Specific uptake of tumor necrosis factor-alpha is involved in growth control of Trypanosoma brucei

Abstract

Trypanosoma brucei is lysed by tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent way, involving specific binding of the cytokine to a trypanosomal glycoprotein present in the flagellar pocket of the parasite. TNF-alpha-gold particles are endocytosed via coated pits and vesicles and are directed towards lysosome-like digestive organelles. The specific uptake of the cytokine by the parasite results in a developmentally regulated loss of osmoregulatory capacity. TNF-alpha specific lysis is prevented when lysis assays are performed at a temperature <26 degrees C, despite uptake of the cytokine. Inhibition of lysis is also observed when a lysosomotropic agent is added during the first 2 h of incubation. Both monomorphic and pleomorphic trypanosomes are lysed but only when isolated during the peak of parasitaemia. Lysis is not observed with early infection stage parasites or procyclic (insect-specific) forms. Anti-TNF-alpha treatment of T. brucei-infected mice reveals a dramatic increase in parasitaemia in the blood circulation, the spleen, the lymph nodes, and the peritoneal cavity. These data suggest that in the mammalian host, TNF-alpha is involved in the growth control of T. brucei.

Figure 1

Lysis of bloodstream forms of T. brucei in function of time (hours) and concentration of TNF-α: 1 U/ml (□), 10 U/ml (▪), 10² U/ml (○), 10³ U/ml (•), 10⁴ U/ml (⋄), 10⁵ U/ml (♦), and 10⁶ U/ml (+). Trypanosomes were isolated from the blood of an infected mouse and incubated for up to 8 h in PSG (pH 8.0) at 30°C. Lysis assays were performed, and the percentage of lysis was calculated as described in Materials and Methods. Each concentration of TNF-α was tested in at least three independent experiments, and a representative experiment is shown.

Figure 2

Morphological analysis by TEM and FSC of TNF-α–induced trypanolysis in function of time (a, 1 h, b, 3 h, c, 4 h, d, 5 h). Purified trypanosomes were incubated for various periods of time in PSG (pH 8.0) in the presence or absence of 10⁵ U/ ml TNF-α. (A) TEM analysis of TNF-α–treated T. brucei parasites. Swelling of organelles and lysis of parasites was recorded from 4 h of incubation onwards (Ac). (B) TEM analysis of nontreated T. brucei parasites. No lysis was observed. (C) FSC of TNF-α–treated T. brucei parasites. A major shift to the left of the FSC spectrum was observed from 4 h of incubation onwards (Cc). Bar, 1 μm.

Figure 2

Morphological analysis by TEM and FSC of TNF-α–induced trypanolysis in function of time (a, 1 h, b, 3 h, c, 4 h, d, 5 h). Purified trypanosomes were incubated for various periods of time in PSG (pH 8.0) in the presence or absence of 10⁵ U/ ml TNF-α. (A) TEM analysis of TNF-α–treated T. brucei parasites. Swelling of organelles and lysis of parasites was recorded from 4 h of incubation onwards (Ac). (B) TEM analysis of nontreated T. brucei parasites. No lysis was observed. (C) FSC of TNF-α–treated T. brucei parasites. A major shift to the left of the FSC spectrum was observed from 4 h of incubation onwards (Cc). Bar, 1 μm.

Figure 3

Binding of ¹²⁵ITNF-α to bloodstream-form and procyclic trypanosomes. (a) Bloodstream-form trypanosomes (▪) and procyclic trypanosomes (□) were incubated in the presence of different molar concentrations of ¹²⁵I-TNF-α. Bloodstream forms were also incubated with ¹²⁵I-TNF-α in the presence of a 100-fold molar excess of cold TNF-α (•). The number of bound molecules per cell was plotted in function of the dose of ¹²⁵I-TNF-α added. (b) Specific ¹²⁵I-TNF-α binding to bloodstream-form trypanosomes. (c) Scatchard plot presentation of the same results. All the results shown are from one representative experiment.

Figure 3

Binding of ¹²⁵ITNF-α to bloodstream-form and procyclic trypanosomes. (a) Bloodstream-form trypanosomes (▪) and procyclic trypanosomes (□) were incubated in the presence of different molar concentrations of ¹²⁵I-TNF-α. Bloodstream forms were also incubated with ¹²⁵I-TNF-α in the presence of a 100-fold molar excess of cold TNF-α (•). The number of bound molecules per cell was plotted in function of the dose of ¹²⁵I-TNF-α added. (b) Specific ¹²⁵I-TNF-α binding to bloodstream-form trypanosomes. (c) Scatchard plot presentation of the same results. All the results shown are from one representative experiment.

Figure 3

Binding of ¹²⁵ITNF-α to bloodstream-form and procyclic trypanosomes. (a) Bloodstream-form trypanosomes (▪) and procyclic trypanosomes (□) were incubated in the presence of different molar concentrations of ¹²⁵I-TNF-α. Bloodstream forms were also incubated with ¹²⁵I-TNF-α in the presence of a 100-fold molar excess of cold TNF-α (•). The number of bound molecules per cell was plotted in function of the dose of ¹²⁵I-TNF-α added. (b) Specific ¹²⁵I-TNF-α binding to bloodstream-form trypanosomes. (c) Scatchard plot presentation of the same results. All the results shown are from one representative experiment.

Figure 4

The binding between TNF-α and total trypanosome lysate (1) or N-glycosidase F–treated trypanosome lysate (2) was analyzed using a biosensor. Sample injection was done at t = 0, and lysate was allowed to bind to a TNF-α coating on aminosilane. Free lysate was removed by a PBS wash after 400 s, and dissociation of the bound lysate was recorded. Both binding and dissociation are measured as a shift in laser reflection angle (Arc sec) as function of time (sec).

Figure 5

Localization by TEM of TNF-α binding and internalization at 30°C by T. brucei. TNF-α was conjugated to 10-nm gold particles as described in Materials and Methods. Cells were incubated with TNF-α–gold particles at a concentration of ∼10⁵ U/ml. (a) TNF-α–gold particles binding in the flagellar pocket (fp) and concentrated in a coated pit formed by its limiting membrane (arrowhead). (b and c) TNF-α– gold particles visible at and near the contact zone between the flagellum and the cell body. (d) TNF-α–gold particles associated with the flagellum. (e) TNF-α–gold particles present in the lumen of the flagellar pocket and two vesicles in the cytoplasm. (f) A coated vesicle (arrowhead) in continuity (arrow) with a vacuole containing TNF-α–gold particles. (g) A vesicle containing TNF-α–gold particles fusing with a vacuole surrounding a cytoplasmic area. (h) TNFα–gold particles present in tubular vesicular structures in close proximity to the coated region of the flagellar pocket membrane (arrowhead). (i) A flattened vesicle (top), and another one with a more electron-dense content, containing TNF-α–gold particles. (j) A dilatation of the collecting membrane system with electron-opaque lumen, containing TNF-α–gold particles, seen in continuity (arrows) with tubular structures. (k) An electron-lucent vacuole containing a few gold particles and an electronopaque vacuole filled with TNF-α–gold particles are visible near the parasite surface. (l) Two groups of TNF-α–gold particles present in a large electron-lucent vacuole surrounding a cytoplasmic area. (m) A large digestive vacuole containing dispersed gold particles. (n) A vacuole surrounding several cytoplasmic areas and containing a group of gold particles (arrow). (o) Flagellar pocket of a procyclic form of T. brucei, whose limiting membrane forms a coated pit (arrow). Observations a–j were done within the first of our TNF-α incubations. Observations k–o were made 2 h after the start of the TNF-α incubation. Bars, 0.1 μm.

Figure 6

TNF-α–mediated lysis of bloodstream forms of T. brucei as function of temperature and time. A lysis assay was performed using a TNF-α concentration of 10⁴ U/ml. Samples were kept at the indicated temperatures for various periods of time, up till the moment that a plateau of TNF-α–specific lysis was reached. The percentage of lysis was calculated as described in Materials and Methods.

Figure 7

Morphological characteristics of temperature dependent TNF-α–mediated lysis of bloodstream forms of T. brucei. Lysis assays were performed for 5 h at 30° and 21°C as described in Materials and Methods, using a TNF-α concentration of 10⁴ U/ml. (a) At 30°C no background lysis was observed in the absence of TNF-α. (b). In the presence of the cytokine, most parasites were lysed after the 5-h incubation period. Remaining cells had a ghostlike appearance (arrow) or showed an abnormal swollen morphology (arrowhead). At 21°C, no signs of altered morphology or lysis were observed in the absence (c) or presence (d) of TNF-α. Bar, 20 μm.

Figure 8

Transmission electron microscopy analysis of intracellular uptake of TNF-α–gold particles and lysis of bloodstream forms of T. brucei, isolated at the early stage and the peak of the parasitaemia. Cells were incubated with TNF-α–gold particles at a concentration of ∼10⁵ U/ml. (a) Peak stage parasites were incubated at 30°C with TNF-α–gold particles for a total period of 4 h as described in Materials and Methods. TNF-α–gold particles are observed in a vacuole of an apparent intact parasite (inset, arrowhead), while other cells are completely lysed. A large disruption in the membrane of the TNF-α–gold containing vesicle is indicated in the inset (arrow). m, mitochondria. (b) Peak stage parasites were incubated at 17°C with TNF-α–gold particles for a total period of 4 h. TNF-α–gold particles are observed in two vacuoles (center inset, arrowhead). No lysed cells were observed under these experimental conditions. (c) Early-stage T. brucei bloodstream forms were incubated with TNF-α–gold particles for a total period of 4 h. TNF-α–gold particles are observed in a vacuole surrounding a cytoplasmic area and in a smaller vesicle (bottom inset, arrowheads). No lysis was observed under these experimental conditions. Bars: (a–c) 1 μm, (inset) 0.1 μm.

Figure 9

Inhibition of TNFα–mediated trypanolysis by NH₄Cl. Lysis assays were carried out as described in Materials and Methods. (a) NH₄Cl effects on lysis of T. brucei as function of concentration. Trypanosomes were incubated at 30°C in the presence of 10⁴ U/ml TNF-α and different concentrations NH₄Cl. (b) Effects of NH₄Cl on trypanolysis as function of addition after preincubation of parasites with TNF-α. Trypanosomes were incubated for 5 h at 30°C in the presence of 10⁴ U/ml TNF-α. Every hour NH₄Cl was added to one sample to a final concentration of 1 mM. The percentage of lysis inhibition was calculated compared to a control lysis of 10⁴ U/ml in the absence of NH₄Cl.

Figure 9

Inhibition of TNFα–mediated trypanolysis by NH₄Cl. Lysis assays were carried out as described in Materials and Methods. (a) NH₄Cl effects on lysis of T. brucei as function of concentration. Trypanosomes were incubated at 30°C in the presence of 10⁴ U/ml TNF-α and different concentrations NH₄Cl. (b) Effects of NH₄Cl on trypanolysis as function of addition after preincubation of parasites with TNF-α. Trypanosomes were incubated for 5 h at 30°C in the presence of 10⁴ U/ml TNF-α. Every hour NH₄Cl was added to one sample to a final concentration of 1 mM. The percentage of lysis inhibition was calculated compared to a control lysis of 10⁴ U/ml in the absence of NH₄Cl.

Figure 10

Trypanolytic activity of TNF-α on both monomorphic and pleomorphic bloodstream forms of T. brucei. Lysis assays were carried out at 30°C in the presence of different concentrations of TNF-α as described in Materials and Methods. (a) Monomorphic T. brucei parasites, isolated at the early stage (▪) and the peak (□) of the parasitaemia, were incubated with TNF-α. The percentage of TNF-α–specific lysis was calculated as described in Materials and Methods. (b) Pleomorphic T. brucei parasites, isolated at the early stage (▪) and peak (□) of the parasitaemia, were incubated with TNF-α, the same way as the monomorphic parasites.

Figure 10

Trypanolytic activity of TNF-α on both monomorphic and pleomorphic bloodstream forms of T. brucei. Lysis assays were carried out at 30°C in the presence of different concentrations of TNF-α as described in Materials and Methods. (a) Monomorphic T. brucei parasites, isolated at the early stage (▪) and the peak (□) of the parasitaemia, were incubated with TNF-α. The percentage of TNF-α–specific lysis was calculated as described in Materials and Methods. (b) Pleomorphic T. brucei parasites, isolated at the early stage (▪) and peak (□) of the parasitaemia, were incubated with TNF-α, the same way as the monomorphic parasites.

Figure 11

TNF-α–mediated lysis of bloodstream forms of T. brucei is preceded by the destruction of their lysosome-like organelles. Both early and late stage trypanosomes were incubated for 3 h at 30°C with the lysosomal marker LysoTracker™ in the absence or presence of 10⁴ U/ml TNF-α. Early stage parasites showed a normal morphology (Aa) and a localized lysosomal staining (Ba). In the presence of TNF-α, both the morphology (Ab) and lysosome staining (Bb) are unaltered. Late stage trypanosomes also show a normal morphology (Ac) and lysosome coloration (Bc) in the absence of TNF-α. In the presence of TNF-α (Ad), some parasites are lysed (arrow) or have an abnormal morphology (arrowhead) and show intracellular diffusion of the lysosome marker (Bd). Bar, 10 μm.

Figure 12

Specific inhibition of TNF-α–mediated lysis of bloodstream forms of T. brucei by anti-TIP antibodies. (a) Freshly isolated bloodstream-form trypanosomes were incubated for up to 8 h in PSG (pH 8.0) at 30°C in the presence of different concentrations of TNF-α. Trypanolysis was calculated as described in Materials and Methods (▪). Lysis was inhibited by preincubation of TNF-α with three different TIP-specific antibodies. The polyclonal antibody (□) as well as both monoclonal antibodies 1E12 (•) and 24C11 (○), all inhibited the TNF-α–specific trypanolysis to approximately the same extent. (b) None of the anti-TIP antibodies inhibited TNF-α–mediated lysis of the TNF-α–sensitive L929 cell line. The same symbols are used as in panel (a).

Figure 12

Specific inhibition of TNF-α–mediated lysis of bloodstream forms of T. brucei by anti-TIP antibodies. (a) Freshly isolated bloodstream-form trypanosomes were incubated for up to 8 h in PSG (pH 8.0) at 30°C in the presence of different concentrations of TNF-α. Trypanolysis was calculated as described in Materials and Methods (▪). Lysis was inhibited by preincubation of TNF-α with three different TIP-specific antibodies. The polyclonal antibody (□) as well as both monoclonal antibodies 1E12 (•) and 24C11 (○), all inhibited the TNF-α–specific trypanolysis to approximately the same extent. (b) None of the anti-TIP antibodies inhibited TNF-α–mediated lysis of the TNF-α–sensitive L929 cell line. The same symbols are used as in panel (a).