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

Magez S, Geuskens M, Beschin A, del Favero H, Verschueren H, Lucas R, Pays E, de Baetselier P - J. Cell Biol. (1997)

Bottom Line: The specific uptake of the cytokine by the parasite results in a developmentally regulated loss of osmoregulatory capacity.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Immunology, Flanders Interuniversity Institute for Biotechnology, Vrije Universiteit Brussel, Belgium.

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.

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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.
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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.

Mentions: To localize TNF-α binding sites on intact parasites and to follow intracellular uptake of TNF-α, the cytokine was conjugated to 10-nm colloidal gold particles and subsequently incubated with monomorphic AnTat 1.1 trypanosomes at 30°C. The bulk of the TNF-α–gold labeling was localized in the flagellar pocket, where beads concentrated in coated pits (Fig. 5 a, arrowhead). Sporadically, TNFα–gold particles were found in association with the flagellum in the flagellar adhesion zone (Fig. 5, b and c), at the entrance of the flagellar pocket, or in association with tiny filamentous material at more distant regions of the flagellum (Fig. 5 d). After binding, gold labeled TNF-α was endocytosed through coated vesicles (Fig. 5 e). Coated vesicles containing TNF-α–gold particles were often seen fusing with larger electrolucent vacuoles wherein a cytoplasmic area was invaginated, assuming cup-like shapes (Fig. 5 f) which might look like rings in TEM (Fig. 5 g). Gold particles were also observed in tubular vesicular structures (Fig. 5, h and i, top) but more frequently in dilatations of the collecting membrane system described by Langreth and Balber (21) which contain electron-dense material (Fig. 5 i, lower part, j and k, middle). In parasites incubated for longer periods (1 or 2 h), gold particles were also localized in larger lysosome-like digestive vacuoles with more (Fig. 5 k, top, and l) or less (Fig. 5 m) electron-lucent contents. These vacuoles often sequestered areas of cytoplasm remaining probably in connection with the surrounding ground cytoplasm. As shown in Fig. 5 n, the vacuole from a partially lysed parasite contains some gold particles (arrow) in addition to several such cytoplasmic areas.


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

Magez S, Geuskens M, Beschin A, del Favero H, Verschueren H, Lucas R, Pays E, de Baetselier P - J. Cell Biol. (1997)

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.
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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.
Mentions: To localize TNF-α binding sites on intact parasites and to follow intracellular uptake of TNF-α, the cytokine was conjugated to 10-nm colloidal gold particles and subsequently incubated with monomorphic AnTat 1.1 trypanosomes at 30°C. The bulk of the TNF-α–gold labeling was localized in the flagellar pocket, where beads concentrated in coated pits (Fig. 5 a, arrowhead). Sporadically, TNFα–gold particles were found in association with the flagellum in the flagellar adhesion zone (Fig. 5, b and c), at the entrance of the flagellar pocket, or in association with tiny filamentous material at more distant regions of the flagellum (Fig. 5 d). After binding, gold labeled TNF-α was endocytosed through coated vesicles (Fig. 5 e). Coated vesicles containing TNF-α–gold particles were often seen fusing with larger electrolucent vacuoles wherein a cytoplasmic area was invaginated, assuming cup-like shapes (Fig. 5 f) which might look like rings in TEM (Fig. 5 g). Gold particles were also observed in tubular vesicular structures (Fig. 5, h and i, top) but more frequently in dilatations of the collecting membrane system described by Langreth and Balber (21) which contain electron-dense material (Fig. 5 i, lower part, j and k, middle). In parasites incubated for longer periods (1 or 2 h), gold particles were also localized in larger lysosome-like digestive vacuoles with more (Fig. 5 k, top, and l) or less (Fig. 5 m) electron-lucent contents. These vacuoles often sequestered areas of cytoplasm remaining probably in connection with the surrounding ground cytoplasm. As shown in Fig. 5 n, the vacuole from a partially lysed parasite contains some gold particles (arrow) in addition to several such cytoplasmic areas.

Bottom Line: The specific uptake of the cytokine by the parasite results in a developmentally regulated loss of osmoregulatory capacity.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Immunology, Flanders Interuniversity Institute for Biotechnology, Vrije Universiteit Brussel, Belgium.

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.

Show MeSH
Related in: MedlinePlus