Cellular immunity of mice to Leishmania donovani in vitro: lymphokine-mediated killing of intracellular parasites in macrophages.

Abstract

Leishmania doovani, an intracellular protozoan, causes kala-azar by parasitizing the macrophages ofits mammalian host. Outbred NCS and CD-i mice develop immunity to this parasite. This immunity was demonstrable when supernatant fluids from cultured splenic lymphocytes were added to infected macrophages. Only the lympholdne preparations from infected mice showed significant leishmanicidal activity. Mice receiving multiple inocula-were more potent producers of leishmanicidal lymphokines than were those receiving single inocula. The expression of leishmanicidal activity in our system required continuous presence of the lympholdne preparation and was independent of trypsin-or neuraminidase-sensitive receptors ofthe macrophages. Light and electron microscopy revealed that, in the presence of lymphokines, macrophages appeared to be "activated," and intraceliular leishmanias developed specific subcellular lesions in the kdnetoplast-mitochondria. A time-course study showed that cultivation of the lymphocytes for 1/2 days completed the release of their leishmanicidal lympholdnes which were heat-labile molecules larger than 50,000 daltons.Leishmanias are trypanosomatid protozoa whose amastigote stage parasitizes the mononuclear series ofthe reticuloendothelial system, resulting in leishmaniasis. In response to infection by different species, susceptible hosts manifest distinct clinical symptoms ranging from self-curing cutaneous lesions to an often-fatal visceral form (1). Immunity of all species of animals is thought to be largely cell-mediated, but supporting evidence for this has been derived from investigations ofthe experimental cutaneous leishmaniases (2, 3). The work of Skov and Twohy (4,5) provided in vivo evidence for the importance of thymusderived lymphocytes in mouse immunity to visceral leishmania (Leishmania donovani).We now provide evidence for the expression ofthis immunity in vitro by soluble lymphokine factors released by splenic lymphocytes derived from infected mice. Some properties of these factors and their effects on intracellular leishmanias in macrophages are characterized. MATERIALS AND METHODSAll chemicals and medium components were purchased from Sigma and GIBCO, respectively.Animals. Outbred NCS and CD-1 mice (at least 25 g in body weight) raised under conventional conditions at the Rockefeller University or Sloan-Kettering Cancer Research Center were used for the present study. Both strains of mice developed immunity to L. donovani, as indicated by examining the parasite burden during the course ofinfection. The number ofparasites in both liver and spleen increased during the initial 30 days and remained stable thereafter.Parasites. Amastigotes ofL. donovani, Sudan-IS strain, were obtained from spleens of Syrian golden hamsters infected intracardially 1-2 months earlier. Isolated amastigotes were intact, viable, and free from host cell contamination (6, 7).Infection of Mice. Aliquots of the amastigote suspension in Hanks' balanced salt solution were injected intraveneously at 50 X 10...

Profile of human T cell response to leishmanial antigens. Analysis by immunoblotting.

“…Identification of antigens that stimulate IFN--y production are further predictive of those antigens that might be involved in immunity. In experimental models, lymphokine production, specifically IFN-'y production, is associated with activation of macrophages and killing of intracellular parasites (31)(32)(33). Antigen-stimulated lymphocytes from patients with cutaneous and mucosal leishmaniasis also produce IFN-y, which can then activate macrophages and effect intracellular killing (2).…”

Identification of antigens recognized by T cells in human leishmaniasis: analysis of T-cell clones by immunoblotting

“…In vitro, IFN-,y has been shown to induce macrophage activation and killing of intracellular Leishmania spp. (5,18,19). IFN-y produced by antigen-stimulated lymphocytes from patients with cutaneous and mucosal leishmaniasis can also activate human macrophages to kill idtracellular organisms (2,22).…”

Oxygen‐dependent microbicidal systems of phagocytes and host defense against intracellular protozoa

“…Several investigators have reported that addition of lymphokines after phagocytosis was completed can activate otherwise susceptible macrophage populations to kill ingested protozoa [31,62,75,76]. Although such activation may proceed by oxygen-independent pathways, in at least two studies the use of inhibitors such as catalase have suggested that killing was via an oxygen-dependent mechanism [3 1,621.…”