Jean-Claude Antoine

The biogenesis and properties of the parasitophorous vacuoles that harbour Leishmania in murine macrophages

Leishmania are protozoan parasites that, as amastigotes, live in the macrophages of mammalian hosts within compartments called parasitophorous vacuoles. These organelles share features with late endosomes/lysosomes and are also involved in the trafficking of several major histocompatibility complex (MHC)-encoded molecules. Improved knowledge of the parasitophorous vacuoles may help clarify how these protozoa persist in their hosts.

Presentation of the Leishmania antigen LACK by infected macrophages is dependent upon the virulence of the phagocytosed parasites.

We have previously demonstrated that murine macrophages (Mϕ) infected with Leishmania promastigotes, in contrast to Mϕ infected with the amastigote stage of these parasites, are able to present the Leishmania antigen LACK (Leishmania homologue of receptors for activated C kinase) to specific, I‐Ad‐restricted T cell hybrids and to the T cell clone 9.1‐2. These T cells react with the LACK (158 – 173) peptide, which is immunodominant in BALB/c mice. Here, we show that the level of stimulation of the LACK‐specific T cell hybridoma OD12 by promastigote‐infected Mϕ is clearly dependent upon the differentiation state of the internalized parasites. Thus, shortly after infection with log‐phase or stationary‐phase promastigotes of L. major or of L. amazonensis, Mϕ strongly activated OD12. The activity was transient and rapidly lost. However, under the same conditions, activation of OD12 by Mϕ infected with metacyclic promastigotes of L. major or of L. amazonensis was barely detectable. At the extreme, Mϕ infected with amastigotes were incapable to stimulate OD12. Thus, the presentation of LACK by infected Mϕ correlates with the degree of virulence of the phagocytosed parasites, the less virulent being the best for the generation/expression of LACK (158 – 173)‐I‐Ad complexes. While the intracellular killing of the parasites appears to be an important condition for the presentation of LACK, it is not the only requisite. The partial or total destruction of intracellular L. amazonensis amastigotes does not allow the presentation of LACK to OD12. A preferential interaction of LACK (158 – 173) with recycling rather than newly synthesized MHC class II molecules does not explain the transient presentation of LACK by Mϕ infected with log‐phase or stationary‐phase promastigotes because brefeldin A strongly inhibited the presentation of LACK to OD12. Taken together, these results suggest that virulent stages of Leishmania, namely metacyclics and amastigotes, have evolved strategies to avoid or minimize their recognition by CD4+ T lymphocytes.

Dendritic cells as host cells for the promastigote and amastigote stages of Leishmania amazonensis: the role of opsonins in parasite uptake and dendritic cell maturation.

In their mammalian hosts, Leishmania are obligate intracellular parasites that mainly reside in macrophages. They are also phagocytosed by dendritic cells (DCs), which play decisive roles in the induction and shaping of T cell-dependent immune responses. Little is known about the role of DCs in the Leishmania life cycle. Here, we examined the ability of mouse bone marrow-derived DCs to serve as hosts for L. amazonensis. Both infective stages of Leishmania (metacyclic promastigotes and amastigotes) could be phagocytosed by DCs, regardless of whether they had previously been experimentally opsonized with either the complement C3 component or specific antibodies. Parasites could survive and even multiply in these cells for at least 72 hours, within parasitophorous vacuoles displaying phagolysosomal characteristics and MHC class II and H-2M molecules. We then studied the degree of maturation reached by infected DCs according to the parasite stage internalised and the type of opsonin used. The cell surface expression of CD24, CD40, CD54, CD80, CD86, OX40L and MHC class II molecules was barely altered following infection with unopsonized promastigotes or amastigotes from nude mice or with C3-coated promastigotes. Even 69 hours post-phagocytosis, a large proportion of infected DCs remained phenotypically immature. In contrast, internalisation of antibody-opsonized promastigotes or amastigotes induced DCs to mature rapidly, as shown by the over-expression of costimulatory, adhesion and MHC class II molecules. Thus, in the absence of specific antibodies (e.g. shortly after infecting naive mammals), infected DCs may remain immature or semi-mature, meaning that they are unable to elicit an efficient anti-Leishmania T cell response. Absence of DC maturation or delayed/incomplete DC maturation could thus be beneficial for the parasites, allowing their establishment and amplification before the onset of immune responses.

Leishmania spp.: on the interactions they establish with antigen-presenting cells of their mammalian hosts

Identification of macrophages as host cells for the mammalian stage of Leishmania spp. traces back to about 40 years ago, but many questions concerning the ways these parasites establish themselves in these cells, which are endowed with potent innate microbicidal mechanisms, are still unanswered. It is known that microbicidal activities of macrophages can be enhanced or induced by effector T lymphocytes following the presentation of antigens via MHC class I or class II molecules expressed at the macrophage plasma membrane. However, Leishmania spp. have evolved mechanisms to evade or to interfere with antigen presentation processes, allowing parasites to partially resist these T cell-mediated immune responses. Recently, the presence of Leishmania amastigotes within dendritic cells has been reported suggesting that they could also be host cells for these parasites. Dendritic cells have been described as the only cells able to induce the activation of naive T lymphocytes. However, certain Leishmania species infect dendritic cells without inducing their maturation and impair the migration of these cells, which could delay the onset of the adaptive immune responses as both processes are required for naive T cell activation. This review examines how Leishmania spp. interact with these two cell types, macrophages and dendritic cells, and describes some of the strategies used by Leishmania spp. to survive in these inducible or constitutive antigen-presenting cells.

Lymphoid cell fractionation on magnetic polyacrylamide-agarose beads

Abstract This paper describes a new method of fractionation of mouse and rat lymphoid cells using as an insoluble support polyacrylamide-agarose spherical beads in which iron oxide particles were trapped (Magnogel) and coated with purified anti-mouse or anti-rat Ig antibodies. The advantage of this method is that both non-adsorbed and adsorbed cells are easily and rapidly recovered due to the magnetic properties of the beads. The viability of the fractionated cells is unaffected and total recovery is high: between 80 and 100%. Surface immunoglobulin-bearing cells and cells containing immunoglobulins in their cytoplasm were searched in the various fractions. 99.6–99.9% of the cells not retained on anti-Ig coated Magnogel were devoid of surface immunoglobulins and were composed of small T lymphocytes and of some immunoglobulin-containing plasma cells. In this fraction, the depletion of the surface Ig-positive cells is of 117–400-fold. The cells adsorbed on the beads were recovered by mechanical stirring followed by the application of a magnet to separate the beads and the cells. The latter were composed of surface Ig-bearing B lymphocytes (62–79%) which were enriched 1.7–2.1-fold, surface Ig-negative cells (21–38%) and some immunoglobulin-containing cells.

Intradermal inoculations of low doses of Leishmania major and Leishmania amazonensis metacyclic promastigotes induce different immunoparasitic processes and status of protection in BALB/c mice.

In order to simulate the natural long term parasitisms which may occur in mammals infected with Leishmania, cutaneous leishmaniases due to Leishmania major or Leishmania amazonensis were induced using a model based on the inoculation of 10-1000 metacyclic promastigotes into the ear dermis of BALB/c mice. The final outcome of these parasitisms was dependent upon the number of inoculated parasites. Only some of the mice inoculated with ten parasites displayed cutaneous lesions, whereas most mice infected with 100 metacyclics and all mice infected with 1000 metacyclics developed progressive lesions. We found, using the latter experimental conditions, that the onset of the pathology was associated with: (a) parasite multiplication in the inoculation site and the draining lymph node correlating with an increase of the lymph node cell number, especially in L. major-infected mice; and (b) the detection of lymph node cells, at least in part CD4(+) T lymphocytes, able to produce high levels of interferon-gamma, interleukin (IL)-4, IL-10 and IL-13. Thereafter, mice infected by L. major harboured few parasites in the ear and had a 100-fold reduction in lymph node parasite load between 23 and 40 weeks post-inoculation. In contrast, the parasite loads of L. amazonensis-infected mice remained stable in the ear and increased in nodes during the same period of time. Only L. major-infected mice that exhibited cutaneous lesions in the primary site were resistant to the re-inoculation of 1000 metacyclic promastigotes, whereas all L. amazonensis-primary infected mice remained susceptible to a second homologous challenge. These results are the first to document that a status of resistance to re-infection, referred to concomitant immunity, is coupled to the development of primary progressive lesions in L. major-infected BALB/c mice. Such a protective status is absent in L. amazonensis-infected BALB/c mice.

Kinetics of the intracellular differentiation of Leishmania amazonensis and internalization of host MHC molecules by the intermediate parasite stages

The establishment of Leishmania in mammals depends on the transformation of metacyclic promastigotes into amastigotes within macrophages. The kinetics of this process was examined using mouse macrophages infected with metacyclic promastigotes of L. amazonensis. The appearance of amastigote characteristics, including large lysosome-like organelles called megasomes, stage-specific antigens, high cysteine protease activity and sensitivity to L-leucine methyl ester, was followed over a 5-day period. Megasomes were observed at 48 h but probable precursors of these organelles were detected at 12h p.i. The promastigote-specific molecules examined were down-regulated within 5 to 12h after phagocytosis whereas the amastigote-specific antigens studied were detectable from 2 to 12-24 h. An increase in the cysteine protease activity and in sensitivity to L-leucine methyl ester of the parasites was detected from 24 h. The data indicate that at 48 h p.i., parasites exhibit several amastigote features but that complete differentiation requires at least 5 days. The appearance of megasomes or of megasome precursors and the rise in cysteine protease activity correlate quite well with the capacity of parasites to internalize and very likely degrade host MHC molecules. The fact that internalization by the parasites of host cell molecules occurs very early during the differentiation process argues for a role of this mechanism in parasite survival.

Megasomes as the targets of leucine methyl ester in Leishmania amazonensis amastigotes.

Certain L-amino acid esters, such as L-leucine methyl ester (Leu-OMe), can kill intracellular and isolated Leishmania amazonensis amastigotes. Killing appears to involve ester trapping and hydrolysis within an acidified parasite compartment (M. Rabinovitch and S. C. Alfieri, 1987, Brazilian Journal of Medical and Biological Research 20, 665-74). We show here by acid phosphatase light microscopic cytochemistry and by ultrastructural morphometry that megasomes, lysosome-like amastigote organelles, are the putative parasite targets of Leu-OMe. This conclusion is supported by the following observations. (a) Control amastigotes displayed a string of electron-dense, acid phosphatase-positive megasomes mostly located in the cellular poles opposite the flagellar pockets. Incubation of the amastigotes with Leu-OMe resulted in concentration-dependent swelling and fusion of the organelles as well as decreased electron density of the internal contents. These changes, which preceded parasite disruption, were followed by the progressive loss of parasite viability and the release of acid phosphatase activity into the medium. (b) Incubation of the amastigotes with L-isoleucine methyl ester, a non-leishmanicidal compound, induced only moderate fusion of the megasomes. (c) Pre-incubation of the parasites with the proteinase inhibitors antipain and chymostatin, previously shown to confer protection from Leu-OMe toxicity, nearly completely prevented the morphological changes of megasomes. (d) Exposure of amastigotes to tryptophanamide (Trp-NH2), the leishmanicidal activity of which is not reduced by antipain and chymostatin, did not result in swelling and fusion of the megasomes. This last finding suggests that different mechanisms underlie the destruction of amastigotes by Trp-NH2 and Leu-OMe.(ABSTRACT TRUNCATED AT 250 WORDS)

Leishmania mexicana: A cytochemical and quantitative study of lysosomal enzymes in infected rat bone marrow-derived macrophages

The cellular localization and activity of the lysosomal enzymes acid phosphatase, trimetaphosphatase, and arylsulfatase were studied in rat bone marrow-derived macrophages infected with Leishmania mexicana amazonensis amastigotes. The specific activity of acid phosphatase normalized for protein content was similar in normal macrophages and in isolated amastigotes, whereas the latter were markedly deficient in trimetaphosphatase and arylsulfatase activities. It is thus likely that trimetaphosphatase and arylsulfatase activities detected in infected macrophages were of host cell origin. The activities of the three enzymes, assayed biochemically, varied independently in the infected macrophages. While arylsulfatase activity was unchanged after infection, the activity of acid phosphatase increased by 19, 40, and 94% at 6, 24, and 48 hr, respectively. Trimetaphosphatase activity rose only slightly during the first 24 hr after infection but increased by 74% at 48 hr. The rise in acid phosphatase activity could be accounted for only partially by multiplication of the amastigotes. Thus, as for trimetaphosphatase, these results suggest enhanced macrophage synthesis of acid phosphatase and/or reduced enzyme degradation by the infected macrophages. The reduction in host cell lysosomes previously described (Ryter et al. 1983; Barbieri et al. 1985) was confirmed but appearance of lysosomal enzyme activity in the parasitophorous vacuole is documented in the present report. Thus, Leishmania do not seem to reduce the amount and the activity of host lysosomal enzymes.

Macrophage subsets harbouring Leishmania donovani in spleens of infected BALB/c mice: localization and characterization.

The purpose of the current study was to characterize parasite‐containing cells located in spleens of BALB/c mice infected with Leishmania donovani. In particular, expression of MHC class II molecules by these cells was examined to determine whether they could potentially act as cells capable of immunostimulating Leishmania‐reactive CD4+ T lymphocytes. To this end, an immunohistological analysis of spleens taken at various time points after infection was undertaken. Using this approach, we observed, in the red pulp, the formation of small cellular infliltrates containing heavily infected macrophages that could be stained with the monoclonal antibodies MOMA‐2 and FA/11. All of them expressed high levels of MHC class II molecules. Parasites were also detected in the white pulp, especially in MOMA‐2+, FA/11+ and MHC class II+ macrophages of the periarteriolar lymphocyte sheath and in MOMA‐2+ marginal zone macrophages. Infected cells were further characterized by fluorescence microscopy after their enrichment by adherence. All infected mononuclear cells recovered by this procedure could be stained with MOMA‐2 and FA/11 and thus very probably belonged to the mononuclear phagocyte lineage. Furthermore, all of them strongly expressed both MHC class II as well as H‐2M molecules, regardless of the time points after infection. Analysis of the parasitophorous vacuoles (PV) by confocal microscopy showed that these compartments were surrounded by a membrane enriched in lysosomal glycoproteins lamp‐1 and lamp‐2, in macrosialin (a membrane protein of prelysosomes recognized by FA/11) and in MOMA‐2 antigen. About 80% of the PV also had MHC class II and H‐2M molecules on their membrane. Altogether, these data indicate that in the spleens of L. donovani‐infected mice, a high percentage of amastigotes are located in macrophages expressing MHC class II molecules and that they live in PV exhibiting properties similar to those of PV detected in mouse bone marrow‐derived macrophages exposed to a low dose of interferon γ (IFN‐γ) and infected in vitro.