Differential inhibition of macrophage microbicidal activity by liposomes.

In vitro culture of murine resident peritoneal macrophages with lymphokine (LK)-rich leukocyte culture fluids induces enhanced microbicidal activity against amastigotes of the protozoan parasite Leishmania tropica. Macrophages infected with Leishmania and treated with LKs after infection acquire the capacity to kill the intracellular parasite within 72 h. When compared with control macrophage cultures treated with medium lacking LKs, 80 to 90% fewer macrophages treated with LKs contained amastigotes. In experiments designed to test liposome delivery of LKs to infected macrophages, addition of multilamellar liposomes composed of phosphatidylcholine and phosphatidylserine (molar ratio, 7:3) completely abrogated LK-induced microbicidal activity. Liposomes containing only phosphatidylcholine were not inhibitory. Inhibition of LK activity by the liposomes occurred regardless of whether the liposomes contained LKs. Liposomal inhibition of activated macrophage effector activity was limited to intracellular killing; LK-induced macrophage extracellular cytolysis (i.e., tumor cytotoxicity) was not affected by liposome treatment. These data indicate that elucidation of the effects of liposome composition on acquired host defense mechanisms may be useful for the design of drug delivery systems that allow expression or augmentation of immunologically induced mechanisms for the intracellular destruction of infectious agents.     

Relationship of ability of phospholipids to stimulate growth and bind to macrophages

Among the phospholipids normally present in mammalian cell membranes, the negatively charged phospholipids, phosphatidylserine (PS), phosphatidylglycerol, and cardiolipin, and the neutral phospholipid phosphatidylethanolamine--but not phosphatidylcholine (PC) or sphingomyelin--were found to induce growth of peripheral macrophages. By use of liposomes prepared from PS, which stimulated growth, and PC, which did not, the relation between growth-stimulating activity and binding of the phospholipids to macrophages was studied. The growth-stimulating activity of PS/PC liposomes decreased with increase in their relative content of PC. The amount of PS bound to macrophages also decreased with increase in the proportion of PC in PS/PC liposomes. These decreases in growth-stimulating and binding activities were both partly recovered by additional incorporation into the PS/PC liposomes of cholesterol, which is also a cell membrane component. Phospholipids that stimulated macrophage growth showed high binding ability to macrophages, whereas those that did not stimulate growth scarcely bound to macrophages. Thus the macrophage growth-stimulating activities of phospholipids correlated well with their ability to bind to macrophages. These liposome models should be useful in elucidating the early mechanism of induction of macrophage growth by lipid materials such as cell debris and lipoproteins.     

Macrophage fatty acid composition and phagocytosis: effect of unsaturation on cellular phagocytic activity.

In order to manipulate the physical properties of the macrophages membrane, methods were developed which potentiated the incorporation of exogenously supplied fatty acids into membrane lipids. Chromatograms of macrophages which were grown in the presence of a variety of fatty acids demonstrated that exogenously supplied unsaturated fatty acids (palmitoleic, oleic, elaidic, linoleic, linolenic and arachidonic acids) were readily incorporated into the cells and selectively altered the fatty acyl composition of macrophage phospholipids. Up to 38% of the total cellular phospholipids were found to be derived from the exogenously added fatty acid supplements. The incorporation of the different fatty acids into cellular phospholipids had striking effects on cellular phagocytic activity. These effects were found to correlate with the degree of unsaturation, and the cis- or trans-double bond configuration. Thus, macrophage phagocytic ingestion rates of 125I-labelled Shigella flexneri were found to alter by more than 2-fold after the cells were cultivated in the presence of cis unsaturated fatty acids.     

Antibodies against phosphatidylinositol and inositol monophosphate specifically inhibit tumour necrosis factor induction by malaria exoantigens.

The active component of the exoantigens of malarial parasites which stimulates macrophages to secrete tumour necrosis factor (TNF) has been shown to depend upon a phospholipid, the activity of which was blocked by phosphatidylinositol (PI) and inositol monophosphate (IMP) in competitive inhibition studies. Antisera made against the exoantigens of Plasmodium yoelii, which inhibited their induction of TNF, were found by an ELISA assay to contain antibody against several other phospholipids. However, the inhibitory antibody was removed specifically by adsorption with liposomes containing PI, but not other phospholipids. Furthermore, PI was the only phospholipid in non-liposomal form which induced the production of inhibitory antisera. Mice immunized with IMP, but not inositol, also produced inhibitory antisera. When incorporated into liposomes several other phospholipids did give rise to inhibitory antibodies but, in contrast to the antisera against parasite exoantigens, PI and IMP, the inhibitory activity was removed by adsorption with heterologous phospholipid liposomes, suggesting that it was directed against a common determinant, presumably the phosphate ester head group. Inhibitory antibodies in the antisera tested were predominantly IgM and titres were not increased after repeated injections. Antisera raised against PI, IMP or the cross-reacting phospholipid liposomes also inhibited TNF secretion by macrophages stimulated by exoantigens of the human parasites P. falciparum and P. vivax, but not by bacterial lipopolysaccharide. These findings confirm our conclusion that exoantigens from these different species contain phosphate bound to inositol in their TNF-inducing moiety.     

Phospholipid epitopes for mouse antibodies against bromelain-treated mouse erythrocytes.

The reactivity of mouse antibodies against bromelain-treated mouse erythrocytes (BrMRBC) with phospholipid epitopes was assessed by ELISA, using four clones of monoclonal anti-BrMRBC antibodies that had idiotypes distinct from one another. The four antibodies could bind to low-density lipoproteins (LDL) from human and chicken, but not to LDL from mouse and rat. As to liposomes of natural phospholipids, all the clones reacted with liposomes of phosphatidylcholine, and some of them could react with liposomes of sphingomyelin, phosphatidylglycerol, phosphatidylic acid or cardiolipin. For liposomes of synthetic phosphatidylcholine with different fatty acids, the length of carbon chains and the number of unsaturated carbon chains of the fatty acids markedly affected the binding of each monoclonal antibody to the liposomes. The addition of dicetyl phosphate or stearylamine to phosphatidylcholine liposomes changed the reactivity of the liposomes. These results support the view that mouse anti-BrMRBC antibodies can recognize appropriately spaced phosphorylcholine residues on the surface of phospholipid liposomes, LDL and cells. The four clones had similar capacities for binding to LDL as well as to BrMRBC, but they had obviously different capacities for binding to phospholipid liposomes; the epitopes on phospholipid liposomes used in the present study were not so perfect as to react well with every anti-BrMRBC antibody.     

Lymphokine activation of J774G8 cells and mouse peritoneal macrophages challenged with Toxoplasma gondii.

In vitro activation of macrophage cell line J774G8 and mouse peritoneal macrophages resulted in oxygen-dependent and oxygen-independent killing of intracellular Toxoplasma gondii. Activation was characterized by oxygen-dependent killing detectable by enhanced lysosome fusion and digestion of T. gondii. The toxoplasmacidal activity of activated J774G8 cells and peritoneal macrophages was prevented by adding the oxygen intermediate scavengers catalase or superoxide dismutase during culture. Activated J774G8 cells and peritoneal macrophages also inhibited replication of those Toxoplasma organisms which survived the initial microbicidal activity. The inhibition of Toxoplasma replication was not significantly affected by exogenous catalase or superoxide dismutase. Peritoneal macrophages from Toxoplasma-immune mice showed similar microbicidal and inhibitory responses, supporting the model that activation leads to destruction of intracellular parasites by two different mechanisms.     

Killing of Leishmania parasites in activated murine macrophages is based on an L-arginine-dependent process that produces nitrogen derivatives.

The experiments described in this report were aimed at determining whether L-arginine (L-arg)-derived nitrogen oxidation products (nitric oxide, nitrous acid, nitrites) are involved in the intracellular killing of Leishmania parasites by activated murine macrophages in vitro. Peritoneal or bone marrow-derived macrophages were infected with L. enriettii or L. major, then activated by exposure to recombinant murine interferon-gamma or to macrophage activating factor (MAF)-rich media in the presence of lipopolysaccharide. Activation of macrophages in regular (i.e., arginine-containing) culture medium led to complete destruction of the microorganisms within 24 h (L. enriettii) or 48 h (L. major), concomitant with accumulation of nitrites (NO2-) in the culture fluids. When macrophage activation was carried out in L-arg-free medium, however, neither parasite killing nor NO2- production was obtained. A similar inhibition of macrophage leishmanicidal activity and of NO2- release was observed using media treated with arginase (which converts L-arg to urea and ornithine), or supplemented with NG-monomethyl-L-arg or guanidine (which inhibit the conversion of L-arg to nitrogen oxidation products). In all these situations, an excellent correlation between the levels of NO2- production by macrophages and intracellular killing of Leishmania was observed, whereas no strict correlation was detectable between leishmanicidal activity and superoxide production. Intracellular parasite killing by activated macrophages could be prevented by addition of iron salts to the incubation fluids. Incubation of free parasites with NaNO2 at acid pH (which permits the production of nitrous acid) led to immobilisation, multiplication arrest, and morphological degeneration of the microorganisms. Similarly, exposure of infected cells to NaNO2 led to killing of the intracellular parasite without affecting macrophage viability. These experiments strongly suggest that the leishmanicidal effect of activated murine macrophages involves the agency of L-arg-derived nitrogen oxidation products.     

Induction of macrophage growth by negatively charged phospholipids

The effects on macrophage growth of seven phospholipids that are normally present in cell membranes were examined. Phosphatidylcholine and sphingomyelin which are the main phospholipid constituents of mammalian cell membranes, had no effect on growth of macrophages. Phosphatidylinositol also had little effect. On the other hand, the relatively minor components phosphatidylserine, phosphatidylglycerol, cardiolipin, and phosphatidic acid, which are negatively charged phospholipids, significantly enhanced macrophage growth. These findings suggest that some acidic phospholipids may increase survival or growth of tissue macrophages when they are ingested by the cells in materials containing phospholipids, such as effete autologous cells or bacteria.     

Unusual fatty acid substitution in lipids and lipopolysaccharides of Helicobacter pylori.

Cellular fatty acids, phospholipid fatty acids, and lipopolysaccharide fatty acids of four strains of Helicobacter pylori were analyzed by gas-liquid chromatography. The presence of myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, 19-carbon cyclopropane fatty acid, beta-hydroxypalmitic acid, and beta-hydroxystearic acid was confirmed. In phospholipids, myristic acid and 19-carbon cyclopropane fatty acid were the major fatty acids. Hydroxy fatty acids and unsaturated fatty acids were not detected or occurred only in small amounts. The major fatty acids of lipopolysaccharides were stearic acid, beta-hydroxypalmitic acid, and beta-hydroxystearic acid. Unsaturated fatty acids and 19-carbon cyclopropane fatty acid were not found. The unusual compositions of H. pylori phospholipid and lipopolysaccharide fatty acids may have important implications for the taxonomy, physicochemical membrane properties, and biological activity of lipopolysaccharides.     

A comparative study of the lipids of plasma membranes of normal cells and those infected and transformed by rous sarcoma virus

Comparative studies of the phospholipids of Rous sarcoma virus (RSV)-transformed chicken embryo cells, uninfected normal cells, and cells infected but not transformed by the transformation-defective mutant of RSV td105, were done. Phospholipids of whole cells as well as those of isolated plasma membranes were analyzed by thin-layer and gas-liquid chromatography. The plasma membrane preparations used were significantly enriched in 5′-nucleotidase enzyme activity over that of whole cell homogenates. Our results indicated that transformation by RSV and infection by td105 do not significantly alter the molar ratios of the various phospholipids compared to uninfected normal cells. However, comparison of the fatty acyl chains of total phospholipid as well as isolated specific phospholipids revealed that the phospholipids of transformed cells have higher percentages of unsaturated fatty acyl chains than those of uninfected normal cells; phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SP) have higher percentages of unsaturated fatty acyl chains, while phosphatidylcholine (PC) and phosphatidylserine (PS) remained unchanged. The percentages of unsaturated fatty acyl chains of PE and PI of td 105-infected cells increased, while that of PC and PS decreased resulting in no overall change. These results are consistent with the concept that altered fluidity of membranes of transformed cells is due to the selection of phospholipids with specific fatty acyl chains by new or modified proteins incorporated into the membranes of transformed and/or virus-producing cells.     

Correlation Between Modification of Membrane Phospholipids and Some Biological Activity of Lymphocytes, Neutrophils and Macrophages

Our study considered the possibility of modifying the functional response of human neutrophils, of mouse lymphocytes and macrophages treated with phospholipids having different polar groups, different isomerisms with saturated and unsaturated fatty acids from C12 to C20 carbon atoms. the results are as follows.

a) Most of the phospholipids containing fatty acids from C12 to C20 cause inhibition of the blastogenic capacity of the polyclonal activators tested.

b) the phospholipids tested cause a decrease in adherence of polymorphonuclear leukocytes with the exception of the phosphatidyl-choline containing saturated and unsaturated fatty acids.

c) A decrease in polymorphonuclear leukocytes migrational capacity almost always occurs.

d) the cells treated with L-phosphatidyl-ethanolamine having fatty acids from C14 to C17 show an increase in chemiluminescence; those treated with phosphatidyl-choline and L-phosphatidyl-glycerol show a decrease of the chemiluminescence; L-phosphatidic acid and L-phosphatidyl-ethanolamine having Microbial fatty acids (FAs) at c16 cause a decrease in the formation of phagolisosomes in the macrophages tested.     

Phospholipid liposomes enhance the infectivity of purified simian virus 40 virions

When simian virus 40 virions purified after treatment with sodium deoxycholate were incubated with the extract of monkey kidney CV-1 cells, infectivity of the virions was enhanced. The infectivity-enhancing activity was recovered from the phospholipid fraction of CV-1 cells. The constructed liposomes composed of phosphatidylserine were able to enhance the infectivity of the purified virions, but those composed either of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, or phosphatidylinositol could not. The liposomes constructed with a mixture of phosphatidylethanolamine and phosphatidylcholine at a ratio of 1:1 (w:w) also enhanced the infectivity of the purified virions. Pretreatment of cells with liposomes either of phosphatidylserine or of phosphatidylethanolamine did not enhance susceptibility of the cells to infection with the purified virions. These observations suggest that the major phospholipids of the cellular membrane, when associated with virions, play a vital role in activation of purified virions.     

Rapid uptake and esterification of arachidonic acid and other fatty acids by microfilariae of Brugia malayi

We have examined the differential incorporation and esterification of exogenous fatty acids by microfilariae of the human filarial parasite Brugia malayi. Microfilariae incubated with 2 nM [3H]arachidonic acid over 1 h rapidly took up this fatty acid. Palmitic, oleic and linoleic acids were also incorporated by parasites. In contrast to these other fatty acids, little incorporated arachidonic acid remained as free fatty acid within microfilariae. Arachidonate was rapidly esterified into phospholipids, with 66% of incorporated arachidonate esterified into phospholipids at 1 min. Esterification of other fatty acids into phospholipids was quantitatively lesser and occurred into phosphatidylcholine and phosphatidylethanolamine. Arachidonate was preferentially esterified into phosphatidylinositol, which constituted only 10% of the total parasite phospholipid pool, and into phosphatidylcholine. By 1 min these two phospholipid classes, respectively, comprised 53% and 43% of [3H]arachidonyl-phospholipids. Neither the microfilarial incorporation of arachidonate nor its esterification into parasite phospholipids could be saturated by noncytotoxic concentrations of up to 600 microM. Microfilariae, which in vivo are exposed to arachidonate in blood, can rapidly, avidly and with high capacity incorporate exogenous arachidonate and esterify it preferentially into specific classes of phospholipids, including phosphatidylinositol. Like many mammalian cells, these phylogenetically distinct metazoan parasites possess efficient means for utilizing host-derived arachidonic acid.     

Correlation Between Modification of Membrane Phospholipids and Some Biological Activity of Lymphocytes,Neutrophils and Macrophages

Abstract

Our study considered the possibility of modifying the functional response of human neutrophils, of mouse lymphocytes and macrophages treated with phospholipids having different polar groups, different isomerisms with saturated and unsaturated fatty acids from C12 to C20 carbon atoms. the results are as follows.

a) Most of the phospholipids containing fatty acids from C12 to C20 cause inhibition of the blastogenic capacity of the polyclonal activators tested.

b) the phospholipids tested cause a decrease in adherence of polymorphonuclear leukocytes with the exception of the phosphatidyl-choline containing saturated and unsaturated fatty acids.

c) A decrease in polymorphonuclear leukocytes migrational capacity almost always occurs.

d) the cells treated with L-phosphatidyl-ethanolamine having fatty acids from C14 to C17 show an increase in chemiluminescence; those treated with phosphatidyl-choline and L-phosphatidyl-glycerol show a decrease of the chemiluminescence; L-phosphatidic acid and L-phosphatidyl-ethanolamine having Microbial fatty acids (FAs) at c16 cause a decrease in the formation of phagolisosomes in the macrophages tested.     

Activation of purified simian virus 40 virions by free amino-group containing phospholipid liposomes

The infectivity of the simian virus 40 (SV40) virions purified after treatment with sodium deoxycholate is activated by mixing, prior to infection, the virions with the liposomes composed of phosphatidylserine or a mixture of phosphatidylethanolamine and phosphatidylcholine (H. Shimura, and G. Kimura (1985), Virology 144, 268-272). The sucrose-CsCl cushion sedimentation analysis of the virions mixed with the liposomes revealed that the density of the radiolabeled virions became lower and that of the radiolabeled liposomes became higher to give a similar range, suggesting the binding of virions with the liposomes. Electron microscopy revealed the side-to-side association of virions with liposomes. The efficiency of adsorption of the virions to monkey kidney BSC-1 cells varied depending on phospholipid types mixed with virions and did not always become high. In the case of phosphatidylethanolamine liposomes, the free amino group in the phospholipid molecule was essential for the activation of the virion infectivity, because mono- and di-methylated phosphatidylethanolamine failed to activate the infectivity. Fluid nature of phospholipids seemed to be necessary also for the infectivity activation, because dipalmitoyl and distearoyl phospholipids did not activate virion infectivity at 37 degrees, the temperature at which the liposomes of these phospholipids are supposed to be in a solid state. Presence of free amino groups and difference in acyl groups of the phospholipids did not influence the adsorption of the virions to cells. These results suggest that events which occur after adsorption of virions to cells are responsible for the activation of the SV40 virion infectivity by the liposomes composed of free amino-group containing phospholipids.     

Effect of macrophage classical (M1) activation on implant-adherent macrophage interactions with Staphylococcus epidermidis: A murine in vitro model system

A model in vitro system was developed for eliciting classical (M1) activation of surface-adherent murine macrophages, which was then used to study the interaction of the M1 macrophages with Staphylococcus epidermidis. Glass substrata were first covalently grafted with a mixture of methoxy- and biotin-terminated silanated polyethylene glycol. Interferon (IFN)-γ and/or lipopolysaccharide (LPS), ligands known to induce the highly microbicidal M1 activation state in macrophages, were biotinylated and immobilized by way of a streptavidin intermediate to the biotin-PEG base substratum. Assessment of mouse bone marrow-derived macrophage (BMDM) interleukin (IL)-12(p40) and nitric oxide response to the fabricated surfaces confirmed that the model system achieved activation of adherent macrophage: IFN-γ-presenting surfaces primed cells for M1 activation, LPS-presenting surfaces elicited innate activation, and surface presenting a combination of IFN-γ and LPS induced M1 activation. The phagocytic and microbicidal capacity of activated, surface-adherent BMDM was evaluated using S. epidermidis, a bacterial species prevalent in implant-associated infections. Results indicate that M1 activation of implant-adherent macrophages trends towards diminishing their phagocytic capacity, but enhances their microbicidal capacity for S. epidermidis.     

Inhibition of interferon-gamma-mediated activation in mouse macrophages treated with lipoarabinomannan.

Lipoarabinomannan (LAM), purified from the cell walls of Mycobacterium leprae and M. tuberculosis, is a potent inhibitor of interferon-gamma (IFN-gamma) mediated activation of macrophages. The capability of LAM to inhibit IFN-gamma activation of macrophages in vitro was dose dependent and required a 24-h pre-exposure. Defective activation was evident as a block in IFN-gamma-induced cytocidal activity for tumour cell targets and microbicidal capacity for intracellular Toxoplasma gondii. Additionally, LAM treatment blocked the induction of surface Ia antigens on peritoneal macrophages by IFN-gamma. The requirement for pretreatment with LAM was further substantiated by the finding that peritoneal macrophages that were activated in vivo were not affected by LAM treatments and retained full microbicidal function. However, once inhibited by LAM treatment in vitro, macrophages remained fully refractory to IFN-gamma activation for up to 5 days in culture. Inhibition of IFN-gamma activation in macrophages treated with LAM was not overcome by 100-fold increases in the dose of IFN-gamma used or by a constant dose of IFN-gamma in combination with 100-fold increases in the level of endotoxin used to trigger cytotoxic activity. The defect in IFN-gamma unresponsiveness was not due to altered receptor function, as control and LAM-treated macrophages showed similar capacity to bind, internalize, and digest radiolabelled IFN-gamma. Based on the in vitro findings reported here, the inhibition of IFN-gamma-mediated macrophage activation by exposure to LAM may contribute to defective macrophage function observed in lepromatous granulomas and thus constitutes an important aspect of pathogenesis in mycobacterioses.     

The microbicidal activity of interferon-gamma-treated macrophages against Trypanosoma cruzi involves an L-arginine-dependent, nitrogen oxide-mediated mechanism inhibitable by interleukin-10 and transforming growth factor-beta.

The present study was carried out to determine the effector mechanism of anti-Trypanosoma cruzi activity by interferon (IFN)-gamma plus lipopolysaccharide (LPS)-treated macrophages. A macrophage cell line (IC-21) that failed to mount an appreciable oxidative burst was nevertheless found able to control T. cruzi growth after exposure to IFN-gamma alone or IFN-gamma plus LPS. Moreover, microbicidal functions of both inflammatory macrophages and IC-21 against T. cruzi was found to be inhibited in the presence of NG-monomethyl-L-arginine (NGMMA), a competitive inhibitor of L-arginine. Addition of supplemental L-arginine to the culture overcame the capacity of NGMMA to block activated macrophage anti-T. cruzi functions. The ability of NGMMA to reverse both parasite growth inhibition and killing by IFN-gamma plus LPS-activated macrophages was found to correlate with the suppression of nitrite accumulation in the culture supernatants. Together, these results implicate the L-arginine-dependent production of nitric oxide in T. cruzi killing by activated macrophages. We also tested the ability of interleukin(IL)-10 and transforming growth factor (TGF)-beta, to block regulation of T. cruzi growth in this system. Both IL-10 and TGF-beta inhibited anti-parasite function by IFN-gamma-activated macrophages, with an optimal dose of 100 units/ml and 0.5 ng/ml, respectively. Moreover, when used in combination, suboptimal doses of IL-10 and TGF-beta were found to produce a synergistic inhibitory effect in the regulation of T. cruzi growth. The ability of IL-10 and TGF-beta to suppress microbicidal function was also positively correlated with inhibition of nitrite generation in macrophage culture supernatants. These results predict an in vivo role for IL-10 and TGF-beta in promoting parasite survival in the face of the host cell-mediated immune response.     

Incorporation of recombinant gamma interferon into liposomes enhances its ability to induce peritoneal macrophage antitoxoplasma activity.

In this study we compared the ability of free- and liposome-incorporated murine recombinant gamma interferon (rIFN-gamma) to enhance peritoneal macrophage H2O2 release and antitoxoplasma activity in vitro. rIFN-gamma was efficiently (37 to 47%) incorporated into multilamellar vesicles composed of phosphatidylglycerol/cholesterol in a 2:1 molar ratio. The amount of rIFN-gamma incorporated into multilamellar vesicles and added to macrophages (0.1 to 1,000 U/ml) was quantitated with [3H]rIFN-gamma. The concentration of liposomal rIFN-gamma required to enhance macrophage H2O2 release (1 U/ml) and maximally inhibit Toxoplasma gondii growth (10 U/ml) was one-tenth the concentration required for free rIFN-gamma (10 and 100 U/ml, respectively). This increase in potency was observed in both thioglycolate-elicited and resident peritoneal macrophages. Control liposomes containing encapsulated buffer had no effect on the potency of free rIFN-gamma. The duration of macrophage activation induced by 24 h of liposomal rIFN-gamma treatment was also considerably longer than that induced by free rIFN-gamma (2 days versus less than 1 day). These data indicate that liposomal rIFN-gamma is more active than free rIFN-gamma as an inducer of macrophage microbicidal properties in vitro. This enhanced activity, combined with the potential for selective delivery of liposomal rIFN-gamma to phagocytic cells in vivo, may improve the therapeutic efficacy of rIFN-gamma in infections characterized by parasitization of phagocytes.     

The activation of vaccinia virus infectivity by the transfer of phosphatidylserine from the plasma membrane

Purified vaccinia virus usually contains a large proportion of noninfectious virus which can be converted to infectious virus by incubation with purified plasma membrane. This activating reaction which is mediated by a heat stable component of the membrane has been studied. A suspension of liposomes containing the lipids extracted from plasma membrane of either KB cells or mouse RBCs activated the noninfectious virus in the same manner as heated plasma membrane. The phospholipid fraction of the KB cell lipids had the activating ability, but neither neutral lipid nor glycolipid fraction activated the virus. Liposomes containing phosphatidylserine activated the virus, whereas other tested phospholipids, including phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, and sphingomyelin had no effect on virus infectivity. Lysolecithin reduced the infectivity. Treatment with isolated plasma membrane or liposomes increased hydrophobicity of the virus slightly, but did not change its density. Analysis of activated and then purified virus showed that all phospholipid species in the coincubated plasma membranes and liposome samples were transferred to the virus. The transfer was not a phospholipid exchange reaction but a one-way net transfer, and took place rapidly at 37° to reach saturation within 1 hr of coincubation. Neither activation of virus nor transfer of phospholipid occurred when the mixture was incubated at a temperature below 8°. The virus has great ability to extract phospholipids from coincubated lipid bilayer membranes, and association with phosphatidylserine gives the virus high infectivity.