeting of liposomes to a defined cell population other than macrophages is possible. Aco-HSA liposomes thus may represent an attractive drug carrier system for treatment of various liver or liver endothelium-associated disorders.

We investigated the in vitro activation of rat liver macrophages to a tumoricidal state with free and liposome-encapsulated immunomodulators. The cytolytic activity of liver macrophages was determined by a radioactivity release assay using murine B16 melanoma cells, labeled with [methyl-3H]thymidine. Exposure of the liver macrophages to concentrations of 50 micrograms of free, nonencapsulated, muramyl dipeptide (MDP) per ml resulted in maximal levels of tumor cell lysis of approximately 20%. Encapsulation of the MDP within liposomes (multilamellar vesicles, 0.3 to 0.5 micron in diameter, consisting of egg phosphatidylcholine, cholesterol, and dicetylphosphate, 4:5:1) not only caused a 500-fold reduction in the amount of MDP required to obtain the same levels of cytolysis but also increased the maximally obtainable level of cytolysis more than 2-fold. A synergistic effect of lipopolysaccharide and free or encapsulated MDP on cytolytic activity was observed when the macrophages were exposed to a combination of the two agents simultaneously. Besides causing tumor cell lysis, activated macrophages were also able to suppress tumor cell proliferation by 80 to 90% as determined by a [methyl-3H]thymidine incorporation assay. With a fixed amount of MDP, encapsulated in different amounts of liposomal lipid, the extent of macrophage activation was found to increase with a larger amount of encapsulating lipid. This increase in macrophage activation may be the result of a sustained intracellular release of encapsulated MDP from the liposomes. Liposome structure and composition will thus be important parameters in the in vivo application of liposomes as carriers of immunoactive substances.

Large unilamellar liposomes (50 to 500 mumol of lipid per kg) were injected i.v. or i.p. into normal and lymphosarcoma-bearing mice. The percentage of the dose remaining in the blood and that accumulated in liver, spleen, and various other organs was measured 4 hr after injection. The results indicate that liposomes cause a dose-dependent saturation of the hepatic and splenic clearance capacities. One day after injection of 10(6) lymphosarcoma cells, the capacity of the tumor-bearing mice to eliminate liposomes from the blood (in a 4-hr period) was inhibited 30 to 50%. This could be ascribed to a decreased activity of the reticuloendothelial system caused by the tumor cells, as was indicated by the simultaneous inhibition of carbon clearance. Six days after injection of the lymphosarcoma cells, the elimination of liposomes from the blood in tumor-bearing mice was restored to the value in normal mice. The possible involvement of tumor cells in the uptake of liposomes by the liver was investigated morphologically after i.v. injection of peroxidase-containing liposomes into lymphosarcoma-bearing mice. Liposome-entrapped peroxidase activity was never observed in the tumor cells. The results presented here indicate that the lymphosarcoma cells do not directly participate in the hepatic accumulation of liposomes, although their mere presence may have significant indirect effects on the elimination of liposomes from the blood and on their tissue distribution.

Highly purified mitochondria from rat liver contain a phospholipase A that catalyzes removal of 2-fatty acids, with a pH optimum above pH 8.0. Lysosomal preparations appeared to have two phospholipases A associated with them, one with a pH optimum at about pH 4.0, the second between pH 6.0 and 7.0. Mitochondrial phospholipase A hydrolyzed exogenous phospholipid as fast as or faster than endogenous phospholipid. The difference in specific radioactivity of (14)C-ethanolamine-labeled endogenous mitochondrial phospholipid before and after incubation indicates that a fraction of mitochondrial phosphatidyl ethanolamine is hydrolyzed more rapidly than the mitochondrial phospholipids as a whole. Acyl bond hydrolysis of exogenous and endogenous phospholipid by mitochondria was stimulated by free fatty acid, Ca(++), or in certain cases, monoacyl phospholipids or by treatments that disrupt the mitochondrial membrane. Of various fatty acids tested, lauric, myristic, oleic, and linoleic were most effective. ADP and ATP inhibited mitochondrial phospholipase, probably because they compete for Ca(++). Mg(++) also behaved as a competitive inhibitor; the effect was overcome by relatively little Ca(++).