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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 the Leishmania immune adherence (IA) reaction in human blood.Aliquots (50 µl) of [111In]-labeled promastigotes (107cells/ml) were mixed with 50 µl heparinized blood and incubated (37°C) for the times indicated. Samples were then fractionated by centrifugation (500×g/3 min) through 72% Percoll; free and erythrocyte-bound parasites were collected separately and [111In] cpm determined in each fraction. The IA kinetic profile is calculated as [111In]-promastigote cpm bound to erythrocytes relative to total [111In]-promastigote cpm at each time point, and expressed as a percentage of maximum binding of triplicate samples from one representative experiment. (A) L. donovani (n = 6), (B) L. amazonensis (n = 6). Insets: plots of L (IAmax/IAmax−IAti) against incubation time (ti), from which kapp values for Leishmania IA reaction were obtained. Blood from three different donors was used and two experiments were performed for each blood sample.
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pntd-0000743-g003: Kinetics of the Leishmania immune adherence (IA) reaction in human blood.Aliquots (50 µl) of [111In]-labeled promastigotes (107cells/ml) were mixed with 50 µl heparinized blood and incubated (37°C) for the times indicated. Samples were then fractionated by centrifugation (500×g/3 min) through 72% Percoll; free and erythrocyte-bound parasites were collected separately and [111In] cpm determined in each fraction. The IA kinetic profile is calculated as [111In]-promastigote cpm bound to erythrocytes relative to total [111In]-promastigote cpm at each time point, and expressed as a percentage of maximum binding of triplicate samples from one representative experiment. (A) L. donovani (n = 6), (B) L. amazonensis (n = 6). Insets: plots of L (IAmax/IAmax−IAti) against incubation time (ti), from which kapp values for Leishmania IA reaction were obtained. Blood from three different donors was used and two experiments were performed for each blood sample.

Mentions: Within seconds of serum contact, nascent C3-opsonized promastigotes bind to CR1 on E and form promastigote-E IA complexes. This interaction has an initial lag time of ∼15 sec followed by a period in which IA complexes are formed at an exponential rate until the reaction reaches completion at 30 to 40 sec of incubation. The IA reaction is so rapid that it gives the impression that promastigote-E binding proceeds before C3 opsonization (Fig. 3A, B). Considering the average number of CR1 molecules per erythrocyte to be 500 [44], the [E-CR1] in this assay is ∼2.1×10−9 M. At equilibrium, L. donovani promastigotes bind ∼180,000 C3 molecules/cell [28]; at the onset of the IA reaction in L. donovani (∼12 sec), there are ∼27,000 C3 molecules (∼15% of maximum binding) bound to promastigotes, indicating a promastigote-bound C3 concentration ([Pm-C3]) of ∼2.2×10−10 M. At the onset of the IA reaction, the [E-CR1]:[Pm-C3] ratio is thus ∼10. In the case of L. amazonensis, at equilibrium promastigotes bind ∼120,000 C3 molecules/cell, and at the onset of the IA reaction (∼15 sec) there are ∼22,000 C3 molecules bound/cell (∼18% of maximum binding); this yields a [Pm-C3] of ∼1.8×10−10 M, and a [E-CR1]:[Pm-C3] ratio of ∼12. In these assays, the [E-CR1] exceeds the [Pm-C3] by ∼10-fold and the reaction proceeds under pseudo-first-order conditions. Regression analysis of the percentage of IA plotted as L (IAmax/IAmax−IAti) against incubation time (ti) yielded kapp values of ∼0.15 sec−1 (L. amazonensis) and ∼0.21 sec−1 (L. donovani), and k+1 constants of ∼0.8×108 M−1 sec−1±0.02×108 M−1 sec−1 (L. amazonensis) and ∼1×108 M−1 sec−1±0.04×108 M−1 sec−1 (L. donovani). The average k+1 for the Leishmania IA reaction was thus ∼9×107 M−1 sec−1±0.4×107 M−1 s−1.


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 the Leishmania immune adherence (IA) reaction in human blood.Aliquots (50 µl) of [111In]-labeled promastigotes (107cells/ml) were mixed with 50 µl heparinized blood and incubated (37°C) for the times indicated. Samples were then fractionated by centrifugation (500×g/3 min) through 72% Percoll; free and erythrocyte-bound parasites were collected separately and [111In] cpm determined in each fraction. The IA kinetic profile is calculated as [111In]-promastigote cpm bound to erythrocytes relative to total [111In]-promastigote cpm at each time point, and expressed as a percentage of maximum binding of triplicate samples from one representative experiment. (A) L. donovani (n = 6), (B) L. amazonensis (n = 6). Insets: plots of L (IAmax/IAmax−IAti) against incubation time (ti), from which kapp values for Leishmania IA reaction were obtained. Blood from three different donors was used and two experiments were performed for each blood sample.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2903471&req=5

pntd-0000743-g003: Kinetics of the Leishmania immune adherence (IA) reaction in human blood.Aliquots (50 µl) of [111In]-labeled promastigotes (107cells/ml) were mixed with 50 µl heparinized blood and incubated (37°C) for the times indicated. Samples were then fractionated by centrifugation (500×g/3 min) through 72% Percoll; free and erythrocyte-bound parasites were collected separately and [111In] cpm determined in each fraction. The IA kinetic profile is calculated as [111In]-promastigote cpm bound to erythrocytes relative to total [111In]-promastigote cpm at each time point, and expressed as a percentage of maximum binding of triplicate samples from one representative experiment. (A) L. donovani (n = 6), (B) L. amazonensis (n = 6). Insets: plots of L (IAmax/IAmax−IAti) against incubation time (ti), from which kapp values for Leishmania IA reaction were obtained. Blood from three different donors was used and two experiments were performed for each blood sample.
Mentions: Within seconds of serum contact, nascent C3-opsonized promastigotes bind to CR1 on E and form promastigote-E IA complexes. This interaction has an initial lag time of ∼15 sec followed by a period in which IA complexes are formed at an exponential rate until the reaction reaches completion at 30 to 40 sec of incubation. The IA reaction is so rapid that it gives the impression that promastigote-E binding proceeds before C3 opsonization (Fig. 3A, B). Considering the average number of CR1 molecules per erythrocyte to be 500 [44], the [E-CR1] in this assay is ∼2.1×10−9 M. At equilibrium, L. donovani promastigotes bind ∼180,000 C3 molecules/cell [28]; at the onset of the IA reaction in L. donovani (∼12 sec), there are ∼27,000 C3 molecules (∼15% of maximum binding) bound to promastigotes, indicating a promastigote-bound C3 concentration ([Pm-C3]) of ∼2.2×10−10 M. At the onset of the IA reaction, the [E-CR1]:[Pm-C3] ratio is thus ∼10. In the case of L. amazonensis, at equilibrium promastigotes bind ∼120,000 C3 molecules/cell, and at the onset of the IA reaction (∼15 sec) there are ∼22,000 C3 molecules bound/cell (∼18% of maximum binding); this yields a [Pm-C3] of ∼1.8×10−10 M, and a [E-CR1]:[Pm-C3] ratio of ∼12. In these assays, the [E-CR1] exceeds the [Pm-C3] by ∼10-fold and the reaction proceeds under pseudo-first-order conditions. Regression analysis of the percentage of IA plotted as L (IAmax/IAmax−IAti) against incubation time (ti) yielded kapp values of ∼0.15 sec−1 (L. amazonensis) and ∼0.21 sec−1 (L. donovani), and k+1 constants of ∼0.8×108 M−1 sec−1±0.02×108 M−1 sec−1 (L. amazonensis) and ∼1×108 M−1 sec−1±0.04×108 M−1 sec−1 (L. donovani). The average k+1 for the Leishmania IA reaction was thus ∼9×107 M−1 sec−1±0.4×107 M−1 s−1.

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