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Toxoplasma gondii is an obligate intracellular parasite that can infect any nucleated host cell by a process of active penetration (7). Acute toxoplasmosis is characterized by dissemination and intracellular growth of the tachyzoite in a variety of host organs. In the innate phase of the immune response, nonspecific inflammatory cells, including neutrophils, monocytes, and dendritic cells, are elicited to the site of infection. The ensuing response of these cells allows for the establishment of the specific cell-mediated immune response that provides for long-term protection in the host against recurrent infection (13). Previous studies have reported that human neutrophils and monocytes become infected by tachyzoites. However, for each cell type there are conflicting reports of the subsequent fate of these intracellular parasites; both parasite stasis (14, 16, 30) and parasite division (15, 19) are described. In a recent study it was shown that there was markedly less uptake and division of tachyzoites in adherent monocytes than in nonadherent monocytes (9). In the present study the response of human peripheral blood leukocytes to tachyzoites was evaluated.

Infection of human leukocytes is parasite dose dependent. Dendritic cells, monocytes, lymphocytes, and neutrophils were isolated from peripheral blood from toxoplasma-seronegative donors under endotoxin-free conditions. To obtain dendritic cells, mononuclear cells were isolated from blood by density gradient centrifugation (2:1 [vol/vol] ratio of blood to 1.07 g of Ficoll-Hypaque per ml; 500 × g for 20 min). Washed cells were plated for 2 h at 37°C in tissue culture flasks, and then nonadherent cells were removed. The adherent cells were cultured in medium supplemented with 2,000 U of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; generously supplied by Immunex, Seattle, Wash.) and 20 ng of recombinant human interleukin-4 (rhIL-4; PeproTech Inc., Rocky Hill, N.J.) per ml. Every 3 days the medium in these flasks was supplemented with rhGM-CSF and rhIL-4. At day 8, the cells demonstrating dendritic cell morphology (nonadherent with projections) were harvested, and their phenotype was characterized by flow cytometry as described previously (27).

View larger version (125K): [in this window] [in a new window]   FIG. 1.   Infection of host cells is parasite dose dependent. Tachyzoites were added to monocytes, neutrophils, and lymphocytes (5 × 105/500 µl in 24-well tissue culture plates) or in vitro-cultured dendritic cells (27) (1.4 × 105/140 µl in 96-well tissue culture plates) at various cell-to-tachyzoite ratios. Triplicate cytospin preparations (100,000 cells centrifuged for 5 min at 700 rpm using a Shandon Cytospin 3) of each infected cell type were made 2 and 24 h postinfection. Cells were fixed and stained with Diff-Quik, and 200 to 300 cells were counted per slide. For each cell the number of tachyzoites per vacuole was determined. Cells with multiple vacuoles were scored for the highest number of tachyzoites per single vacuole (i.e., as if they contained a single vacuole), and the cell-to-tachyzoite ratios where multiple vacuoles occurred were recorded. Results from 24 h postinfection are reported as the mean + standard deviations of triplicate samples for lymphocytes, neutrophils, and monocytes and as the mean of duplicate samples for dendritic cells. Results are representative of two donors.

Monocytes and dendritic cells are permissive to rapid parasite division. Tachyzoites divide through a type of binary fission termed endodygeny, and a division time of 5 to 9 h is characteristic of any toxoplasma strain (21). To determine whether leukocytes were permissive to intracellular parasite division, cells infected overnight were scored for numbers of parasites per vacuole. Photomicrographs of these infected leukocytes are presented in Fig. 2. Large vacuoles containing small pieces of parasites, consistent with phagocytosis of parasites lysed extracellularly, were seen for a minority of monocytes and neutrophils, particularly at higher multiplicities of infection (Fig. 2A and C). The infected leukocytes were quantified for numbers of intracellular parasites. Figure 3 shows parasite division for each type of leukocyte. There is a striking difference between the parasite division time in dendritic cells and monocytes and that in neutrophils and lymphocytes. While dendritic cells and monocytes support the characteristic rapid parasite division time (21) with four to eight parasites per vacuole, neutrophils and lymphocytes are less permissive for parasite division, harboring mainly single parasites. For example, at a cell-to-parasite ratio of 1:4, 92 and 36% of monocytes and dendritic cells, respectively, had vacuoles containing two, four, or eight parasites (Fig. 3). In contrast, 46% of neutrophils were uninfected, 49% had one tachyzoite per vacuole, and 5% had two tachyzoites per vacuole (Fig. 3). At the same multiplicity of infection 81% of lymphocytes were uninfected, 15% had one tachyzoite per vacuole, and 4% had two tachyzoites per vacuole (Fig. 3). Between 18 and 48 h of infection monocytes and dendritic cells lysed following the egress of viable intracellular tachyzoites. For monocytes, the number of tachyzoites per vacuole was dependent on the multiplicity of infection.

View larger version (129K): [in this window] [in a new window]   FIG. 2.   Photomicrographs of cytospin preparations of tachyzoite-infected host cells. Host cells were incubated for 24 h with tachyzoites at a cell-to-parasite ratio of 1:4. Arrowheads indicate parasites. Arrows indicate vacuoles containing digesting tachyzoites. Magnification, ×630. Bar = 2 µm. (A) Monocytes showing rapid tachyzoite division, as evidenced by up to eight tachyzoites per vacuole (see arrowheads). (B) Dendritic cells showing rapid tachyzoite division. (C) Neutrophils showing slow tachyzoite division, as seen by fewer than two tachyzoites per vacuole. rbc, red blood cells. (D) Lymphocytes showing slow tachyzoite division, even though tachyzoites in the contaminating monocyte were dividing rapidly. Photomicrographs were made using Kodak Elite chrome ASA 100 slide film, and 35-mm slides were scanned into Adobe Photoshop files using a SprintScan 35 (Polaroid Corp., Cambridge, Mass.).

View larger version (48K): [in this window] [in a new window]   FIG. 3.   Intracellular tachyzoites divide at a rapid rate in monocytes and dendritic cells but not in neutrophils or lymphocytes. Host cells were incubated with parasites at various multiplicities of infection for 24 h and the number of tachyzoites per host intracellular vacuole was determined by light microscopy of cytospin preparations. Results are reported as the cumulative percentage of cells that are uninfected (), or have one (), two (), four (), or eight () tachyzoites per vacuole. Results are representative of two donors.

Parasites in infected neutrophils have a longer division time. The majority of infected neutrophils contained one whole parasite per vacuole (Fig. 2C) and the intracellular parasite was often distended. To determine whether distended parasites could have been dividing, cytospins of infected neutrophils were fixed in 2% formaldehyde for 30 min and permeabilized in ice-cold acetone for 15 min. Parasites were stained with a tachyzoite-specific antibody (30 min of incubation with 10 µg of fluorescein isothiocyanate-conjugated anti-SAG-1 rabbit polyclonal immunoglobulin G, made in our laboratory, per ml), and cell nuclei were stained with propidium iodide (5 min of incubation with 2.5 µg/ml in phosphate-buffered saline). Cells were examined using a Zeiss Axiophot microscope equipped with a fluorescein isothiocyanate-tetramethyl rhodamine isocyanate filter set. For 24-h-infected neutrophils, all distended tachyzoites contained two daughter nuclei, consistent with endodyogeny (21). Moreover, when the samples were examined 48 h postinfection, the frequency of infected neutrophils containing two tachyzoites per vacuole was found to be increased (data not shown). These results suggest that parasite division time in neutrophils is markedly longer than the typical 5 to 9 h seen for nonadherent monocytes and dendritic cells. Similar results were seen for intracellular tachyzoites of lymphocytes (data not shown).

Parasites in infected neutrophils can reinfect fibroblasts, where they show a rapid division time. To determine whether parasites that exhibit a longer division time in neutrophils also have a longer division time in a more permissive cell type, infected neutrophils (sorted free of extracellular parasites using flow cytometry) were added to human fibroblasts. For these experiments neutrophils were infected overnight with parasites at a cell-to-parasite ratio of 1:1 and then stained for cell surface expression of Fc receptor III (murine monoclonal antibody 3G8 supernatant; generous gift of M. Fanger) and examined by flow cytometry. Fc receptor III-stained neutrophils fell into two populations, a brightly stained viable population and a dimly stained nonviable population. A gate was set on the brightly stained cells, and cells were sorted using a FacStar flow cytometer (Becton Dickinson) under sterile conditions. Cytospin preparations of sorted brightly stained cells showed them to be 100% viable, 12% tachyzoite infected, and free of extracellular tachyzoites, whereas dimly stained cells were 100% nonviable and uninfected. Serial dilutions of brightly stained neutrophils were added to small flasks of confluent human fibroblasts and incubated at 37°C. After 4 days, fibroblasts were examined for plaques caused by tachyzoite infection and division (24). Incubation of fibroblasts with 120,000 infected neutrophils (10⁶ total neutrophils) resulted in the formation of 25,000 plaques. The efficiency of plating, i.e., the ability of extracellular PLK strain tachyzoites to make plaques, was 50%. Hence, at least 40% of the intracellular tachyzoites within neutrophils were infective. Moreover, the majority of infected fibroblasts contained four to eight parasites per vacuole.