I – Animals
Animals naturally infected (ANI) with L. chagasi
Five mongrel dogs of unknown age were obtained from the City of Santa Luzia (suburban area of Belo Horizonte, City Hall Zoonosis Department), MG, Brazil. All dogs were positive for Leishmania by indirect immunofluorescence (IIF), complement fixation tests (CFT) and ELISA (enzyme-linked immunosorbent assay). Animals were maintained with food and water "ad libitum". All animal studies were performed under the guidance and approval of the institute's animal welfare committee under the supervision of a certified veterinarian. The experimental protocol using dogs was approved by CETEA (Comitê de ética em experimentação animal – UFMG), number 034/2004.
Preparing parasites for canine experimental infection
Promastigote forms of Leishmania (Leishmania) chagasi strain MHOM/BR/1972/BH6 were maintained in hamsters and cultivated in vitro with NNN/MEM (Gibco – BRL – USA) with 10% inactivated fetal calf serum (FCS) (Cultilab – SP – BR) supplemented with 2 mM glutamine, 100 UI/ml penicillin G and 100 μg/ml streptomycin, and maintained at 23°C in a FANEM® incubator (model 347). The Leishmania promastigotes in stationary phase were washed once with phosphate buffered saline (PBS), centrifuged at 200 g for 10 min/18°C and resuspended to 1 × 107 parasites/ml in PBS.
Experimental canine infection
Experimental infection was carried out at HERTAPE CALIER SAÚDE ANIMAL S/A (Juatuba, MG). A group of 3-month-old laboratory-bred beagles (3 males and 2 females) were inoculated intravenously (saphenous vein) with 1 × 107 promastigotes/ml resuspended in PBS. The infection was confirmed parasitologically and seroconversion was evident after 90 days post-infection. A second group of 5-month-old male L. chagasi seronegative beagles that served as uninfected controls were inoculated with sterile PBS at the time of L. chagasi inoculation of the experimentally infected animals (AEI).
II – In vitroassays
Obtaining canine monocytes and monocyte-derived macrophages
Peripheral blood (120 ml) was collected from the external jugular vein into heparanized tubes. To isolate peripheral blood mononuclear cells (PBMC), the blood was centrifuged at 300 g for 10 min at room temperature. Plasma was separated and blood cells were resuspended in PBS (1:1 proportion), overlaid on Ficoll solution (HISTOPAQUE 1077 – SIGMA) at a ratio of 1 Ficoll:2 blood, and centrifuged at 200 g for 40 min at 4°C. PBMC were separated, resuspended and washed twice in PBS (1:1 proportion), centrifuging at 300 g for 10 min at 4°C. They were resuspended in 8 ml RPMI-1640 (GIBCO, CARLSBAD, US) supplemented with 10% FCS (CUTILAB), 200 mM L-glutamine, 10 mM pyruvate, 10 mM non-essential amino acids, 7.5% w/v sodium bicarbonate (Gibco, Carlsbad, USA), 50 IU/ml penicillin and 50 μl/100 ml streptomycin. This medium was adjusted to pH 7.4.
In order to obtain monocyte-derived macrophages, another part of the peripheral blood suspension was transferred to Teflon flasks (NalgeNunc, Rochester, USA), supplemented with 10% autologous canine serum and 20% DE GMCSF (granulocyte-macrophage colony-stimulating factor), and cultured at 37°C with 5% CO2 (Forma Scientific Incubator, Waltham, USA). The medium was changed to remove non-adherent cells 24 h later and the culture was maintained under the same conditions for 10 days, changing the medium every 3 days. After 10 days in culture [21] the cells formed a monolayer, as observed by phase contrast microscopy. The macrophage-containing Teflon flasks were placed on ice for 30 min and agitated to harvest the cells. The cells were washed with cold PBS, as described by Bueno et al. (2005) [13] with modifications: we supplemented the medium with GMCSF obtained from L-cells (mouse fibroblasts) and autologous canine serum [18]. The cells were then were transferred to falcon tubes (50 ml), centrifuged at 600 g for 15 min and resuspended in 1 ml complete RPMI-1640. The concentration of the suspension was adjusted to 3 × 106 viable cells/ml and counted in a Newbauwer chamber.
Obtaining canine peritoneal macrophages
To obtain the peritoneal macrophages, the dogs were anesthetized with 2.5% Thiopental sodium (1 ml/kg) and sacrificed using a lethal dose (0.3 ml/Kg) of T-61® (Intervet). The animals were positioned in decumbency and the peritoneal cavity was disinfected using iodine-alcohol and washed with sterile saline, as described by Gonçalves et al. (2005) [12]. The washed peritoneal cell suspensions were adjusted to 3 × 106 cells/ml in culture medium (D-10 - DMM + 10% FCS + L-glutamine + penicillin-streptomycin).
Preparing parasites for assays for binding and survival in monocyte-macrophages and peritoneal macrophages
Leishmania (Leishmania) chagasi parasites (stationary phase – MHOM/BR/1972/BH6) were adjusted to 5 × 107 cells/ml in "phagocytosis buffer" (PB) culture medium (equal parts of Dulbecco's Modified Eagle Medium and Medium 199 supplemented with 1% BSA and 12.5 mM HEPES) [18].
Leishmania binding assays
The interactions between Leishmania chagasi promastigotesand canine peritoneal monocytes, monocyte-derived macrophages and peritoneal macrophages were measured as follows. Cells were added to 24-well plates containing 3 × 105 cells/100 μl. All binding assays were performed in triplicate in a PB consisting of equal parts of medium 199 and Dulbecco's modified Eagle medium (Mediatech) supplemented with 1% BSA [18]. Assays performed in the presence of serum were carried out at a concentration of 5% of AKRJ C5-deficient mouse serum (C5D) [22] with a final volume of 400 μl in each well.
The use of C5D serum obviates any problems associated with complement-mediated lysis of the parasites [1]. Promastigote forms of L. chagasi (3 × 106 or 5 × 106/well) were added to the monolayer cells. After 50 min incubation at 35°C, unbound promastigotes were removed by thorough washing. The monolayers and bound promastigotes were fixed with 2.5% glutaraldehyde and stained with 10% Giemsa. The number of promastigotes bound per well was deduced using an optical microscope, assuming 100 monocytes-macrophages/well. All the experiments were done in triplicate [20].
Survival assay
Assays to measure the internalization of promastigotes into canine monocyte-derived macrophages and peritoneal macrophages and visualization of amastigotes were performed in a similar way to the binding assays after the Leishmania had interacted with the cells for 48 h. The monolayers with intracytoplasmatic amastigotes in the macrophages were fixed with 2.5% glutaraldehyde and stained with 10% Giemsa. The number of parasitized macrophages and intracellular amastigotes was deduced by optical microscopy, assuming 100 macrophages/well. All experiments were done in triplicate [20].
Staining of parasites and binding assay for flow Cytometric analysis
Promastigote forms of Leishmania were resuspended in 1 ml PBS (5 × 107 parasites/ml) with 1 μl of CFSE (carboxyfluorescein diacetate succinimidyl ester – Molecular Probes C-1157) from a 2.8 μg/ml stock in DMSO (dimethyl sulfoxide), and incubated in a 37°C water bath for 10 min in the dark as described by Gonçalves et al. (2005) [12]. The CFSE-stained Leishmania chagasi promastigotes were allowed to interact with canine cells as follows: (1) monocytes obtained from five dogs naturally infected with L. chagasi; (2) peritoneal macrophages from three naturally-infected dogs and one experimentally-infected dog. These interactions took place in polypropylene tubes to avoid cell adhesion. Canine cells (100 μl, 3 × 105 cells) and 100 μl CFSE stained-Leishmania chagasi (5 × 106 cells) were combined in the tubes and maintained for 45–60 min at 37°C in a 5% CO2 atmosphere. The assays were performed in the presence of either normal serum or a final concentration of 5% C5-deficient mouse serum (from AKR/J mice) [22].
Specific staining with anti-CR3
Canine cells obtained as described above were stained with anti-CR3 (CD11b/CD18) rat anti-human CD18-RPE antibody (Serotec), which cross-reacts with canine cells. Nonconjugated, purified mouse anti-canine CD11b (Serotec) was conjugated using a Zenon tricolor kit (Molecular Probes – Z-25080), as described by the manufacturer, with the "C" compound that represents a 647 nm emission band and can be used with CFSE and R Phycoerythrin (RPE). The cells were incubated with labeled antibody solutions for 20 min at 4°C. After staining, the preparations were washed with 0.1% sodium azide in PBS, fixed with 200 μl 2% formaldehyde in PBS and kept at 4°C until data were acquired by flow cytometry (FACSVantage, Becton & Dickinson, San José, CA, USA).
Binding analysis by flow cytometry
The cells were run on an analytical flow cytometer equipped with a laser emitting at 488 nm (FACSVantage, Becton-Dickinson, San Diego, CA, USA). Whole cells were distinguished from fragments by gating based on the forward and side scatter signals. CFSE-stained promastigotes bound to macrophages were identified by their fluorescence intensities relative to those of uninfected cells using the FL1 (green) detector. Both the frequency of cells associated with promastigote forms of Leishmania and the intensity of cells associated with Leishmania were determined as described previously [12].
Statistical analysis
Results are given as a complete randomized design and the means from each group were compared using Student's t test; p values less than 0.05 were considered significant. All analyses were carried out using Prism 3.0 software.