Intracellular replication of Leishmania tropica in mouse peritoneal macrophages: comparison of amastigote replication in adherent and nonadherent macrophages.

Intracellular replication of Leishmania tropica was assessed in mouse peritoneal macrophage cultures. L. tropica replicated poorly in macrophage monolayers: both the percentage of cells infected and the number of intracellular amastigotes decreased with time in culture. In contrast, nonadherent macrophages supported continuous replication of the parasite, and intracellular amastigotes increased more than 10-fold in these cultures over 8 days.     

Natural killer activity in experimental cutaneous leishmaniasis

Studies were performed to determine the role of natural killer (NK) cell cytotoxicity in experimental cutaneous leishmaniasis. Analysis of a possible correlation between in vitro NK cell activity and in vivo susceptibility to Leishmania mexicana infection showed that there is no relationship between the degree of NK reactivity to YAC-1 lymphoma cells and in vivo leishmania growth. It was also observed that spleen lymphoid cells from mice with high NK activity did not cause an increase in isotope release by the macrophage permanent cell line J774G8-1 previously infected with the parasite and supporting its growth. Mice infected with L. mexicana manifested an increased NK activity to YAC-1 cells but not to leishmania-infected J774G8-1 tumor macrophages. The lack of effect of NK cell activity is discussed with regard to the role of NK cells in immune mechanisms to intracellular parasites.     

Stage-specific, strain-specific, and cross-reactive antigens of Leishmania species identified by monoclonal antibodies.

The fusion of NS-1 myeloma cells with spleen cells from mice chronically infected with Leishmania tropica resulted in nine clones of hybridomas producing monospecific antibodies to membrane antigens of L. tropica. One of the antibodies (L-5-85) bound specifically to the promastigote form of the parasite, and the remaining eight recognized antigens shared by the promastigote and amastigote of L. tropica. Four of the antibodies (L-5-16, L-5-34, L-5-44, and L-2-3F11) detected parasite antigens on the surface of L. tropica-infected macrophages. Common antigens shared by L. tropica, L. mexicana, and L. donovani were identified as well as one antigen apparent on most Leishmania spp. and present also in Crithidia fasciculata. Two monoclonal antibodies (L-5-27 and L-5-41) were found to bind only to strains of L. tropica from simple cutaneous leishmaniasis. A special property shared by these two antibodies was the inhibition of parasite growth in macrophages in vitro.     

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.     

Immunomodulatory role of interleukin-10 in visceral leishmaniasis: defective activation of protein kinase C-mediated signal transduction events

Leishmania donovani, an intracellular protozoan parasite, challenges host defense mechanisms by impairing the signal transduction of macrophages. In this study we investigated whether interleukin-10 (IL-10)-mediated alteration of signaling events in a murine model of visceral leishmaniasis is associated with macrophage deactivation. Primary in vitro cultures of macrophages infected with leishmanial parasites markedly elevated the endogenous release of IL-10. Treatment with either L. donovani or recombinant IL-10 (rIL-10) inhibited both the activity and expression of the Ca2+-dependent protein kinase C (PKC) isoform. However, preincubation with neutralizing anti-IL-10 monoclonal antibody (MAb) restored the PKC activity in the parasitized macrophage. Furthermore, we observed that coincubation of macrophages with rIL-10 and L. donovani increased the intracellular parasite burden, which was abrogated by anti-IL-10 MAb. Consistent with these observations, generation of superoxide (O2-) and nitric oxide and the release of murine tumor necrosis factor-alpha were attenuated in response to L. donovani or rIL-10 treatment. On the other hand, preincubation of the infected macrophages with neutralizing anti-IL-10 MAb significantly blocked the inhibition of nitric oxide and murine tumor necrosis factor-alpha release by the infected macrophages. These findings imply that infection with L. donovani induces endogenous secretion of murine IL-10, which in turn facilitates the intracellular survival of the protozoan and orchestrates several immunomodulatory roles via selective impairment of PKC-mediated signal transduction.     

Liposomal Cholesterol Delivery Activates the Macrophage Innate Immune Arm To Facilitate Intracellular Leishmania donovani Killing

Leishmania donovani causes visceral leishmaniasis (VL) by infecting the monocyte/macrophage lineage and residing inside specialized structures known as parasitophorous vacuoles. The protozoan parasite has adopted several means of escaping the host immune response, with one of the major methods being deactivation of host macrophages. Previous reports highlight dampened macrophage signaling, defective antigen presentation due to increased membrane fluidity, and the downregulation of several genes associated with L. donovani infection. We have reported previously that the defective antigen presentation in infected hamsters could be corrected by a single injection of a cholesterol-containing liposome. Here we show that cholesterol in the form of a liposomal formulation can stimulate the innate immune arm and reactivate macrophage function. Augmented levels of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI), along with proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), corroborate intracellular parasite killing. Cholesterol incorporation kinetics is favored in infected macrophages more than in normal macrophages. Such an enhanced cholesterol uptake is associated with preferential apoptosis of infected macrophages in an endoplasmic reticulum (ER) stress-dependent manner. All these events are coupled with mitogen-activated protein (MAP) kinase activation, while inhibition of such pathways resulted in increased parasite loads. Hence, liposomal cholesterol is a potential facilitator of the macrophage effector function in favor of the host, independently of the T-cell arm.     

Leishmania-derived murine monocyte chemoattractant protein 1 enhances the recruitment of a restrictive population of CC chemokine receptor 2-positive macrophages

Transgenic Leishmania parasites that encode the murine chemokine monocyte chemoattractant protein 1 (MCP-1) were generated. These parasites transcribed MCP-1 mRNA and secreted MCP-1 protein. Infection of BALB/c, C57BL/6, or MCP-1 knockout (KO) mice with these parasites resulted in minimal lesion development with fewer parasites in the infected foot, lymph node, and spleen compared to wild-type-infected mice. In contrast, transgenic parasites caused substantial lesions with relatively high numbers of parasites in CC chemokine receptor 2 (CCR2) KO mice, indicating that the parasites are viable and healthy and that the lack of lesion development is CCR2 dependent. Prior infection of mice with transgenic parasites offered no protection to subsequent wild-type L. major challenge, suggesting that the transgenic parasites are controlled by an early innate immune response. Consistent with innate immunity, flow cytometry of cells from the ears of mice infected with transgenic parasites revealed an increase in the number of CCR2-positive macrophages by day 7 postinfection. The enumeration of transgenic parasites in ear lesions demonstrated a significant reduction in parasite numbers, which coincided with the increased CCR2-positive macrophage migration. CCR2-positive macrophages isolated from ears of mice infected with transgenic parasites contained virtually no parasites. In vitro studies revealed that optimal parasite killing required the recruitment of CCR2-positive macrophages, followed by stimulation with a combination of both MCP-1 and gamma interferon (IFN-gamma). This work suggests that the parasite-derived MCP-1 can recruit a restrictive population of CCR2-positive macrophages into lesions that can be optimally stimulated by MCP-1 and IFN-gamma to efficiently kill Leishmania parasites.     

Interaction of Leishmania donovani promastigotes with human monocyte-derived macrophages: parasite entry, intracellular survival, and multiplication.

Leishmania donovani promastigotes were incubated with human monocyte-derived macrophages in vitro to assess the role of macrophages in the early stage of visceral leishmaniasis. Adherent mononuclear cells, obtained from nonimmune human donors, were cultivated on glass cover slips for 5 days and then incubated with axenically grown promastigotes in the presence of heat-inactivated autologous serum. Promastigotes attached to macrophages with either their flagellar or aflagellar ends, and macrophage pseudopodia formed around them. Intracellular parasites were identified within phagocytic vacuoles by electron microscopy, and the parasites assumed a form similar to that of amastigotes obtained from infected hamster spleens. Initially, 67 +/- 5% of the macrophages were infected with a mean of 4.2 +/- 0.7 parasites per infected cell. After 6 days of incubation, 79 +/- 7% of the macrophages were infected with 15.9 +/- 3.2 parasites per infected cell. The total number of parasites per monolayer increased from 4.8 +/- 0.8 x 10(5) to 1.8 +/- 0.4 x 10(6) (P less than 0.05). Dividing parasites were identified in macrophage vacuoles by electron microscopy. Human monocyte-derived macrophage vacuoles by electron microscopy. Human monocyte-derived macrophages can phagocytize promastigotes, allow the conversion of promastigotes to an amastigote-like state, and support intracellular multiplication.     

Survival of immunoglobulin G-opsonized Toxoplasma gondii in nonadherent human monocytes.

Toxoplasma gondii is a protozoan parasite that is able to penetrate human monocytes by either passive uptake during phagocytosis or active penetration. It is expected that immunoglobulin G (IgG) opsonization will target the parasite to macrophage Fc gamma receptors for phagocytic processing and subsequent degradation. Antibody-opsonized T. gondii tachyzoites were used to infect nonadherent and adherent human monocytes obtained from the peripheral blood of seronegative individuals. The infected monocytes were evaluated for the presence of intracellular parasites and the degree of parasiticidal activity. A marked difference in both the numbers of infected macrophages and numbers of parasites per 100 macrophages was observed in the nonadherent cells when compared with those of the adherent cell population. When macrophage Fc gamma receptors were down-modulated, opsonized tachyzoites retained their ability to penetrate the host cell at a rate similar to that observed for unopsonized parasites. These results suggest that antibody opsonization of T. gondii does not prevent active penetration of human monocytes by the parasite and, furthermore, has little effect on intracellular replication of the parasite.     

Myeloid-derived suppressor cells help protective immunity to Leishmania major infection despite suppressed T cell responses

Th1/Th2 cytokines play a key role in immune responses to Leishmania major by controlling macrophage activation for NO production and parasite killing. MDSCs, including myeloid precursors and immature monocytes, produce NO and suppress T cell responses in tumor immunity. We hypothesized that NO-producing MDSCs could help immunity to L. major infection. Gr1(hi)(Ly6C(hi)) CD11b(hi) MDSCs elicited by L. major infection suppressed polyclonal and antigen-specific T cell proliferation. Moreover, L. major-induced MDSCs killed intracellular parasites in a NO-dependent manner and reduced parasite burden in vivo. By contrast, treatment with ATRA, which induces MDSCs to differentiate into macrophages, increased development of lesions, parasite load, and T cell proliferation in draining LNs. Altogether, these results indicate that NO-producing MDSCs help protective immunity to L. major infection, despite suppressed T cell proliferation.     

Expression of Leishmania antigen on the surface membrane of infected human macrophages in vitro.

Resolution of leishmaniasis is associated with host immunological responsiveness to parasite antigens. In clinical disease, leishmania are found as amastigotes contained with macrophages. We investigated the possibility that Leishmania antigens are expressed on the infected macrophage surface by reacting infected macrophages with antibody to Leishmania. In vitro-infected human monocyte-derived macrophages were labelled with antibody to amastigotes when examined with immunofluorescent or immunoelectron microscopic techniques. Infected macrophages were poorly labelled by antibody to promastigotes (insect forms of Leishmania). Certain antisera that reacted with the surface membranes of amastigotes did not label the infected macrophage surface. These results indicate that human macrophages infected in vitro express Leishmania amastigote antigen(s) on their surface membranes, that such antigen(s) may not be present in large quantities in promastigotes, and that certain antigen(s) on the amastigote surface are not expressed on the surface membranes of infected macrophages.     

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.     

Cell contact-mediated macrophage activation for antileishmanial defence: mapping of the genetic restriction to the I region of the MHC.

Employing a murine model of cutaneous leishmaniasis, we observed that draining popliteal lymph node Lyt-1+2- T cells from C57BL/6 mice with Leishmania major (also called L. tropica major) footpad infections activate macrophages in vitro to kill intracellular Leishmania amastigotes. This antileishmanial effect requires direct contact between T cell effectors and infected macrophage targets. Furthermore, it neither is strictly lymphokine mediated nor causes damage to infected macrophages; and it is antigen specific. Syngeneic combinations of L. major sensitized T cells and target macrophages result in maximum antileishmanial effects whereas allogeneic combinations result in minimum effects. The present study examined the nature of this genetic restriction and specifically investigated whether it was under the control of Major Histocompatibility Complex (MHC) gene products. Using combinations of L. major sensitized T cells and infected macrophages obtained from different inbred and congenic mouse strains in the in vitro antileishmanial assay, we determined that antileishmanial effects occur optimally when the target and effector cells are derived from mice with the same I-A haplotype. These results suggest that I-A encoded gene products play a role in the genetic restriction of contact mediated activation of macrophages for antileishmanial defence. We postulate that an antigen-Ia complex may provide for close physical interactions between effector and target cells and may approximate putative T cell membrane-bound activating molecules (as yet unidentified) with the macrophage plasma membrane. Such an association might result in a more efficient delivery system for macrophage activating factors than is afforded by soluble lymphokine mediators.     

Impairment of the oxidative metabolism of mouse peritoneal macrophages by intracellular Leishmania spp.

When stimulated in vitro with macrophage-activating factor or lipopolysaccharide, mouse peritoneal macrophages acquire the capacity to develop a strong respiratory burst when they are triggered by membrane-active agents. The presence of intracellular parasites of the genus Leishmania (L. enriettii, L. major) significantly inhibited such activity, as measured by chemiluminescence, reduction of cytochrome c and Nitro Blue Tetrazolium, and hexose monophosphate shunt levels. On the contrary, inert intracellular particles such as latex beads strongly increased the macrophage respiratory burst, suggesting that the Leishmania-linked inhibition resulted from a specific parasite effect. Impairment of macrophage oxidative metabolism by intracellular Leishmania spp. was a function of the number of infecting microorganisms and was more pronounced in macrophages infected with living than with dead parasites. Moreover, the metabolic inhibition was less apparent in L. enriettii-infected macrophages that were exposed to both macrophage-activating factor and lipopolysaccharide, i.e., conditions leading to complete parasite destruction. The mechanisms of respiratory burst inhibition by intracellular Leishmania spp. are unclear, but these observations suggest that such effects may contribute significantly to intracellular survival of the microorganisms.     

Cytophilic and Opsonic Antibodies in Visceral Leishmaniasis in Mice

Although acquired immunity to leishmaniasis is generally considered to be cell mediated, humoral factors may be partially responsible. The present study showed that antisera from C57BL/6J mice superinfected with Leishmania donovani contained cytophilic antibody and opsonins for both the amastigote and promastigote stages of the parasite. Macrophages treated with mouse hyperimmune serum in an in vitro macrophage culture system bound statistically significantly more parasites at 4°C (and subsequently phagocytized them at 37°C) than did macrophage cultures treated with control serum. The percentages of antibody-treated macrophages bearing and containing parasites were also significantly greater than the percentages of control serum-treated macrophages bearing and containing parasites, respectively. These differences persisted in cultures during a 9-day observation period when sera from mice killed 10 or 11 days after superinfection were used. However, when sera from mice killed 24 days after superinfection were tested with amastigotes, by day 9 the number of parasites and the percentage of cells parasitized in the culture decreased to control values or significantly below them. Thioglycolate-stimulated macrophages treated with hyperimmune serum bound more amastigotes at 4°C than did stimulated macrophages treated with control serum. Activated macrophages also demonstrated increased nonspecific binding of amastigotes. Treatment of macrophages with trypsin reduced both cytophilic antibody-specific and nonspecific binding of amastigotes. The demonstration of in vitro effects of anti-leishmanial antibody from superinfected mice might indicate a possible role for humoral antibody in immunity to leishmaniasis in mice.     

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.     

Skin dendritic cells in murine cutaneous leishmaniasis

Studies of the immunopathogenesis of Leishmania major-induced murine cutaneous leishmaniasis provide a framework for understanding the evolution of L. major infection of skin in humans and the foundation for rationale vaccine design. Experiments in which infection is initiated with “supraphysiologic” numbers of parasites clearly identify Th-derived type I cytokines as essential participants in macrophage activation and macrophage nitric oxide production as prerequisite for parasite control. Dendritic cells, rather than macrophages, appear to be responsible for L. major-specific Th priming in these studies. Recent studies of murine cutaneous leishmaniasis in a model system in which infection is initiated with lower, more physiologic numbers of parasites confirm many of the important findings obtained in “high dose” inoculation models, but important differences have been noted. The low dose inoculation model should ultimately provide insights into mechanisms that are responsible for dendritic cell recruitment into leishmania lesions, mechanisms that facilitate parasite acquisition by skin dendritic cells and cellular interactions that eventuate in T cell priming and lesion involution.     

In vitro suppression of lymphocyte blastogenic response to mitogen and antigen by Leishmania tropica.

Cell-mediated immune (CMI) responses are important in the immunity against Leishmania spp. infection in man. However, an infection continues to persist for a limited or indefinite period of time in spite of demonstrable CMI. The factors which allow the infection to persist in the presence of the CMI are hitherto unknown. Evidence is presented here that Leishmania tropica or their products suppress the in vitro proliferative response of normal human lymphocytes to mitogen and specific antigens. The suppressive effect of L. tropica is neither due to a direct toxic action on lymphocytes nor to competition for nutrients or antigens. In vivo such an immunosuppressive effect could both facilitate macrophage parasitization and the intracellular survival of L. tropica, even after the CMI develops to processed L. tropica antigen. Persistence of infection is seen in many other bacterial, viral and fungal infections. The in vitro suppressive effect of L. tropica on the immune response observed in our study therefore becomes relevant to the understanding of the host-parasite interaction, which may determine the eventual outcome of infection in many other intracellular infections.