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Kinetic analysis of ex vivo human blood infection by Leishmania.

Moreno I, Domínguez M, Cabañes D, Aizpurua C, Toraño A - PLoS Negl Trop Dis (2010)

Bottom Line: C3-promastigote binding is a key step in opsonization; nascent C3-promastigotes are the substrate for two simultaneous reactions, C3-promastigote immune adherence (IA) to erythrocytes and complement-mediated promastigote killing.The k(+1) for IA was 75-fold greater than that for promastigote killing, showing that IA facilitates promastigote endocytosis and circumvents lysis.At 5 min post-infection, when reaction velocity is still linear and promastigote concentration is not limiting, 17.4% of granulocytes and 10.7% of monocytes had bound promastigotes, of which approximately 50% and approximately 25%, respectively, carried surface-bound (live) or internalized (live and dead) leishmanias.

View Article: PubMed Central - PubMed

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

ABSTRACT
The leishmanioses, vector-borne diseases caused by the trypanosomatid protozoan Leishmania, are transmitted to susceptible mammals by infected phlebotomine sand flies that inoculate promastigotes into hemorrhagic pools created in host skin. We assumed that promastigotes are delivered to a blood pool, and analyzed early promastigote interactions (0-5 min) with host components, which lead to parasite endocytosis by blood leukocytes, and to host infection. Promastigotes were incubated with NHS or with heparinized blood in near-physiological conditions, and we used cell radioimmunoassay and flow cytometry to measure the on-rate constants (k(+1)) of promastigote interactions with natural opsonins and erythrocytes. We obtained quantitative data for parasitized cells to determine the time-course of promastigote binding and internalization by blood leukocytes. In these reactions, promastigotes bind natural opsonins, immune adhere to erythrocytes and activate complement cytolysis, which kills approximately 95% of promastigotes by 2 min post-infection. C3-promastigote binding is a key step in opsonization; nascent C3-promastigotes are the substrate for two simultaneous reactions, C3-promastigote immune adherence (IA) to erythrocytes and complement-mediated promastigote killing. The k(+1) for IA was 75-fold greater than that for promastigote killing, showing that IA facilitates promastigote endocytosis and circumvents lysis. At 5 min post-infection, when reaction velocity is still linear and promastigote concentration is not limiting, 17.4% of granulocytes and 10.7% of monocytes had bound promastigotes, of which approximately 50% and approximately 25%, respectively, carried surface-bound (live) or internalized (live and dead) leishmanias. Of other leukocyte types, 8.5% of B cells bound but did not internalize promastigotes, and T cells, NK cells and CD209(+) dendritic cells did not bind parasites. These data show that, once in contact with blood, promastigote invasion of human leukocytes is an extremely rapid and efficient reaction, and suggest that the IA reaction constitutes a central strategy for this parasite in subverting host innate immune defenses.

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Kinetics of leukocyte binding and internalization of L. amazonensis and L. donovani promastigotes in human blood.Aliquots of heparinized human blood were infected with CMFDA-labeled L. amazonensis or L. donovani promastigotes and incubated (37°C) for various times (0–60 min). Samples were then processed as indicated in Methods. To calculate the percentage of granulocytes and monocytes that bound and internalized parasites, cells were gated by SSC vs. FSC and plotted independently in a secondary plot of SSC vs. FL1 (green, 530 nm). The percentage of cells that internalized promastigotes was determined using trypan blue to quench the extracellular fluorescence emitted by cell-bound complement-killed CMFDA-labeled promastigotes. (A) For L. amazonensis promastigotes: (a) Time course of granulocyte binding and internalization (•) or internalization (○); (b) data from inset (shaded area) in (a); (c) time course of monocyte binding and internalization (•) or internalization (○); (d) data from inset (shaded area) in (c). (B) Data as above (a–d) are shown for L. donovani. Results from seven (L. donovani) and ten (L. amazonensis) experiments with blood from six donors.
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pntd-0000743-g006: Kinetics of leukocyte binding and internalization of L. amazonensis and L. donovani promastigotes in human blood.Aliquots of heparinized human blood were infected with CMFDA-labeled L. amazonensis or L. donovani promastigotes and incubated (37°C) for various times (0–60 min). Samples were then processed as indicated in Methods. To calculate the percentage of granulocytes and monocytes that bound and internalized parasites, cells were gated by SSC vs. FSC and plotted independently in a secondary plot of SSC vs. FL1 (green, 530 nm). The percentage of cells that internalized promastigotes was determined using trypan blue to quench the extracellular fluorescence emitted by cell-bound complement-killed CMFDA-labeled promastigotes. (A) For L. amazonensis promastigotes: (a) Time course of granulocyte binding and internalization (•) or internalization (○); (b) data from inset (shaded area) in (a); (c) time course of monocyte binding and internalization (•) or internalization (○); (d) data from inset (shaded area) in (c). (B) Data as above (a–d) are shown for L. donovani. Results from seven (L. donovani) and ten (L. amazonensis) experiments with blood from six donors.

Mentions: The time course of granulocyte and monocyte binding of promastigotes was linear in the first 5 to 10 min of infection. Promastigote concentration subsequently became limiting and the kinetics followed a hyperbolic course; the binding reaction was complete by 60 min (Fig. 6). After 30 min incubation, 39.2% of granulocytes (∼94% of total binding) had already bound L. amazonensis and 45.1% (∼90% of total binding) had bound L. donovani promastigotes. At this time, 23.5% of monocytes (∼76% of total binding) had bound L. amazonensis and 20.2% (∼75% of total binding), L. donovani promastigotes. We analyzed leukocyte binding and promastigote internalization in the period between 0 and 5 min, when the velocity of promastigote binding is proportional with time and the cytolytic activity of complement on Leishmania is probably not yet complete (Fig. 6A,B; insets). After 3 min incubation, the ratio of cells that bound∶internalized promastigotes was 12.9∶5.4% for granulocytes and 8.9∶2% for monocytes (mean value of L. amazonensis and L. donovani); after 5 min, these values were 17.4∶8.4% for granulocytes and 10.7∶2.6% for monocytes.


Kinetic analysis of ex vivo human blood infection by Leishmania.

Moreno I, Domínguez M, Cabañes D, Aizpurua C, Toraño A - PLoS Negl Trop Dis (2010)

Kinetics of leukocyte binding and internalization of L. amazonensis and L. donovani promastigotes in human blood.Aliquots of heparinized human blood were infected with CMFDA-labeled L. amazonensis or L. donovani promastigotes and incubated (37°C) for various times (0–60 min). Samples were then processed as indicated in Methods. To calculate the percentage of granulocytes and monocytes that bound and internalized parasites, cells were gated by SSC vs. FSC and plotted independently in a secondary plot of SSC vs. FL1 (green, 530 nm). The percentage of cells that internalized promastigotes was determined using trypan blue to quench the extracellular fluorescence emitted by cell-bound complement-killed CMFDA-labeled promastigotes. (A) For L. amazonensis promastigotes: (a) Time course of granulocyte binding and internalization (•) or internalization (○); (b) data from inset (shaded area) in (a); (c) time course of monocyte binding and internalization (•) or internalization (○); (d) data from inset (shaded area) in (c). (B) Data as above (a–d) are shown for L. donovani. Results from seven (L. donovani) and ten (L. amazonensis) experiments with blood from six donors.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2903471&req=5

pntd-0000743-g006: Kinetics of leukocyte binding and internalization of L. amazonensis and L. donovani promastigotes in human blood.Aliquots of heparinized human blood were infected with CMFDA-labeled L. amazonensis or L. donovani promastigotes and incubated (37°C) for various times (0–60 min). Samples were then processed as indicated in Methods. To calculate the percentage of granulocytes and monocytes that bound and internalized parasites, cells were gated by SSC vs. FSC and plotted independently in a secondary plot of SSC vs. FL1 (green, 530 nm). The percentage of cells that internalized promastigotes was determined using trypan blue to quench the extracellular fluorescence emitted by cell-bound complement-killed CMFDA-labeled promastigotes. (A) For L. amazonensis promastigotes: (a) Time course of granulocyte binding and internalization (•) or internalization (○); (b) data from inset (shaded area) in (a); (c) time course of monocyte binding and internalization (•) or internalization (○); (d) data from inset (shaded area) in (c). (B) Data as above (a–d) are shown for L. donovani. Results from seven (L. donovani) and ten (L. amazonensis) experiments with blood from six donors.
Mentions: The time course of granulocyte and monocyte binding of promastigotes was linear in the first 5 to 10 min of infection. Promastigote concentration subsequently became limiting and the kinetics followed a hyperbolic course; the binding reaction was complete by 60 min (Fig. 6). After 30 min incubation, 39.2% of granulocytes (∼94% of total binding) had already bound L. amazonensis and 45.1% (∼90% of total binding) had bound L. donovani promastigotes. At this time, 23.5% of monocytes (∼76% of total binding) had bound L. amazonensis and 20.2% (∼75% of total binding), L. donovani promastigotes. We analyzed leukocyte binding and promastigote internalization in the period between 0 and 5 min, when the velocity of promastigote binding is proportional with time and the cytolytic activity of complement on Leishmania is probably not yet complete (Fig. 6A,B; insets). After 3 min incubation, the ratio of cells that bound∶internalized promastigotes was 12.9∶5.4% for granulocytes and 8.9∶2% for monocytes (mean value of L. amazonensis and L. donovani); after 5 min, these values were 17.4∶8.4% for granulocytes and 10.7∶2.6% for monocytes.

Bottom Line: C3-promastigote binding is a key step in opsonization; nascent C3-promastigotes are the substrate for two simultaneous reactions, C3-promastigote immune adherence (IA) to erythrocytes and complement-mediated promastigote killing.The k(+1) for IA was 75-fold greater than that for promastigote killing, showing that IA facilitates promastigote endocytosis and circumvents lysis.At 5 min post-infection, when reaction velocity is still linear and promastigote concentration is not limiting, 17.4% of granulocytes and 10.7% of monocytes had bound promastigotes, of which approximately 50% and approximately 25%, respectively, carried surface-bound (live) or internalized (live and dead) leishmanias.

View Article: PubMed Central - PubMed

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

ABSTRACT
The leishmanioses, vector-borne diseases caused by the trypanosomatid protozoan Leishmania, are transmitted to susceptible mammals by infected phlebotomine sand flies that inoculate promastigotes into hemorrhagic pools created in host skin. We assumed that promastigotes are delivered to a blood pool, and analyzed early promastigote interactions (0-5 min) with host components, which lead to parasite endocytosis by blood leukocytes, and to host infection. Promastigotes were incubated with NHS or with heparinized blood in near-physiological conditions, and we used cell radioimmunoassay and flow cytometry to measure the on-rate constants (k(+1)) of promastigote interactions with natural opsonins and erythrocytes. We obtained quantitative data for parasitized cells to determine the time-course of promastigote binding and internalization by blood leukocytes. In these reactions, promastigotes bind natural opsonins, immune adhere to erythrocytes and activate complement cytolysis, which kills approximately 95% of promastigotes by 2 min post-infection. C3-promastigote binding is a key step in opsonization; nascent C3-promastigotes are the substrate for two simultaneous reactions, C3-promastigote immune adherence (IA) to erythrocytes and complement-mediated promastigote killing. The k(+1) for IA was 75-fold greater than that for promastigote killing, showing that IA facilitates promastigote endocytosis and circumvents lysis. At 5 min post-infection, when reaction velocity is still linear and promastigote concentration is not limiting, 17.4% of granulocytes and 10.7% of monocytes had bound promastigotes, of which approximately 50% and approximately 25%, respectively, carried surface-bound (live) or internalized (live and dead) leishmanias. Of other leukocyte types, 8.5% of B cells bound but did not internalize promastigotes, and T cells, NK cells and CD209(+) dendritic cells did not bind parasites. These data show that, once in contact with blood, promastigote invasion of human leukocytes is an extremely rapid and efficient reaction, and suggest that the IA reaction constitutes a central strategy for this parasite in subverting host innate immune defenses.

Show MeSH
Related in: MedlinePlus