Studies on the mechanisms of macrophage activation. I. Destruction of intracellular Leishmania enriettii in macrophages activated by cocultivation with stimulated lymphocytes.

When cultures of normal mouse peritoneal macrophages were infected with the intracellular protozoan parasite Leishmania enrietti, the micro-organism was found to survive intracellularly for several days, apparently without multiplication. However, exposure of infected macrophages to certain stimuli led to rapid parasite killing and digestion, providing a sensitive assay with which the mechanisms of macrophage activation can be studied. Microbicidal activity was induced by incubation of macrophages with syngeneic spleen lymphocytes, which were stimulated either by allogeneic cells in mixed lymphocyte culture (MLC) or by the plant lectin concanavalin A (Con A). Cocultivation with MLCs led to parasite killing within 48-72 h, whereas exposure of infected cells to Con A-stimulated lymphocytes resulted in substantial destruction of the micro-organism within less than 24 h, an effect which was dependent on the presence of thymus-derived lymphocytes and was inhibited by alpha methyl-mannoside. Incubation with Con A-stimulated lymphocytes also led to lysis of part of the macrophage monolayer. However, parasite killing did not result from decreased macrophage survival, as destruction of the micro-organism was highest under culture conditions which were the least detrimental to the phagocytes. Conversely, excess numbers of Con A-stimulated lymphocytes were less efficient at inducing macrophage activation and displayed marked toxicity to the macrophage monolayer. When spleen cells were stimulated by Con A at concentrations above 10 mug/ml, a decrease was noted in the capacity of macrophages to destroy the parasite, probably reflecting a toxicity of the lectin for lymphocytes resulting in impaired activating capacity.

Antibody-induced movement of membrane components of Leishmania enriettii.

Incubation in vitro of viable Leishmania enriettii with antibodies from infected or immune guinea pigs and a fluorescein-labeled antiguinea pig Ig conjugate induced aggregation of surface antigens to form a "cap" over the anterior pole of the amastigote and over both the anterior and posterior poles of the promastigote form of the parasite. Cap formation occurred only with optimum quantities of guinea pig antibodies and was inhibited by low temperature and the metabolic inhibitors, sodium azide and iodoacetamide. The aggregated antigens were rapidly lost from the surface of the parasite but reappeared after 3 h of incubation at 23 degrees C. This phenomenon of ligand-induced membrane antigen movement is apparently similar to that described in mammalian cells, and may represent the first stage of the interaction between host antibodies and the surface membrane of protozoal parasites.

Mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell 1-type CD4+ T cell response.

Mice with homologous disruption of the gene coding for the ligand-binding chain of the interferon (IFN) gamma receptor and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in the differentiation of functional CD4+ T cell subsets in vivo and resistance to infection. Wild-type 129/Sv/Ev mice are resistant to infection with this parasite, developing only small lesions, which resolve spontaneously within 6 wk. In contrast, mice lacking the IFN-gamma receptor develop large, progressing lesions. After infection, lymph nodes (LN) and spleens from both wild-type and knockout mice showed an expansion of CD4+ cells producing IFN-gamma as revealed by measuring IFN-gamma in supernatants of specifically stimulated CD4+ T cells, by enumerating IFN-gamma-producing T cells, and by Northern blot analysis of IFN-gamma transcripts. No biologically active interleukin (IL) 4 was detected in supernatants of in vitro-stimulated LN or spleen cells from infected wild-type or deficient mice. Reverse transcription polymerase chain reaction analysis with primers specific for IL-4 showed similar IL-4 message levels in LN from both types of mice. The IL-4 message levels observed were comparable to those found in similarly infected C57BL/6 mice and significantly lower than the levels found in BALB/c mice. Anti-IFN-gamma treatment of both types of mice failed to alter the pattern of cytokines produced after infection. These data show that even in the absence of IFN-gamma receptors, T helper cell (Th) 1-type responses still develop in genetically resistant mice with no evidence for the expansion of Th2 cells.

Antibody-induced movement of protozoan surface membrane antigens.

Direct and indirect immunofluorescence techniques were used to study the effects of host antibodies on surface membrane antigens of viable Leishmania parasites in vitro. Antisera to L. enriettii and L. tropica caused surface membrane antigens of these parasites to aggregate, move toward the poles of the parasite, and to eventually disappear. This sequence did not occur at low temperature or in the presence of some metabolic inhibitors. Antigen subsequently reappeared on the parasite membrane. These phenomena appear to be similar to those described in mammalian cells. Antibodies may therefore effect an alteration in the surface membrane structure of living protozoan parasites.

Mechanisms of protective immunity in experimental cutaneous leishmaniasis of the guinea-pig. III. Inhibition of leishmanial lesion in the guinea-pig by delayed hypersensitivity reaction to unrelated antigens.

The purpose of this investigation was to determine whether prior induction of a non-specific delayed reaction at a site of leishmanial infection could modify the course of infection. Groups of animals were made hypersensitive to either DNCB or BCG and a delayed reaction was elicited by corresponding antigen in one or both ears when an infective dose of L. enriettii was inoculated. With various experimental designs the following results were obtained: (a) induction of delayed reaction by DNCB or BCG inhibited the development of leishmanial lesions; (a) the protection was effective only when delayed reaction occurred at the site of infection; (c) to be effective, the reaction had to be continuously present at the site of infection for at least 3--4 weeks; (d) lesions developed normally, in the absence of delayed reaction, in DNCB-tolerant animals treated with DNCB; (E) a protective delayed reaction did not completely eliminate the parasites from the host tissues, since metastatic lesions appeared later at ectopic areas; (f) the suppressed development of a lesion did not confer resistance to reinfection dose of the parasite. It is concluded that cell-mediated immunity plays an important role in healing leishmanial lesions in the guinea-pig and that the final effector mechanism may be sought in the non-specific microbicidal capacity of activated macrophages. The relevance of leishmania-specific delayed reaction in the course of the disease is discussed.

Mechanisms of protective immunity in experimental cutaneous leishmaniasis of the guinea-pig. II. Selective destruction of different Leishmania species in activated guinea-pig and mouse macrophages.

Macrophages activation as the effector mechanism in destroying L. enriettii in the guinea-pig, and L. tropica in the mouse, was tested in vitro. Activated guinea-pig macrophages, with enhanced anti-Listeria capacity had no effect on the survival of intracellular L. enriettii, irrespective of the antigen used. Activated mouse macrophages, on the other hand, destroyed ingested L. enriettii within 24-48 hr but had no effect on L. tropica during the same time period. It is suggested that the pathogenicity of a Leishmania parasite in a given host depends on the ability of the parasite to survive in the host's activated macrophages. The possible mechanisms by which L. enriettii evades destruction in activated guinea-pig macrophages are discussed.

Mechanisms of protective immunity in experimental cutaneous leishmaniasis of the guinea-pig. I. Lack of effects of immune lymphocytes and of activated macrophages.

Leishmania enriettii is an obligatory intracellular protozoan parasite which infects guinea-pigs and resides in macrophages. Subcutaneous inoculation produces a skin infection which heals spontaneously and leaves the animal immune to reinfection. Experiments have been performed to explore the mechanisms of parasite destruction in the recovering and immune animal. Using quantitative techniques to assess parasite survival it was found that L. enriettii is not killed in vitro in macrophages from immune guinea-pigs. Inocubation of monolayers of parasitized macrophages with lymphocytes from Leishmania-immune animals had no effect on the intracellular parasites. Finally, macrophages activated to destroy Listeria monocytogenes did not impair intracellular survival of L. enriettii. The possible significance of these findings in explaining the course of infection is discussed.

Analysis of the cellular parameters of the immune responses contributing to resistance and susceptibility of mice to infection with the intracellular parasite, Leishmania major.

Although the course of infection induced by L.major in mice is influenced by several factors, including the parasite virulence, the macrophage permissiveness to this parasite and response to T cell-produced lymphokines, this review has been restricted to summarizing, the recent data concerning the T-cell responses generated during infection and their effect on the disease process. Experimental evidence strongly suggests that T-cell responses play a fundamental role in resistance and susceptibility of mice to infection with L.major. It appears that resolution of lesion and exacerbation of disease result from the activity of distinct specific CD4+ T cells. There is a consensus of opinion that CD4+ T cells from the TH1 functional phenotype are generally endowed with protective function through their secreted lymphokines (e.g. IFN-gamma). However, some evidence exists that other lymphokines (e.g. TNF) might be involved in resolution of lesions. Results exist which indicate that some TH1 CD4+ T cells also contribute to susceptibility to infection. Their specificity differs from that of protective TH1 cells in the sense that these T cells might recognize parasite antigens not appropriately presented by parasitized macrophages and therefore, although releasing IFN-gamma, would not be able to concentrate this lymphokine on the surface of macrophages containing multiplying L.major. It appears that parasite-specific TH2 cells play an important role, through the IL-4 that they produce, in the severe disease seen in BALB/c mice. Determining the mechanisms responsible for the expansion of TH2 cells in genetically susceptible mice as well as assessing whether or not some parasite antigens are preferentially recognized by TH1 and TH2 cells are areas of investigation of prime importance for the rational design of a vaccine against leishmaniasis. Several observations indicate that CD8+ T cells have a role in the resolution of lesions induced by this parasite. Precise investigation of the mechanism(s) accounting for their beneficial effect might depend upon our ability to derive and maintain in vitro homogenous populations and clones of L.major-specific CD8+ T cells.

Aggravation of experimental cutaneous leishmaniasis in mice by administration of interleukin 3.

Previous studies from our laboratory have shown that some in vitro maintained Leishmania major-specific L3T4+ T cells were capable of exacerbating cutaneous leishmaniasis after adoptive transfer to normal syngeneic mice. Results presented in this report show that these cells released substantial amounts of interleukin 3 (IL 3) and granulocyte-macrophage colony-stimulating factors after specific stimulation in vitro. In order to assess the involvement of such lymphokines in the exacerbation of cutaneous leishmaniasis by these L3T4+ T cells, the effect of the administration of important doses of IL 3 on the course of infection with L. major was investigated. The treatment of genetically susceptible BALB/c mice with IL 3 resulted in an enhancement of the size of lesions and favored the multiplication of parasites at anatomical sites distant from the primary lesion. Although IL 3 did not modify the development of lesions in genetically resistant CBA mice, this lymphokine promoted the growth of Leishmania in lymph node draining the lesion. Finally, the addition of IL 3 to macrophages parasitized in vitro enhanced the survival of intracellular Leishmania major.

Exacerbation of experimental murine cutaneous leishmaniasis with CD4+ Leishmania major-specific T cell lines or clones which secrete interferon-gamma and mediate parasite-specific delayed-type hypersensitivity.

Leishmania major-specific T cell lines were derived from mice sensitized to the parasite. The cells were of the CD4+ T cell lineage and, upon adoptive transfer, were found to be capable of inducing parasite-specific delayed-type hypersensitivity. Adoptive transfer of these L. major-specific T cells to syngeneic recipients which were either normal, T cell deficient or B cell and antibody deficient led to exacerbation of infection upon subsequent challenge with L. major. This suggested that host T cells, B cells and antibody were not required for the L. major-specific T cells to exert their exacerbative effect on the course of cutaneous leishmaniasis. Additional studies revealed that the adoptive transfer of graded doses of these L. major-specific T cells always resulted in exacerbation of infection. Study of the localization pattern of the cells following transfer showed that they migrate preferentially to the site of the lesions. Furthermore, although the induction phase of this phenomenon was immunologically specific, its effector phase was not. Finally, T cell clones were derived from the L. major-specific T cell lines. The T cell clones were phenotypically and functionally identical to the T cell lines from which they were derived. Adoptive transfer of these parasite-specific T cell clones to normal syngeneic recipients induced an exacerbated course of infection with L. major. Interestingly, when these cloned T cells were specifically activated in vitro, the cells produced interleukin 2 and interferon-gamma, but no interleukin 4, indicating that they belong to the murine Th1 subset of CD4+ T cells.

Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response.

Mice with homologous disruption of the gene coding for either the p35 subunit or the p40 subunit of interleukin-12 (IL-12) and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in resistance to infection and the differentiation of functional CD4+ T cell subsets in vivo. Wild-type 129/Sv/Ev mice are resistant to infection with L. major showing only small lesions which resolve spontaneously within a few weeks and develop a type 1 CD4+ T cell response. In contrast, mice lacking bioactive IL-12 (IL-12p35-/- and IL-12p40-/-) developed large, progressing lesions. Whereas resistant mice were able to mount a delayed-type hypersensitivity (DTH) response to Leishmania antigen, susceptible BALB/c mice as well as IL-12-deficient 129/Sv/Ev mice did not show any DTH reaction. To characterize the functional phenotype of CD4+ T cells triggered in infected wild-type mice and IL-12-deficient mice, the expression of mRNA for interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in purified CD4+ lymph node cells was analyzed. Wild-type 129/Sv/Ev mice showed high levels of mRNA for IFN-gamma and low levels of mRNA for IL-4 which is indicative of a Th1 response. In contrast, IL-12- deficient mice and susceptible BALB/c mice developed a strong Th2 response with high levels of IL-4 mRNA and low levels of IFN-gamma mRNA in CD4+ T cells. Similarly, lymph node cells from infected wild-type 129 mice produced predominantly IFN-gamma in response to stimulation with Leishmania antigen in vitro whereas lymph node cells from IL-12-deficient mice and susceptible BALB/c mice produced preferentially IL-4. Taken together, these results confirm in vivo the importance of IL-12 in induction of Th1 responses and protective immunity against L. major.