Abstract
BackgroundThe Leishmania promastigote-macrophage interaction occurs through the association of multiple receptors on the biological membrane surfaces. The success of the parasite infection is dramatically dependent on this early interaction in the vertebrate host, which permits or not the development of the disease. In this study we propose a novel methodology using flow cytometry to study this interaction, and compare it with a previously described "in vitro" binding assay.MethodsTo study parasite-macrophage interaction, peritoneal macrophages were obtained from 4 dogs and adjusted to 3 × 106 cells/mL. Leishmania (Leishmania) chagasi parasites (stationary-phase) were adjusted to 5 × 107 cells/mL. The interaction between CFSE-stained Leishmania chagasi and canine peritoneal macrophages was performed in polypropylene tubes to avoid macrophage adhesion. We carried out assays in the presence or absence of normal serum or in the presence of a final concentration of 5% of C5 deficient (serum from AKR/J mice) mouse serum. Then, the number of infected macrophages was counted in an optical microscope, as well as by flow citometry. Macrophages obtained were stained with anti-CR3 (CD11b/CD18) antibodies and analyzed by flow citometry.ResultsOur results have shown that the interaction between Leishmania and macrophages can be measured by flow cytometry using the fluorescent dye CFSE to identify the Leishmania, and measuring simultaneously the expression of an important integrin involved in this interaction: the CD11b/CD18 (CR3 or Mac-1) β2 integrin.ConclusionFlow cytometry offers rapid, reliable and sensitive measurements of single cell interactions with Leishmania in unstained or phenotypically defined cell populations following staining with one or more fluorochromes.
Highlights
The Leishmania promastigote-macrophage interaction occurs through the association of multiple receptors on the biological membrane surfaces
Flow cytometry was used to analyze the interaction between canine peritoneal macrophages and L. chagasi promastigotes, stained with the fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) (Fig. 1), and compared to a conventional methodology ("in vitro" binding-assays)
L. chagasi promastigotes were stained with the intracellular dye CFSE and the efficiency of the staining procedure evaluated trough flow cytometry (A) and conventional fluorescence microscopy (B)
Summary
The Leishmania promastigote-macrophage interaction occurs through the association of multiple receptors on the biological membrane surfaces. The success of the parasite infection is dramatically dependent on this early interaction in the vertebrate host, which permits or not the development of the disease. Leishmaniasis is a disease resulting from infection by protozoa of the genus Leishmania, which affects man and several other species of mammals. Upon inoculation of the host, promastigotes are phagocytized by skin macrophages, where they transform into aflagellar ovoid bodies known as amastigotes. The parasite or infected macrophages, may spread and give rise to secondary infections in distant organs, producing distinct clinical forms of the disease [9]. The pathogenesis of Leishmania infections depends to a large extent on the relationship between the parasites and host macrophages
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