Localization and activity of different lysosomal proteases in rat macrophages infected by Leishmania amazonensis

Leishmania are protozoans of the trypanosomatidae family that cause human infections. The amastigote form of Leishmania is an obligate intracellular parasite of mononuclear phagocytes that multiplies within parasitophorous vacuoles (pv) of phagolysosomal origin. To investigate the strategies which allow Leishmania to withstand these potentially cytotoxic conditions, the distribution and activities of various lysosomal peptidases in rat macrophages infected or uninfected with Leishmania amazonensis amastigotes were studied. Specific immunoglobulins against cathepsins (cat.) B, H, L and D were used to localize these endopeptidases by immunocytochemistry. Results showed that most or even all of the secondary lysosomes in the host cell fuse with parasite-filled phagosomes, leading to translocation of the proteases in the parasitophorous vacuoles. A further study consisted in assays of five protease activities: dipeptidylpeptidases (DPP) I and II (exopeptidases), cat. B, cat. H and cat. D. Infection of macrophages was followed by a gradual increase in all these protease activities except for DPP II. These increases were apparently not related to parasite protease activities. It seems that infection by Leishmania amazonensis is followed by increased synthesis and/or reduced catabolism of host cell lysosomal proteases or alternatively by inactivation of endogenous inhibitors. Amastigote infectivity may be related, at least in part, to the development of mechanisms that allow the parasite to withstand unfavorable environmental conditions.     

Parasitophorous vacuoles of Leishmania amazonensis-infected macrophages maintain an acidic pH.

Leishmania amastigotes are intracellular protozoan parasites of mononuclear phagocytes which reside within parasitophorous vacuoles of phagolysosomal origin. The pH of these compartments was studied with the aim of elucidating strategies used by these microorganisms to evade the microbicidal mechanisms of their host cells. For this purpose, rat bone marrow-derived macrophages were infected with L. amazonensis amastigotes. Intracellular acidic compartments were localized by using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine as a probe. This indicator, which can be detected by light microscopy by using immunocytochemical methods, mainly accumulated in perinuclear lysosomes of uninfected cells, whereas in infected cells, it was essentially localized in parasitophorous vacuoles, which thus appeared acidified. Phagolysosomal pH was estimated quantitatively in living cells loaded with the pH-sensitive endocytic tracer fluoresceinated dextran. After a 15- to 20-h exposure, the tracer was mainly detected in perinuclear lysosomes and parasitophorous vacuoles of uninfected and infected macrophages, respectively. Fluorescence intensities were determined from digitized video images of single cells after processing and automatic subtraction of background. We found statistically different mean pH values of 5.17 to 5.48 for lysosomes and 4.74 to 5.26 for parasitophorous vacuoles. As for lysosomes of monensin-treated cells, the pH gradient of parasitophorous vacuoles collapsed after monensin was added. This very likely indicates that these vacuoles maintain an acidic internal pH by an active process. These results show that L. amazonensis amastigotes are acidophilic and opportunistic organisms and suggest that these intracellular parasites have evolved means for survival under these harsh conditions and have acquired plasma membrane components compatible with the environment.     

Localization of major histocompatibility complex class II molecules in phagolysosomes of murine macrophages infected with Leishmania amazonensis

Leishmania-infected macrophages are potential antigen-presenting cells for CD4+ T lymphocytes, which recognize parasite antigens bound to major histocompatibility complex class II molecules (Ia). However, the intracellular sites where Ia and antigens may interact are far from clear, since parasites grow within the modified lysosomal compartment of the host cell, whereas Ia molecules seem to be targeted to endosomes. To address this question, the expression and fate of Ia molecules were studied by immunocytochemistry in Leishmania amazonensis-infected murine macrophages stimulated with gamma interferon. In uninfected macrophages, Ia molecules were localized on the plasma membrane and in perinuclear vesicles, but they underwent a dramatic redistribution after infection, since most of the intracellular staining was then associated with the periphery of the parasitophorous vacuoles (p.v.) and quite often polarized towards amastigote-binding sites. The Ii invariant chain, which is transiently associated with Ia during their intracellular transport, although well expressed in infected macrophages, apparently did not reach the p.v. Similar findings were observed with macrophages from mice either resistant or highly susceptible to Leishmania infection. In order to determine the origin of p.v.-associated Ia, the fate of plasma membrane, endosomal, and lysosomal markers, detected with specific antibodies, was determined after infection. At 48 h after infection, p.v. was found to exhibit a membrane composition typical of mature lysosomes. Overall, these data suggest that (i) Ia located in p.v. originate from secondary lysosomes involved in the biogenesis of this compartment or circulate in several endocytic organelles, including lysosomes and (ii) p.v. could play a role in antigen processing and presentation. Alternatively, the presence of high amounts of Ia in p.v. could be due to a Leishmania-induced mechanism by means of which this organism may evade the immune response.     

Depletion of secondary lysosomes in mouse macrophages infected withLeishmania mexicana amazonensis: A cytochemical study

Leishmania amastigotes lodge and multiply within parasitophorous vacuoles, which can fuse with secondary lysosomes of the host macrophages. This study examines the effect of infection with amastigotes ofL. mexicana amazonensis on the secondary lysosomes of mouse macrophage cultures. The cultures were stained for the activities of two lysosomal enzyme markers, acid phosphatase and arylsulfatase, and the light microscopic observations were supplemented by electron microscopy. Nearly all noninfected macrophages contained numerous stained secondary lysosomes. The number of such lysosomes was markedly reduced 24 h postinfection, and the reduction persisted for at least 10 days. Stained secondary lysosomes reppeared after the amastigotes were destroyed by exposure of the cultures to phenazine methosulfate or by placing them at 37.5° C. The depletion of lysosomes shown by cytochemical methods may reflect a high rate of fusion of the lysosomes with the parasitophorous vacuoles, exceeding the rate of formation of new secondary lysosomes. Alternatively, the parasites may inhibit the synthesis of lysosomal hydrolases, or the assembly or formation of primary or secondary lysosomes.     

Biogenesis of Leishmania major-harboring vacuoles in murine dendritic cells

In mammalian hosts, Leishmania sp. parasites are obligatory intracellular organisms that invade macrophages and dendritic cells (DC), where they reside in endocytic organelles termed parasitophorous vacuoles (PV). Most of the present knowledge of the characteristics of PV harboring Leishmania sp. is derived from studies with infected macrophages. Since DC play a key role in host resistance to leishmaniasis, there is a need to understand the properties and biogenesis of PV in Leishmania sp.-infected DC. Therefore, we determined the acquisition of endosomal and lysosomal molecules by Leishmania major-containing compartments in DC at different maturation stages, using fluorescence labeling and confocal microscopy. The results show that newly formed phagosomes in DC rapidly develop into late endosomal compartments. However, the small GTPase Rab7, which regulates late fusion processes, was found only in PV of mature bone marrow-derived DC (BMDC); it was absent in immature BMDC, suggesting an arrest of their PV biogenesis at the stage of late endosomes. Indeed, fusion assays with endocytic tracers demonstrated that the fusion activity of L. major-harboring PV toward lysosomes is higher in mature BMDC than in immature BMDC. The inhibition of PV-lysosome fusion in DC is dependent upon the viability and life cycle stage of the parasite, because live promastigotes blocked the fusion almost completely, whereas killed organisms and amastigotes induced a considerable level of fusion activity. The differences in the fusion competences of immature and mature DC may be relevant for their distinct functional activities in the uptake, transport, and presentation of parasite antigens.     

Multiplication of Leishmania in human macrophages in vitro.

To facilitate in vitro studies of the immunology of human leishmaniasis, we developed a method of growing pathogenic Leishmania in human monocyte-derived macrophages. After 6 days of incubation, adherent mononuclear cells were infected with Leishmania donovani amastigotes obtained from infected hamster spleen cells or with L. tropica amastigotes obtained from infected BALB/c tissue mouse footpad. Forty-eight percent of the macrophages were initially infected, with a mean of 3.0 amastigotes per infected macrophage. After 6 days of incubation, 59% of macrophages were infected and contained 8.8 amastigotes per infected macrophage, representing 2.9-fold multiplication. Electron microscopy revealed the presence of dividing parasites within phagolysosomes. These observations indicate that Leishmania survive and multiply within human monocyte-derived macrophages despite fusion of secondary lysosomes with the parasitophorous vacuole.     

Antigen presentation by Leishmania mexicana-infected macrophages: activation of helper T cells by a model parasite antigen secreted into the parasitophorous vacuole or expressed on the amastigote surface

Leishmania are protozoan parasites which invade mammalian macrophages and multiply as amastigotes in phagolysosomes (parasitophorous vacuoles). Using L. mexicana and bone marrow-derived macrophages (BMM), the question is addressed whether infected BMM induced to express major histocompatibility complex class II molecules can present defined antigens to specific T helper type 1 cells. As a model antigen, a membrane-bound acid phosphatase (MAP), a minor protein associated with intracellular vesicles in amastigotes, was either overexpressed at the surface of the parasites or overexpressed in a soluble form leading to antigen secretion into the parasitophorous vacuole. Presentation of MAP epitopes by these three types of amastigotes was then compared for macrophages containing live parasites or amastigotes inactivated by drug treatment. It is shown that surface-exposed and secreted MAP can be efficiently presented to T cells by macrophages harboring live amastigotes. Therefore, the parasitophorous vacuole communicates by vesicular membrane traffic with the plasmalemma of the host cell. The intracellular MAP of wild-type cells or the abundant lysosomal cysteine proteinases are not or only inefficiently presented, respectively. After killing of the parasites, abundant proteins such as overexpressed MAP and the cysteine proteinases efficiently stimulate T cells, while wild-type MAP levels are not effective. We conclude that intracellular proteins of intact amastigotes are not available for presentation, while after parasite inactivation, presentation depends on antigen abundance and possibly stability. The cell biological and possible immunological consequences of these results are discussed.     

In vitro light and electron microscope studies on different virulent promastigotes ofLeishmania donovani in hamster peritoneal macrophages

Summary Hamster peritoneal macrophages were infected with arivulent and virulent promastigotes of aL. donovani strain using various ratios (11; 110) of parasites and peritoneal cells. Light microscope studies have shown that there was a significant difference in the number of parasites taken up by phagocytic cells between the macrophage cultures infected with avirulent and virulent promastigotes at 4 h as well as during the following 14 days of infection. In both virulent groups the number of amastigotes were sharply increased. However, the surviving parasites were eliminated continuously when the macrophage cultures were infected with avirulent parasites. Electron microscope examinations of the different infected macrophage cultures did not show any difference in the localization of the surviving parasites. At one and 24 h post-infection, parasites have been observed in typical parasitophorous vacuoles. However, by day 4, 7, and 14 post-infections, the majority of intact parasites were surrounded by a four-laminar membrane without a space between parasite and vacuole membrane. Besides, some amastigotes were seen in large parasitophorous vacuoles. It seemed as if some of these amastigotes were trying to leave the parasitophorous vacuoles. In all cases acid phosphatase could be demonstrated in the parasitophorous vacoules and around the parasites indicating that the lysosomes of the host cell have been fused with the parasitophorous vacuole. It is indicated that the virulentLeishmania parasites are more resistant to the digestive system of the macrophages.     

Liposomal chemotherapy in visceral leishmaniasis: An ultrastructural study of an intracellular pathway

The intracellular fate of liposomes administered intracardially was examined in the liver and spleen of hamsters experimentally infected withLeishmania donovani. Separate groups of animals were treated with liposomes containing either an antileishmanial agent, a colloidal gold marker, or saline. Ultrastructural examinations of lysosomal interactions with the parasitophorous vacuole and with phagocytized liposomes were made. Lysosomes readily fused with the parasitophorous vacuoles but appeared to have little effect on the parasite, possibly due to the production of enzyme inhibitors. Liposomes rapidly became localized in lysosomes subsequent to endocytosis by macrophages. Morphologic evidence suggested that secondary lysosomes containing liposomal residues then fused with the parasitophorous vacuole. Aspects of one possible pathway are discussed which may account for the greatly enhanced effectiveness of liposomal chemotherapy for experimental visceral leishmaniasis.     

Leishmania RAB7: characterisation of terminal endocytic stages in an intracellular parasite

Leishmania species are intracellular parasites that inhabit a parasitophorous vacuole (PV) within host macrophages and engage with the host endo-membrane network to avoid clearance from the cell. Intracellular Leishmania amastigotes exhibit a high degree of proteolytic/lysosomal activity that may assist degradation of MHC class II molecules and subsequent interruption of antigen presentation. As an aid to further analysis of the endosomal/lysosomal events that could facilitate this process, we have characterised a Leishmania homologue of the late endosomal marker, Rab7, thought to be involved in the terminal steps of endocytosis and lysosomal delivery. The Leishmania major Rab7 (LmRAB7) protein is expressed throughout the life-cycle, shows 73 and 64% identity to Trypanosoma cruzi and Trypanosoma brucei Rab7s (TcRAB7 and TbRAB7), respectively, and includes a kinetoplastid-specific insertion. The recombinant protein binds GTP and polyclonal antibodies raised against this antigen recognise structures in the region of the cell between the nucleus and kinetoplast. By immunoelectron microscopy of axenic amastigotes, Leishmania mexicana Rab7 (LmexRAB7) is found juxtaposed to and overlapping membrane structures labelled for the megasomal marker, cysteine proteinase B, confirming a late-endosomal/lysosomal localisation.     

Receptor-mediated entry of peroxidases into the parasitophorous vacuoles of macrophages infected with Leishmania Mexicana amazonensis

Leishmania amastigotes, obligatory parasites of macrophages, lodge and multiply within long-lived phagolysosomelike "parasitophorous vacuoles" (PV). The glycoprotein horseradish peroxidase (HRP) was shown, by light and electron microscopic cytochemistry, to enter the PVs of rat in vitro-derived bone marrow macrophages infected with Leishmania mexicana amazonensis. Uptake was obtained both in preinfected macrophages incubated with HRP and in macrophages pulsed with HRP, infected, and further incubated in ligand-free medium. Peroxidase positive and negative PVs could coexist in the same macrophages. Infected macrophages commonly displayed fewer labeled secondary lysosomes than noninfected cells. Lactoperoxidase (LP) was also shown, by light microscopy, to enter the PVs of rat macrophages. Uptake of HRP and of LP was blocked by mannan, supporting the mannose receptor mediated recognition of these ligands. Transfer of HRP to PVs was much less efficient in resident mouse peritoneal macrophages, even at 50 X higher ligand concentrations. Such macrophages expressed negligible mannose receptor function. The efficient mannan-inhibitable uptake of HRP by rat marrow macrophages was confirmed biochemically. Bulk HRP uptake in infected and noninfected cultures was found to be similar. Peroxidases should be useful in further studies of endocytosis by Leishmania-infected macrophages and in the development of lysosomotropic macrophage-targeted drug carriers.     

Freeze-fracture and cytochemistry study of the interaction between Leishmania mexicana amazonensis and macrophages

The process of interaction between macrophages and promastigote and amastigote forms of Leishmania mexicana amazonensis was analyzed using freeze fracture and cytochemistry. The promastigotes inside endocytic vacuoles of macrophages presented an altered distribution of intramembranous particles and a wavy aspect of the plasma membrane. However, amastigotes did not show such alterations. The membrane alterations are probably caused by intracellular cell lysis of the promastigotes by the macrophages. An accumulation of intramembranous particles was seen in the plasma membrane of amastigote forms in the area of adhesion to the macrophages. The parasitophorous vacuole membrane had intramembranous particles randomly distributed. The enzyme activity of Mg++-ATPase, 5'-nucleotidase and NAD(P)H-oxidase was cytochemically detected, at the ultrastructural level, in normal mouse peritoneal macrophages and in macrophages infected with Leishmania mexicana amazonensis. Mg++-ATPase and 5'-nucleotidase are uniformly distributed throughout the macrophage's plasma membrane but were not detected in the membrane lining endocytic vacuoles containing ingested parasites (parasitophorous vacuole). NAD(P)H-oxidase activity was seen in those portions of the macrophage's plasma membrane which enter in direct contact with parasites and also in association with the membrane of the parasitophorous vacuole. The amount of reaction product, indicative of NAD(P)H-oxidase activity, was larger in macrophages which interacted with the promastigote than in those which interacted with the amastigote form of L. mexicana amazonensis. Concanavalin A binding sites and anionic sites of the macrophage's surface, labeled before the interaction, are not interiorized together with the parasites, however, are observed in endocytic vacuoles which do not contain parasites.     

Leishmania pifanoi amastigotes avoid macrophage production of superoxide by inducing heme degradation

Whereas infections of macrophages by promastigote forms of Leishmania mexicana pifanoi induce the production of superoxide, infections by amastigotes barely induce superoxide production. Several approaches were employed to gain insight into the mechanism by which amastigotes avoid eliciting superoxide production. First, in experiments with nitroblue tetrazolium, we found that 25% of parasitophorous vacuoles (PVs) that harbor promastigotes are positive for the NADPH oxidase complex, in contrast to only 2% of PVs that harbor amastigotes. Second, confocal microscope analyses of infected cells labeled with antibodies to gp91phox revealed that this enzyme subunit is found in PVs that harbor amastigotes. Third, in immunoblots of subcellular fractions enriched with PVs from amastigote-infected cells and probed with antibodies to gp91phox, only the 65-kDa premature form of gp91phox was found. In contrast, subcellular fractions from macrophages that ingested zymosan particles contained both the 91- and 65-kDa forms of gp91phox. This suggested that only the immature form of gp91phox is recruited to PVs that harbor amastigotes. Given that gp91phox maturation is dependent on the availability of heme, we found that infections by Leishmania parasites induce an increase in heme oxygenase 1 (HO-1), the rate-limiting enzyme in heme degradation. Infections by amastigotes performed in the presence of metalloporphyrins, which are inhibitors of HO-1, resulted in superoxide production by infected macrophages. Taken together, we propose that Leishmania amastigotes avoid superoxide production by inducing an increase in heme degradation, which results in blockage of the maturation of gp91phox, which prevents assembly of the NADPH oxidase enzyme complex.     

Isolation of the intracellular stage of Trypanosoma cruzi and its interaction with mouse macrophages in vitro.

Interaction between Trypanosoma cruzi spheromastigotes (amastigotes) and mouse macrophages was studied. The spheromastigotes, isolated from the spleens of infected mice, were incorporated and digested by the macrophages. By use of horseradish peroxidase labeling of the macrophage lysosomes we showed that fusion of lysosomes with phagocytic vacuoles containing T. cruzi occurred. Parasites showing alterations indicative of digestion were seen inside the phagocytic vacuoles. Our results suggest that intracellular sphermastigotes of T. cruzi, isolated from the spleens of infected mice, and not able to induce a protective infection in mouse macrophages maintained in vitro.     

Subverted transferrin trafficking in Leishmania- infected macrophages

The intracellular fate of human transferrin (HTf) in macrophages infected by Leishmania was investigated. Binding of HTf-gold complexes at 4 °C was competitively inhibited by native holoHTf but not by apoHTf. Infected and uninfected macrophages displayed rather distinct HTf trafficking. Pulse-chase experiments using uninfected macrophages loaded with 15-nm gold-conjugated bovine serum albumin (BSA) and then incubated with 5-nm gold-conjugated HTf revealed a remarkable segregation of these tracers in distinct compartments. Nevertheless, Leishmania-infected macrophages presented extensive particle colocalization at both 60 min and 18 h. Light and electron microscopy immunolabeling indicated that HTf was delivered to the parasitophorous vacuole, formed patches on the amastigote surface, and was endocytosed via the flagellar pocket. Double-staining assays showed the colocalization of biotinylated HTf and its receptor in association with the parasitophorous vacuole. To approach the Tf-binding sites of amastigotes we performed HTf-fluorescein isothiocyanate (FITC) assays. Staining was diffuse at 4 °C and punctate at 35 °C, and only the former was sensitive to ethidium bromide, indicating an eventual temperature-dependent endocytic process. Within parasites, HTf was found in cysteine-proteinase-rich structures, suggesting that the protein can be endocytosed by intracellular amastigotes and sorted to the parasite endosomal-lysosomal compartments rather than being recycled. The treatment of infected macrophages with holoHTf, but not apoHTf, promoted the parasite's intracellular survival. These results suggest that Leishmania amastigotes can exploit and subvert the host-cell endocytic system and indicate the role of Tf-carried iron in the outcome of leishmanial infection. Received: 20 May 1998 / Accepted: 18 June 1998     

Polyketide synthase (PKS) reduces fusion of Legionella pneumophila-containing vacuoles with lysosomes and contributes to bacterial competitiveness during infection

L. pneumophila-containing vacuoles (LCVs) exclude endocytic and lysosomal markers in human macrophages and protozoa. We screened a L. pneumophila mini-Tn10 transposon library for mutants, which fail to inhibit the fusion of LCVs with lysosomes by loading of the lysosomal compartment with colloidal iron dextran, mechanical lysis of infected host cells, and magnetic isolation of LCVs that have fused with lysosomes. In silico analysis of the mutated genes, D. discoideum plaque assays and infection assays in protozoa and U937 macrophage-like cells identified well established as well as novel putative L. pneumophila virulence factors. Promising candidates were further analyzed for their co-localization with lysosomes in host cells using fluorescence microscopy. This approach corroborated that the O-methyltransferase, PilY1, TPR-containing protein and polyketide synthase (PKS) of L. pneumophila interfere with lysosomal degradation. Competitive infections in protozoa and macrophages revealed that the identified PKS contributes to the biological fitness of pneumophila strains and may explain their prevalence in the epidemiology of Legionnaires’ disease.     

Biochemical analysis of proteins and lipids found in parasitophorous vacuoles containing <Emphasis Type="Italic">Leishmania amazonensis</Emphasis>

One fundamental step of Leishmania-macrophage interaction is the phase of parasite internalization through an endocytic process, with the formation of the parasitophorous vacuole (PV). The present study analyzed this process using two approaches. First, to investigate the host cell proteins which take part in this compartment, the macrophage surface was biotinilated and allowed to interact with both Leishmania forms, the PV was then isolated, and the biotinilated proteins were analyzed by Western blot. The results obtained showed that the isolated PV from macrophages infected for 60 min with infective promastigotes displayed high molecular weight proteins, 220 kDa and 180 kDa, contrary to the isolated PV obtained from amastigotes. The isolated PV from amastigotes, after 60 min interaction, displayed a faint, biotinilated protein profile, in contrast to the PV containing amastigote which, after 30 min interaction, displayed a strong protein profile in the range of 120 kDa and 40-60 kDa. The biotinilated protein profile may represent proteins distributed in the PV membrane and may also correspond to biotinilated proteins incorporated by the intracellular parasite, as observed by confocal microscopy. In a second approach, to investigate the PV phospholipid composition, macrophages were incubated with (32)P, allowed to interact with the parasites, and the isolated PV was then processed for phospholipid analysis by thin layer chromatography and scintillation counting. An increase in the levels of lysophosphatidylcholine was observed in infected macrophages. The isolated PV from infective promastigotes and amastigotes, after 60 min interaction, displayed high levels of phosphatidylcholine. Then the PV was ruptured and the intravacuolar parasite's (32)P phospholipid composition was analyzed by TLC; and labeling of the parasites was found, suggesting that phospholipids from the macrophage are transferred to the parasite. Taken together, the results obtained show that several proteins and phospholipids found in the plasma membrane of the macrophage are also found in the PV compartment.     

Cathepsin L maturation and activity is impaired in macrophages harboring M. avium and M. tuberculosis

Mycobacterium tuberculosis-infected macrophages demonstrate diminished capacity to present antigens via class II MHC molecules. Since successful class II MHC-restricted antigen presentation relies on the actions of endocytic proteases, we asked whether the activities of cathepsins (Cat) B, S and L-three major lysosomal cysteine proteases-are modulated in macrophages infected with pathogenic Mycobacterium spp. Infection of murine bone marrow-derived macrophages with either Mycobacterium avium or M. tuberculosis had no obvious effect on Cat B or Cat S activity. In contrast, the activity of Cat L was altered in infected cells. Specifically, whereas the 24-kDa two-chain mature form of active Cat L predominated in uninfected cells, we observed an increase in the steady-state activity of the precursor single-chain (30 kDa) and 25-kDa two-chain forms of the enzyme in cells infected with either M. avium or M. tuberculosis. Pulse-chase analyses revealed that maturation of nascent, single-chain Cat L into the 25-kDa two-chain form was impaired in infected macrophages, and that maturation into the 24-kDa two-chain form did not occur. Consistent with these data, M. avium infection inhibited the IFNgamma-induced secretion of active two-chain Cat L by macrophages. Viable bacilli were not required to disrupt Cat L maturation, suggesting that a constitutively expressed mycobacterial component was responsible. The absence of the major active form of lysosomal Cat L in M. avium- and M. tuberculosis-infected macrophages may influence the types of T cell epitopes generated in these antigen-presenting cells, and/or the rate of class II MHC peptide loading.     

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.     

Nitric oxide-mediated proteasome-dependent oligonucleosomal DNA fragmentation in Leishmania amazonensis amastigotes

Resistance to leishmanial infections depends on intracellular parasite killing by activated host macrophages through the L-arginine-nitric oxide (NO) metabolic pathway. Here we investigate the cell death process induced by NO for the intracellular protozoan Leishmania amazonensis. Exposure of amastigotes to moderate concentrations of NO-donating compounds (acidified sodium nitrite NaNO(2) or nitrosylated albumin) or to endogenous NO produced by lipopolysaccharide or gamma interferon treatment of infected macrophages resulted in a dramatic time-dependent cell death. The combined use of several standard DNA status analysis techniques (including electrophoresis ladder banding patterns, YOPRO-1 staining in flow cytofluorometry, and in situ recognition of DNA strand breaks by TUNEL [terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling] assay) revealed a rapid and extensive fragmentation of nuclear DNA in both axenic and intracellular NO-treated amastigotes of L. amazonensis. Despite some similarities to apoptosis, the nuclease activation responsible for characteristic DNA degradation was not under the control of caspase activity as indicated by the lack of involvement of cell-permeable inhibitors of caspases and cysteine proteases. In contrast, exposure of NO-treated amastigotes with specific proteasome inhibitors, such as lactacystin or calpain inhibitor I, markedly reduced the induction of the NO-mediated apoptosis-like process. These data strongly suggest that NO-induced oligonucleosomal DNA fragmentation in Leishmania amastigotes is, at least in part, regulated by noncaspase proteases of the proteasome. The determination of biochemical pathways leading up to cell death might ultimately allow the identification of new therapeutic targets.