Effects of negatively charged lipids on phagocytosis of liposomes opsonized by complement

Ingestion of liposomes opsonized by specific antibody plus complement was investigated in vitro. Although the antibodies alone (IgM) did not have an opsonizing effect, in the presence of such antibodies uptake and ingestion of liposomes by mouse peritoneal macrophages was enhanced 5- to 10-fold by addition of complement. Phagocytosis of complement-opsonized liposomes was strongly dependent on the charge of the liposomal lipids. The presence of a negatively charged (i.e., acidic) lipid profoundly suppressed the uptake of the liposomes. Each of three acidic liposomal lipids, phosphatidylserine, phosphatidylinositol and dicetyl phosphate, suppressed liposome uptake. We conclude that opsonization of liposomes with complement greatly stimulates ingestion of liposomes by murine macrophages. However, most of the opsonic enhancement conferred by complement can be prevented by the presence of negatively charged membrane lipids.     

Immune opsonin-independent phagocytosis by pulmonary macrophages

The uptake of albumin-coated latex particles by hamster pulmonary macrophages (PM) in vitro was investigated by using a new technique that combined flow cytometry and fluorescence microscopy to differentiate and quantitate bound vs ingested particles. In the absence of serum, PM avidly bound and ingested particles, whereas phagocytosis by hamster polymorphonuclear leukocytes (PMN) was less marked. In the presence of serum, phagocytosis by PM was slightly depressed, whereas phagocytosis by PMN was stimulated more than 10-fold. The binding of particles to PM in the absence of serum was pH, temperature, and trypsin sensitive and was dependent on the presence of extracellular Ca++ but not Mg++. The ingestion of particles by this immune opsonin-independent pathway was also temperature sensitive but was not affected by either pH or extracellular Ca++. Particle ingestion, but not binding, was inhibited by cytochalasin D and the divalent cation ionophore A23187.     

Pharmacokinetics of stealth versus conventional liposomes: effect of dose

Liposomes which substantially avoid uptake into the mononuclear phagocyte system (MPS), termed Stealth liposomes, have recently been formulated (Allen, T.M. and Chonn, A., (1987) FEBS Lett. 223, 42-46). The pharmacokinetics of stealth liposomes as a function of liposome dose and a comparison to conventional liposome pharmacokinetics, was the subject of the present study. We have examined the tissue distribution of two different formulations of stealth liposomes, i.e., sphingomyelin:egg phosphatidylcholine:cholesterol:monosialoganglioside GM1 (SM:PC:CHOL:GM1) 1:1:1:0.2 and SM:PC:CHOL:polyethylene glycol distearoylphosphatidylethanolamine (PEG(1990)-DSPE) 1:1:1:0.2, and compared them with the tissue distributions seen for a liposomal formulation which is avidly removed from circulation by the cells of the MP system (PC:CHOL, 2:1). Tissue distribution in mice was examined over a 100-fold concentration range (0.1 to 10 mumol phospholipid/mouse) and at several time points over a 48 h time period. Liposome size ranged from 92-123 nm in diameter for all compositions. Clearance from blood of PC:CHOL liposomes following intravenous administration showed a marked dose dependence (i.e., saturation-type or Michaelis-Menten kinetics), with MPS uptake decreasing and % of injected dose in blood increasing as dose increased, over the entire dosage range. Injection of stealth liposomes, on the other hand, resulted in % of injected doses of liposomes in MPS, blood and carcass which were dose-independent and log-linear (first order kinetics) over the entire dosage range. The doses of stealth liposomes containing PEG(1900)-DSPE required for MPS saturation was higher than 10 mumol phospholipid/mouse or 400 mumol/kg. The dosage-independence of the pharmacokinetics of stealth liposomes and their lack of MPS saturation within the therapeutic dose range are two more assets, in addition to the prolonged circulation half-lives, leading towards their eventual use as drug delivery systems in the clinic.     

Quantitative studies of phagocytosis by alveolar macrophages

The initial rate of phagocytosis by rabbit alveolar macrophages of paraffin oil emulsions stabilized with albumin was markedly increased by prior incubation of the emulsion with serum. The active component(s) of serum was non-dialyzable and heat-labile and was absent from zymosan-treated serum. Magnesium and calcium were both required for the maximal rate of phagocytosis. At 4 °C or in the presence of 1 mM N-ethylmaleimide, the rate of phagocytosis was less than 2% of the control (37° C) rate. The initial rates of phagocytosis of this emulsion by alveolar macrophages from rabbits injected with Freund's adjuvant were not demonstrably different from those observed with macrophages from normal rabbits. Per of mg of cell protein, polymorphonuclear leukocytes ingested serum-treated emulsion more rapidly than did macrophages, but per cell the rates were not different.     

High-activity radio-iodine labeling of conventional and stealth liposomes

A new method to label preformed liposomes with high activities of radiohalogenated compounds has been developed. It uses activated esters of simple synthetic molecules that may be readily halogenated, such as Bolton-Hunter reagent (BH), and arginine-containing liposomes. BH, in the form of an activated ester, crosses the liposome membrane to react with arginine inside the liposomes, as demonstrated by thin-layer chromatography and by the fact that saline-containing liposomes, or hydrolyzed BH or the water soluble sulfo-BH afforded only marginal encapsulation yields. Under optimized conditions, between 37 and 55 degrees C, 62 +/- 4% (mean +/- SD) of radiolabeled BH were consistently encapsulated in the liposomes within 30 min. In molar amounts, this corresponds to a mean of 56 nmol of BH per micromol of lipids. Based on achievable specific activity, up to 2.8 GBq of iodine-131 could be entrapped per micromol of lipids. Leakage of radioactivity was very low, with less than 5% of the encapsulated activity released within 6 days at 4 degrees C in phosphate-buffered saline and less than 50% within 24 h in human serum at 37 degrees C. The labeling stability, and the fact that both conventional and PEGylated liposomes can be readily labeled with high doses of radioactivity, will make this technique useful for in vivo targeting applications, such as tumor detection (using iodine-123 or iodine-124) or therapy (with iodine-131 or astatine-211).     

Phagocytosis of liposomes by human alveolar macrophages

The ability of cells recovered from lung lavages to phagocytose liposomes has been investigated.Inulin (¹⁴C-labelled), entrapped in multilamellar immunoglobulin-G coated liposomes with ³H-labelled cholesterol as the lipid phase marker, was fed to the recovered cells. Fifteen patients with diffuse interstitial pulmonary disease (DIPD), prior to steroid treatment, and eight normal controls were lavaged for the study. Uptake was found for both groups and it was concluded that the liposomes enter the cells predominantly via endocytosis. Follow-up lavage, three months after the initial lavage, was repeated on three patients receiving 60 mg prednisone per day. A decrease in the uptake of liposomes was observed after steroid treatment.     

Internalization of paramagnetic phosphatidylserine-containing liposomes by macrophages

ABSTRACT: BACKGROUND: Inflammation plays an important role in many pathologies, including cardiovascular diseases, neurological conditions and oncology, and is considered an important predictor for disease progression and outcome. In vivo imaging of inflammatory cells will improve diagnosis and provide a read-out for therapy efficacy. Paramagnetic phosphatidylserine (PS)-containing liposomes were developed for magnetic resonance imaging (MRI) and confocal microscopy imaging of macrophages. These nanoparticles also provide a platform to combine imaging with targeted drug delivery. RESULTS: Incorporation of PS into liposomes did not affect liposomal size and morphology up to 12 mol% of PS. Liposomes containing 6 mol% of PS showed the highest uptake by murine macrophages, while only minor uptake was observed in endothelial cells. Uptake of liposomes containing 6 mol% of PS was dependent on the presence of Ca2+ and Mg2+. Furthermore, these 6 mol% PS-containing liposomes were mainly internalized into macrophages, whereas liposomes without PS only bound to the macrophage cell membrane. CONCLUSIONS: Paramagnetic liposomes containing 6 mol% of PS for MR imaging of macrophages have been developed. In vitro these liposomes showed specific internalization by macrophages. Therefore, these liposomes might be suitable for in vivo visualization of macrophage content and for (visualization of) targeted drug delivery to inflammatory cells.     

Quantifying complement-mediated phagocytosis by human monocyte-derived macrophages

The present study demonstrates that SRBC can be opsonized with untreated human serum such that lysis by active complement components is minimal but sufficient opsonization occurs to permit high rates of complement-mediated phagocytosis. Phagocytosis of SRBC opsonized with 2% whole human serum by human monocyte-derived macrophages was quantified in a colourimetric assay. Ingestion of SRBC was shown to occur solely via complement receptors because no phagocytosis was observed when SRBC were coated with heat- inactivated human serum, phagocytosis was augmented by the phorbol ester, PMA, and phagocytosis was inhibited by a protein kinase C (PKC)-specific inhibitor RO 31-8220. This method was used to demonstrate directly that HIV-1 infection of human monocyte-derived macrophages inhibits complement-mediated phagocytosis and will provide a useful tool for pharmacological investigations on complement-mediated phagocytosis by adherent macrophages.     

Pulmonary intravascular macrophages and hemodynamic effects of liposomes in sheep

We studied the effects of liposomes on the pulmonary circulation of sheep and found a close correlation between liposome retention in the lung and the intravascular macrophages. A test dose of liposomes (5.5 mumol of total lipids) injected intravenously transiently increased pulmonary arterial pressure from 24 +/- 2 to 55 +/- 16 (SD) cmH2O. The pulmonary arterial pressure responses were dose dependent and reproducible. The rise in pulmonary arterial pressure was blocked completely by indomethacin and 75% by a thromboxane synthase inhibitor. Systemic arterial thromboxane B2 concentration increased from a base-line level of less than 50 pg/ml to 250 +/- 130 pg/ml at the peak of the pressor response. Larger doses of liposomes (220 mumol of total lipids) infused intravenously over 1 h increased pulmonary arterial pressure maximally within the first 15 min. Lymph flow increased and lymph protein concentration decreased, suggesting venoconstriction. Over half (62.4 +/- 15.7%) of 111In-labeled liposomes remained in the lung after 2 h. Fluorescence and transmission electron microscopy showed that greater than 90% of the liposomes were associated with mononuclear cells in the lumen of the alveolar wall microvessels. We conclude that liposomes affect pulmonary arterial pressure transiently by a mechanism involving the arachidonate cascade, principally thromboxane. Our observations suggest that a population of pulmonary intravascular macrophages is likely to be the source of the thromboxane and the pulmonary hemodynamic and lymph dynamic changes that occur in a dose-dependent fashion, although interactions between liposomes, leukocytes, or endothelial cells, in addition to the macrophages, have not been completely ruled out. We believe this is the first demonstration that pulmonary intravascular macrophages may be the source of the arachidonate metabolites rather than endothelial cells, neutrophils, or perivascular interstitial cells.     

Phosphatidylserine-mediated phagocytosis of anticancer drug-treated cells by macrophages

Apoptotic cells are rapidly phagocytosed and eliminated from the organism. Although cancer cells apoptose when treated with anticancer drugs, how those cells are recognized by phagocytic cells has remained unclear. The human leukemia cell line Jurkat was cultured with doxorubicin or bufalin and induced to undergo apoptosis accompanied by phosphatidylserine externalization. When apoptotic Jurkat cells were mixed with mouse peritoneal macrophages, efficient phagocytosis was observed. Apoptosis and phagocytosis of Jurkat cells were both inhibited by Z-VAD-FMK, and phagocytosis was significantly reduced in the presence of phosphatidylserine-containing liposomes. These results suggest that anticancer drugs induce apoptosis-dependent and phosphatidylserine-mediated phagocytosis in cancer cells.     

Enhancing and suppressive effects of macrophages on T-lymphocyte stimulation in vitro.

The immunoregulatory effect of peritoneal and splenic macrophages on Con A-stimulated mouse splenic T lymphocytes was investigated in vitro using [¹²⁵I]UdR incorporation as a measure of lymphocyte proliferation. [¹²⁵I]UdR incorporation was enhanced by the addition of increasing numbers of splenic or low doses of peritoneal adherent cells to macrophagedepleted splenic lymphocytes. The addition of increasing numbers of peritoneal macrophages beyond 5–10%, however, proportionally suppressed T-cell proliferation. Activated splenic macrophages obtained from mice 6 days after infection with Listeria monocytogenes were suppressive, whereas macrophages obtained from immune donors 9–10 days after infection were not, so that a chronological association appeared to exist between macrophage activation and immunosuppression. The addition of 2-mercaptoethanol to the cell cultures increased [¹²⁵I]UdR incorporation without affecting the stimulatory and suppressive effects of splenic and peritoneal macrophages, respectively. Heat-killed and freeze-thawed macrophages lost their capacity to enhance or inhibit lymphocyte transformation. Macrophages treated with mitomycin C to inhibit DNA synthesis retained their regulatory functions. These studies suggest differential regulatory roles for spleen versus peritoneal macrophages on T-lymphocyte responses to Con A stimulation in vitro.     

Inhibitory effects of various synthetic substrates for aminopeptidases on phagocytosis of immune complexes by macrophages

The inhibitory effects of various 7-(aminoacyl)-4-methylcoumarinylamides (AA-MCA's), synthetic substrates for aminopeptidases, on phagocytosis of immune complexes by guinea pig peritoneal macrophages were investigated by measuring the intracellular uptake of sensitized 51Cr-sheep erythrocytes and 125I-alpha-amylase complexed with homologous IgG2 antibody. Among the AA-MCA's examined, MCA compounds of hydrophobic amino acids (Phe, Tyr, Leu, and Pro) were found to inhibit the intracellular uptake and digestion of immune complexes. Sucrose density gradient centrifugation of the homogenates of macrophages treated with the inhibitors for 1 h at 37 degrees C showed that they modulated the lysosomes, resulting in a decrease in buoyant density of the organella. These effects of the inhibitors on the buoyant density of the lysosomes as well as on the phagocytic activity of macrophages disappeared upon removal of the inhibitors from the cells by washing. Since none of 7-amino-4-methylcoumarin, L-phenylalanine, and bestatin methyl ester could significantly inhibit the phagocytosis of immune complexes by macrophages, the MCA compounds of hydrophobic amino acids appear to inhibit the phagocytosis as a consequence of their lysosomotropic nature.     

Induction of apoptosis in macrophages by cationic liposomes

The effects of liposomes on apoptosis in macrophages were evaluated from DNA content and DNA fragmentation. Cationic liposomes composed of different kinds of cationic lipids induced apoptosis in mouse splenic macrophages and the macrophage-like cell line, RAW264.7 cells. Generation of reactive oxygen radicals from macrophages treated with cationic liposomes was detected using flow cytometry, and further apoptosis was inhibited by the addition of oxidant scavenger, N-acetylcysteine. From these findings, the production of reactive oxygen species may be important in the regulation of apoptosis induced by cationic liposomes.     

Protein synthesis by attached pulmonary macrophages effect of phagocytosis

We studied the effect of phagocytosis of polystyrene latex beads on protein synthesis by pulmonary macrophages. To do this we determine the specific radioactivity of extracellular and intracellular free phenylalanine and of phenylalanine released from tRNA and used this information in calculating the rates of protein synthesis. Phagocytosis resulted in an increased rate of protein synthesis irrespective of which precursor specific radioactivity was used in the calculation. The rate of protein synthesis was increased per μg polyribosomal RNA; but there was no increase in the amount of polyribosomal RNA in phagocytizing macrophages. The increase in the rate of protein synthesis (1.4-fold) was almost identical to the increase (1.3-fold) in the rate of ribosome transit in phagocytizing compared to nonphagocytizing macrophages. The decreased ribosome transit time during phagocytosis occurred without a fall in the average molecular weight of macrophage proteins. We conclude that phagocytosis increases the rate of protein synthesis in attached pulmonary macrophages and that this increased rate of synthesis can be accounted for almost completely by an increased rate of polypeptide chain elongation and/or termination.