Modifications of glycosylation patterns in macrophages upon activation.

Activated macrophages, in contrast to inflammatory and resident macrophages, are able to inhibit the growth of intracellular pathogens and tumor cells. In order to understand the adaptative changes which allow macrophages to express antitumor activity, we compared, among several parameters, the glycoproteins of cytotoxic and non-cytotoxic macrophages. After activation of mouse peritoneal macrophages by two stimuli applied in a sequence (trehalose dimycolate in vivo, lipopolysaccharide in vitro), we observed that: (1) surface sialic acid residues (labeled by tritiated borohydride after treatment of intact cells in culture by periodate) were reduced by 37%; (2) total sialic acid, as measured by an adaptation to HPLC of the thiobarbituric assay, was reduced by 30%. Variations in the intensity of the labeling after periodate/borohydride treatment were especially pronounced for a few high-molecular-weight glycoproteins. Analysis of glycopeptides indicated that the reduction of sialylation was accompanied by a slight increase in the relative importance of high mannose-type oligosaccharides (glycopeptides sensitive to endoglycosidase H or retained on concanavalin A-Sepharose) but did not affect the ratio of the various anionic species separated on QAE-Sephadex. A reduced sialylation of glycans after activation may facilitate interactions of macrophages with microbes and tumor cells.

Preparation and characterisation of liposomes containing mannosylated phospholipids capable of targetting drugs to macrophages.

We have prepared liposomes from mannosylated phosphatidylmyo-inositol, derived from mycobacteria, and cholesterol. The size of the particles so formed could be controlled by membrane filtration. The vesicles encapsulated a significant amount of aqueous phase (about 8 microliter per mg phospholipid). Markers of the liposomal membrane and aqueous phase rapidly associated with mouse peritoneal macrophages and, more slowly, with rat alveolar macrophages. The uptake was saturable at high liposome concentrations, although phagocytosis of latex particles of the same mean diameter was not saturable at these concentrations. An excess of unlabelled liposomes composed of phosphatidylcholine and phosphatidylserine, which were also taken up readily by macrophages, did not inhibit the uptake of mannosylated liposomes. The uptake of fluorescent mannosylated bovine serum albumin was inhibited by these liposomes, suggesting a specific interaction with the macrophage mannose-fucose receptor. We conclude that this type of liposome would be useful for the delivery of immunomodulators to reticuloendothelial cells.

Transmembrane potential variations accompanying the PMA-triggered O-2 and H2O2 release by mouse peritoneal macrophages.

Stimulation by PMA of Streptococci-elicited macrophages induced a transient membrane depolarization preceding the onset of detectable O-2 production. Mice-resident peritoneal macrophages were unresponsive to PMA for both activities. The PMA-triggered membrane depolarization seemed to be independent from O-2 production because inhibition of membrane depolarization by EGTA had no effect on rates of O-2 or H2O2 release and rate of antimycin A insensitive O2 uptake by Streptococci-elicited macrophages. The portion of O2 uptake recovered as O-2 was found to be 1/3. The rate of O-2 release was twice the rate of H2O2 production (1.1 nmol H2O2.min-1 X 10(6) macrophages-1).

A novel muramyl peptide derivative stimulates tumoricidal activity of macrophages and antibody production by B cells.

A novel analog of MDP, the 3'-iodo-4'-azido-L-phenylalanine methyl ester derivative of N-acetyl-L-alanyl-D-isoglutamine, has been prepared. This compound is capable of activating macrophages to the tumoricidal state and increasing the specific immune response of B cells. It thus appears to exhibit similar biological activities to MDP. Moreover, this compound is of potential interest for receptor photolabelling studies.

[Stoichiometry of calcium transport by sarcoplasmic reticulum: utilization of p-nitrophenylphosphate as an energy donor]. / Stoechiométrie du transport du calcium par le reticulum sarcoplasmique: utilisation du p-nitrophénylphosphate comme donneur d'énergie.

We have studied, under different conditions and through the hydrolysis of p-nitrophenylphosphate, the stoechiometry of the active transport of calcium by sarcoplasmic reticulum. According to a minimal kinetic model, it is possible to explain the variation of the coupling factor n(t) during the calcium accumulation in absence of precipitating agent. Under the described experimental conditions, at the beginning of the experiment, the coupling factor's value is slightly above 2.

Regulation of plasminogen activator secretion in mouse peritoneal macrophages. I. - Role of serum studied by a new spectrophotometric assay for plasminogen activators.

A chromogenic tripeptide - H-D-Val-Leu-Lys-p-nitroanilide-substrate of plasmin, can be used to follow plasminogen activation by an activator such as urokinase or the activator secreted by mouse peritoneal macrophages (thioglycolate-elicited). The acceleration of p-nitroaniline production is proportional to the initial rate of plasmin formation from plasminogen. Thus, at a given plasminogen concentration, this acceleration is proportional to the activator concentration. The acceleration can be evaluated from the spectrophotometer trace recording at 405 nm the appearance of p-nitroaniline, either by means of a computer program or by a plot of delta A405 vs.t2. The sensitivity of this assay allows detection of 0.003 CTA units of urokinase. Thioglycollate-elicited mouse peritoneal macrophages secrete plasminogen activator into the extracellular medium during in vitro cultivation only after a contact with serum.

Binding of nucleotides ATP and ADP to sarcoplasmic reticulm: study by rate of dialysis.

Binding of the nucleotides ATP and ADP by preparations of sarcoplasmic reticulum was investigated by the method of flow dialysis. For ATP, experimental data could not be analyzed directly in terms of binding since a significant though small amount of hydrolysis could be observed even in presence of EDTA. ADP binding could be analyzed and gave a dissociation constant of 10-20 muM at neutral pH, and a stoichiometry of 0.35 - 0.45 per mole ATPase. The possible significance of this stoichiometry is discussed. Similar experiments were performed after ethoxyformylation of sarcoplasmic reticulum which inhibits the Ca2+ dependnet ATPase activity. The results confirmed the inhibition of ATP hydrolysis and pointed to a considerably reduced affinity for nucleotides. The method based on the measurement of dialysis rates is convenient, and accurate enough to detect the effects of chemical modification on sarcoplasmic reticulum membranes.

Sequence dependency of the internalization and distribution of phosphorothioate oligonucleotides in vascular smooth muscle cells.

Antisense studies imply the utilization of oligonucleotides (ODN) for sequence-specific down-regulation of genes. This usually consists in assessing antisense sequences versus control sequences (mismatched, inverted, scrambled, randomized or any sequence unrelated to the relevant target). Even though the investigated biological effect (knockdown of an unwanted protein) is observed only with the antisense sequence and weakly, if at all, with any of the control sequences, this is a necessary but not a sufficient condition to demonstrate an antisense effect. Indeed, biochemical parameters such as stability, uptake and subcellular compartmentalization of ODN in a given cellular system are most often sequence-dependent processes. In this work, a series of phosphorothioate ODN of different lengths and sequences were evaluated as to their binding, internalization and subcellular distribution properties in vascular smooth muscle cells. In addition to membrane binding and nuclear accumulation, the partition of ODN in the cytosol of cells was measured by a method based upon controlled permeabilization of the plasma membrane, permitting the recovery of the cytosolic content with minimal damage to the membranes of the endocytic vesicles and lysosomes. We found that the tested ODN showed striking differences in their uptake and distribution in smooth muscle cells. Our results gave rise to the problem of validating the observed biological effects when different sequences of ODN were compared. Cellular studies such as the one presented in this work could help in choosing the proper control sequences among ODN exhibiting similar cell interactions as compared to the antisense sequences. Moreover, this method could be useful for the selection of antisense sequences that can be efficiently internalized and preferentially distributed in the appropriate compartments in cells for in vitro antisense studies.

Use of a simple fractionation method to evaluate binding, internalization and intracellular distribution of oligonucleotides in vascular smooth muscle cells.

Antisense oligonucleotides (ODN) are potent molecules that could be used to inhibit the synthesis of a protein specifically if delivered to the appropriate compartments (cytoplasm and nucleus) of the cell under study. We present here a simple method providing access to the fractions of internalized ODN available in the cytosolic and nuclear compartments. Cells are incubated with appropriately labeled ODN, either naked or vectorized. They are then washed and treated with pronase to remove species bound to the surface of the cell. Digitonin is added at a low concentration to induce leakage of the cytosol, which is collected. Endosomes and lysosomes are then lysed with Triton X100, and their contents, recovered by centrifugation. The crude nuclei comprising the pellet are purified by ultracentrifugation through a 2M sucrose cushion. Lactate dehydrogenase, fluorescent transferrin and cathepsin B are used as cytosolic, endosomal and lysosomal markers respectively. For vascular smooth muscle cells, the use of digitonin under optimal conditions (0.008% w/v, 4 degrees C for 5 min) resulted in more than 88% plasma membrane permeabilization, with less than 12% of endosomes and 5% of lysosomes lysed. We mainly studied a 3'-tritiated 20-mer ODN sequence complementary to the AUG region of the mRNA for the insulin-like growth factor 1 receptor, with either a phosphodiester (PO-ODN) or a phosphorothioate (PS-ODN) backbone. Cellular processing was evaluated with and without 25 kDa polyethylenimine (PEI) as a carrier. After 2.5 h of incubation at 37 degrees C, 100 times as much naked PS-ODN as naked PO-ODN was bound to the cell surface and internalized. Complexation with PEI dramatically increased both binding, by a factor of 10 and internalization by a factor of 80 of PO-ODN and, to a lesser extent, of PS-ODN. The intracellular distributions of naked PO-ODN and PS-ODN were similar. The radioactivity accumulated in nuclei accounted for about 15-20% of an intracellular radioactivity. A large proportion (about 60%) of intracellular radioactivity remained associated with the endocytic compartment. Complexation with PEI completely changed intracellular distributions: the nuclear fraction increased to 70% for PS-ODN. The fractionation method proposed, facilitating study of the subcellular distribution of the ODN, could also be used under appropriate circumstances, to study variations in cytosolic ODN content.

Involvement of nitric oxide synthase in antiproliferative activity of macrophages: induction of the enzyme requires two different kinds of signal acting synergistically.

Activated rodent macrophages inhibit micro-organism and tumour cell growth through a high output of nitric oxide; generated by an isoform of nitric oxide synthase which is induced, for example, in murine macrophages, by concomitant stimulation with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). We show here that LPS could be replaced as a co-stimulant by the mycobacterial derivative muramyl dipeptide (MDP) in macrophages, and by interleukin-1 (IL-1) in EMT-6 adenocarcinoma cells. Moreover, our results indicate that nitric oxide synthase RNA synthesis required either simultaneous or sequential exposure to IFN-gamma and MDP/IL-1; whereas exposure to MDP/IL-1 followed by exposure to IFN-gamma was ineffective. Thus, two kinds of signal could be distinguished: IFN-gamma on the one hand, acting first in an irreversible way, and LPS, MDP, IL-1 on the other hand, which seemed to be permanently required for continuous transcription of the nitric oxide synthase gene.

Kinetic modelling of the nitric oxide gradient generated in vitro by adherent cells expressing inducible nitric oxide synthase.

Inducible nitric oxide (NO) synthase produces a long-lasting NO flux which can exert cytotoxic effects on target cells. A prerequisite for the understanding of the molecular basis of NO action is quantitative data on the availability of this small neutral radical molecule at both the spatial and temporal levels. The limits of NO availability depend on the respective rates of NO production, diffusion and autoxidation by molecular oxygen. Kinetic modeling of these processes has been performed for a widely used experimental system consisting of a monolayer of adherent cells cultured in vitro for hours in unstirred culture medium. It appears that: (i) the maximal NO concentration in the culture is in the immediate vicinity of the monolayer, where target cells will sediment; (ii) the steady-state NO concentration in this area is lower than 4 to 5 microM; and (iii) measurements of nitrite/nitrate or citrulline accumulation in the bulk cell medium culture during a given time period significantly underestimate (by a factor of up to 3 to 4) the true rate of NO synthesis at the level of the producer cell. This rate can be, nevertheless, easily estimated from the rate of production of the stable NO synthase products.

Arachidonate metabolism triggered in primed macrophages by signals inducing antitumor activity.

The acquisition of antitumoral functions by mouse peritoneal macrophages is controlled by the addition of activating agents (lipopolysaccharide (LPS), muramyldipeptide (MDP) or A23187), on appropriately primed macrophages. The release of eicosanoids during this activation step was examined by radio-HPLC. We demonstrated that the induction of antitumor activity in primed macrophages by LPS or MDP was associated with the release of 20:4 derivatives; arachidonic acid was metabolized predominantly via the cyclooxygenase pathway to PGE2 and thromboxane. The production of PGE2, quantified by an enzyme immunoassay, was sustained and important (up to 20 ng/ml/h/10(6) macrophages). However, PGE2 and thromboxane did not seem essential to the activation process: induction of antitumor activity took place and was even enhanced in the presence of indomethacin, whereas it was decreased by exogenous PGE2. During culture in vitro, primed macrophages released spontaneously significant amounts of 20:4 metabolites and became unresponsive to activation stimuli. Again indomethacin had a positive effect: it protected primed macrophages against this loss of activability. Cyclooxygenase metabolites released in response to activating stimuli or spontaneously seem to trigger deactivation pathways.

High-performance liquid chromatographic analysis of the unusual pathway of oxidation of L-arginine to citrulline and nitric oxide in mammalian cells.

A very unusual pathway of the oxidation of L-arginine to citrulline and nitric oxide has been discovered recently in cytotoxic macrophages. In an attempt to detect molecules generated through this metabolic pathway, a fast radio high-performance liquid chromatographic method was developed to analyse the whole set of radiolabelled L-arginine-derived metabolites produced by mammalian cells after appropriate induction. A new intermediate which might be NG-hydroxy-L-arginine was found.

N omega-hydroxy-L-arginine, a reactional intermediate in nitric oxide biosynthesis, induces cytostasis in human and murine tumor cells.

Conversion of L-arginine to L-citrulline and nitric oxide (NO) by NO synthase induced in the murine EMT-6 cells resulted in the release of a large amount of the stable reactional intermediate N omega-hydroxy-L-arginine into the extracellular medium. We have prepared [3H]N omega-hydroxy-L-arginine biosynthetically, and shown that, after its uptake, this molecule can induce cytostasis in NO synthase-deficient P-815 and U-937 tumor cells. This long-lived intermediate could behave as a supplier of NO or other toxic molecules in cell-cell interactions.

[A simple method for the study of cytosolic content of oligonucleotides in cells]. / Une méthode simple pour étudier le contenu cytosolique en oligonucléotides dans les cellules.

Antisense oligonucleotides are currently used for the specific control of the expression of a selected gene. Their putative targets are located in the cytoplasm (messenger RNA) or the nucleus (pre-messenger RNA or DNA). This approach is conditioned by the presence of the antisense molecule inside the cell at sufficient concentrations and in the appropriate compartments. We propose in this paper a simple method for the study of the cytosolic content of internalized oligonucleotides. This method is based on the selective permeabilization of the plasmic membrane by the detergent digitonin. By complexing to membrane cholesterol, the detergent creates pores through which soluble and diffusible species can escape outside the cells. The selectivity of membrane permeabilization was controlled by using compartment markers: lactate dehydrogenase (LDH) for cytosol, dextrane-rhodamine (DEX) and hexosaminidase (HAM) for endocytic vesicles and lysosomes, respectively. Optimal digitonin concentrations and incubation times have been defined to reach the following pattern of membrane permeabilization: LDH > 80%; DEX and HAM < 15%. The method was applied to monitor the quantity of extractible oligonucleotides from cells after endocytosis. The results showed that phosphodiester and phosphorothioate oligomers are readily available in the cytosol (60-50% of the internalized species), whereas those bearing a hydrophobic moiety (fluorescein, cholesterol) are less diffusible probably owing to membrane binding. Internalization and cytosol partition were found to depend on the chemical nature of the oligonucleotide, and also on the sequence and the cell type. This method could be useful for the selection of antisense molecules that exhibit the best internalization and distribution in cells, and for a more appropriate choice of control sequences in antisense studies.

Enhancement of in vitro and in vivo antitumor activity by cord factor (6-6'-dimycolate of trehalose) administered suspended in saline.

The antitumor activity of trehalose-6,6'-dimycolate (TDM) dispersed in saline was studied in vitro and in vivo. In vitro, macrophages from TDM-treated mice entirely abolish, even at a 1.25:1 effector/target cell ratio, the (3H)TdR incorporation of P815 mastocytoma cells to the culture in which they were added. In vivo, the number of P815 cells harvested 10 days after their injection into TDM-treated mice was reduced by a factor higher than 50. The slopes of the radiolabel elimination of TDM-treated mice injected with 10(7) 125I-UdR-labelled L1210 leukemic cells were greater than those of untreated controls. The interest of such a compound which seems to be one of the active structures responsible for the antitumor activity of BCG is its efficiency when administered dispersed in saline.

Stimulation of thymocyte mitogenic protein secretion and of cytostatic activity of mouse peritoneal macrophages by trehalose dimycolate and muramyldipeptide.

Immunomodulators of bacterial origin, such as muramyl-dipeptide (MDP) and trehalose dimycolate, are able to stimulate some important biological functions of macrophages, such as their capacity to secrete a monokine, the thymocyte mitogenic protein (TMP), and to limit the growth of mastocytoma cells in vitro. Adherent cells from the peritoneal cavity of untreated mice do not secrete significant amounts of TMP and are not cytostatic. In contrast, adherent peritoneal cells from mice injected with trehalose dimycolate (emulsified in water) secrete TMP and are strongly cytostatic for P815 cells. Trehalose dimycolate is also active when added in vitro: (a) it enhances the cytostatic action of thioglycollate-elicited macrophages; (b) alone or in sequence with MDP, it induces an appreciable cytostatic activity in resident macrophages; (c) it limits the decline of the strong cytostatic action of trehalose-dimycolate-elicited macrophages occurring during in vitro cultivation. MDP also stimulates the cytostatic activity of the macrophages in vitro. The most interesting effect of MDP is its capacity to induce a strong secretion of TMP after a short contact (1 h) with elicited macrophages.

Nitric oxide biosynthesis, nitric oxide synthase inhibitors and arginase competition for L-arginine utilization.

Nitric oxide (NO) is a recently discovered mediator produced by mammalian cells. It plays a key role in neurotransmission, control of blood pressure, and cellular defense mechanisms. Nitric oxide synthases (NOSs) catalyze the oxidation of L-arginine to NO and L-citrulline. NOSs are unique enzymes in that they possess on the same polypeptidic chain a reductase domain and an oxygenase domain closely related to cytochrome P450s. NO and superoxide formation as well as NOS stability are finely regulated by Ca2+/calmodulin interactions, by the cofactor tetrahydrobiopterin, and by substrate availability. Strong interactions between the L-arginine-metabolizing enzymes are clearly demonstrated by competition between NOSs and arginases for L-arginine utilization, and by potent inhibition of arginase activity by N(omega)-hydroxy-L-arginine, an intermediate in the L-arginine to NO pathway.