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Immune adherence-mediated opsonophagocytosis: the mechanism of Leishmania infection.

Domínguez M, Toraño A - J. Exp. Med. (1999)

Bottom Line: We propose that adaptation to the immune adherence mechanism aids Leishmania survival, promoting rapid promastigote phagocytosis by leukocytes.This facilitates host colonization and may represent the parasite's earliest survival strategy.In light of this mechanism, it is unlikely that infection-blocking vaccines can be developed.

View Article: PubMed Central - PubMed

Affiliation: Servicio de Inmunología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, E-28220 Madrid, Spain.

ABSTRACT
To mimic the sandfly pool feeding process and characterize the cellular and biochemical events that occur during the early stages of promastigote-host interaction, we developed an ex vivo model of human blood infection with Leishmania promastigotes. Within 30 s of blood contact, Leishmania promastigotes bind natural anti-Leishmania antibodies, which then activate the classical complement pathway and opsonization by the third component of complement. The opsonized promastigotes undergo an immune adherence reaction and bind quantitatively to erythrocyte CR1 receptors; opsonized Leishmania amastigotes also bind to erythrocytes. Progression of infection implies promastigote transfer from erythrocytes to acceptor blood leukocytes. After 10 min of ex vivo infection, 25% of all leukocytes contain intracellular parasites, indicating that blood cells are the early targets for the invading promastigotes. We propose that adaptation to the immune adherence mechanism aids Leishmania survival, promoting rapid promastigote phagocytosis by leukocytes. This facilitates host colonization and may represent the parasite's earliest survival strategy. In light of this mechanism, it is unlikely that infection-blocking vaccines can be developed.

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Promastigote opsonization kinetics in NHS. (A)  Promastigote complement activation pathway in NHS (closed  symbols) and Mg-EGTA–treated  NHS (open symbols). Duplicate  aliquots (107 cells) of L. donovani  (•, ○) and L. amazonensis (▴,  ▵) promastigotes were incubated at 37°C in 25% NHS or 10  mM EGTA/7 mM Mg2Cl-treated 25% NHS, for 0, 0.25,  0.5, 1, 2, 3, 4, 5, 7.5, 10, and 15  min. Promastigotes were then  washed twice by centrifugation  (11,000 g, 1 min) in cold PFS  and incubated 1 h on ice with  mAb [125I]SIM 27-49. After incubation, promastigotes were washed twice (11,000 g, 1 min) and [125I]SIM 27-49 cpm determined. C3 deposition is expressed as a percentage of the point of maximum [125I]SIM 27-49 binding. Data are the mean value of duplicate samples taken from one representative  experiment of three performed. (B) IgM binding kinetics to L. donovani and L. amazonensis promastigotes. Duplicate aliquots (107 cells) of L. donovani (•)  and L. amazonensis (▴) promastigotes were incubated at 37°C in 25% NHS for 0, 0.25, 0.5, 1, 2, 3, 4, 5, 7, and 10 min. After reaction, promastigotes  were washed three times (11,000 g, 1 min) with cold PFS and incubated for 1 h on ice with 125I-goat anti-μ antibody. Results are expressed as the percentage of IgM binding relative to the point of maximum binding. Each point value represents the mean of duplicate samples taken from a representative  experiment of five performed.
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Figure 3: Promastigote opsonization kinetics in NHS. (A) Promastigote complement activation pathway in NHS (closed symbols) and Mg-EGTA–treated NHS (open symbols). Duplicate aliquots (107 cells) of L. donovani (•, ○) and L. amazonensis (▴, ▵) promastigotes were incubated at 37°C in 25% NHS or 10 mM EGTA/7 mM Mg2Cl-treated 25% NHS, for 0, 0.25, 0.5, 1, 2, 3, 4, 5, 7.5, 10, and 15 min. Promastigotes were then washed twice by centrifugation (11,000 g, 1 min) in cold PFS and incubated 1 h on ice with mAb [125I]SIM 27-49. After incubation, promastigotes were washed twice (11,000 g, 1 min) and [125I]SIM 27-49 cpm determined. C3 deposition is expressed as a percentage of the point of maximum [125I]SIM 27-49 binding. Data are the mean value of duplicate samples taken from one representative experiment of three performed. (B) IgM binding kinetics to L. donovani and L. amazonensis promastigotes. Duplicate aliquots (107 cells) of L. donovani (•) and L. amazonensis (▴) promastigotes were incubated at 37°C in 25% NHS for 0, 0.25, 0.5, 1, 2, 3, 4, 5, 7, and 10 min. After reaction, promastigotes were washed three times (11,000 g, 1 min) with cold PFS and incubated for 1 h on ice with 125I-goat anti-μ antibody. Results are expressed as the percentage of IgM binding relative to the point of maximum binding. Each point value represents the mean of duplicate samples taken from a representative experiment of five performed.

Mentions: Complement activation by Leishmania promastigotes is believed to occur mainly through the alternative pathway (3). The rapid kinetics observed for the IA reaction suggested that this is not the case, for which the activation pathway of the complement cascade by L. amazonensis and L. donovani promastigotes was studied. Classical pathway activation requires both Ca2+ and Mg2+, whereas the alternative pathway requires only Mg2+; differential cation chelation thus permits identification of the activation pathway. In NHS adjusted to 10 mM EGTA and 7 mM MgCl2, Ca2+ is effectively chelated but free Mg2+ is available for alternative pathway activation. The course of C3 deposition on the promastigote surface is shown in the presence of 25% NHS or Mg-EGTA–treated 25% NHS (Fig. 3 A). After 2–3 min of incubation, C3 binding reaches a maximum and remains unchanged for the remainder of the time course. In contrast, C3 deposition in Mg-EGTA–treated serum follows a slower course that varies depending on the Leishmania species studied.


Immune adherence-mediated opsonophagocytosis: the mechanism of Leishmania infection.

Domínguez M, Toraño A - J. Exp. Med. (1999)

Promastigote opsonization kinetics in NHS. (A)  Promastigote complement activation pathway in NHS (closed  symbols) and Mg-EGTA–treated  NHS (open symbols). Duplicate  aliquots (107 cells) of L. donovani  (•, ○) and L. amazonensis (▴,  ▵) promastigotes were incubated at 37°C in 25% NHS or 10  mM EGTA/7 mM Mg2Cl-treated 25% NHS, for 0, 0.25,  0.5, 1, 2, 3, 4, 5, 7.5, 10, and 15  min. Promastigotes were then  washed twice by centrifugation  (11,000 g, 1 min) in cold PFS  and incubated 1 h on ice with  mAb [125I]SIM 27-49. After incubation, promastigotes were washed twice (11,000 g, 1 min) and [125I]SIM 27-49 cpm determined. C3 deposition is expressed as a percentage of the point of maximum [125I]SIM 27-49 binding. Data are the mean value of duplicate samples taken from one representative  experiment of three performed. (B) IgM binding kinetics to L. donovani and L. amazonensis promastigotes. Duplicate aliquots (107 cells) of L. donovani (•)  and L. amazonensis (▴) promastigotes were incubated at 37°C in 25% NHS for 0, 0.25, 0.5, 1, 2, 3, 4, 5, 7, and 10 min. After reaction, promastigotes  were washed three times (11,000 g, 1 min) with cold PFS and incubated for 1 h on ice with 125I-goat anti-μ antibody. Results are expressed as the percentage of IgM binding relative to the point of maximum binding. Each point value represents the mean of duplicate samples taken from a representative  experiment of five performed.
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Figure 3: Promastigote opsonization kinetics in NHS. (A) Promastigote complement activation pathway in NHS (closed symbols) and Mg-EGTA–treated NHS (open symbols). Duplicate aliquots (107 cells) of L. donovani (•, ○) and L. amazonensis (▴, ▵) promastigotes were incubated at 37°C in 25% NHS or 10 mM EGTA/7 mM Mg2Cl-treated 25% NHS, for 0, 0.25, 0.5, 1, 2, 3, 4, 5, 7.5, 10, and 15 min. Promastigotes were then washed twice by centrifugation (11,000 g, 1 min) in cold PFS and incubated 1 h on ice with mAb [125I]SIM 27-49. After incubation, promastigotes were washed twice (11,000 g, 1 min) and [125I]SIM 27-49 cpm determined. C3 deposition is expressed as a percentage of the point of maximum [125I]SIM 27-49 binding. Data are the mean value of duplicate samples taken from one representative experiment of three performed. (B) IgM binding kinetics to L. donovani and L. amazonensis promastigotes. Duplicate aliquots (107 cells) of L. donovani (•) and L. amazonensis (▴) promastigotes were incubated at 37°C in 25% NHS for 0, 0.25, 0.5, 1, 2, 3, 4, 5, 7, and 10 min. After reaction, promastigotes were washed three times (11,000 g, 1 min) with cold PFS and incubated for 1 h on ice with 125I-goat anti-μ antibody. Results are expressed as the percentage of IgM binding relative to the point of maximum binding. Each point value represents the mean of duplicate samples taken from a representative experiment of five performed.
Mentions: Complement activation by Leishmania promastigotes is believed to occur mainly through the alternative pathway (3). The rapid kinetics observed for the IA reaction suggested that this is not the case, for which the activation pathway of the complement cascade by L. amazonensis and L. donovani promastigotes was studied. Classical pathway activation requires both Ca2+ and Mg2+, whereas the alternative pathway requires only Mg2+; differential cation chelation thus permits identification of the activation pathway. In NHS adjusted to 10 mM EGTA and 7 mM MgCl2, Ca2+ is effectively chelated but free Mg2+ is available for alternative pathway activation. The course of C3 deposition on the promastigote surface is shown in the presence of 25% NHS or Mg-EGTA–treated 25% NHS (Fig. 3 A). After 2–3 min of incubation, C3 binding reaches a maximum and remains unchanged for the remainder of the time course. In contrast, C3 deposition in Mg-EGTA–treated serum follows a slower course that varies depending on the Leishmania species studied.

Bottom Line: We propose that adaptation to the immune adherence mechanism aids Leishmania survival, promoting rapid promastigote phagocytosis by leukocytes.This facilitates host colonization and may represent the parasite's earliest survival strategy.In light of this mechanism, it is unlikely that infection-blocking vaccines can be developed.

View Article: PubMed Central - PubMed

Affiliation: Servicio de Inmunología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, E-28220 Madrid, Spain.

ABSTRACT
To mimic the sandfly pool feeding process and characterize the cellular and biochemical events that occur during the early stages of promastigote-host interaction, we developed an ex vivo model of human blood infection with Leishmania promastigotes. Within 30 s of blood contact, Leishmania promastigotes bind natural anti-Leishmania antibodies, which then activate the classical complement pathway and opsonization by the third component of complement. The opsonized promastigotes undergo an immune adherence reaction and bind quantitatively to erythrocyte CR1 receptors; opsonized Leishmania amastigotes also bind to erythrocytes. Progression of infection implies promastigote transfer from erythrocytes to acceptor blood leukocytes. After 10 min of ex vivo infection, 25% of all leukocytes contain intracellular parasites, indicating that blood cells are the early targets for the invading promastigotes. We propose that adaptation to the immune adherence mechanism aids Leishmania survival, promoting rapid promastigote phagocytosis by leukocytes. This facilitates host colonization and may represent the parasite's earliest survival strategy. In light of this mechanism, it is unlikely that infection-blocking vaccines can be developed.

Show MeSH
Related in: MedlinePlus