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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 C3 deposition on L. donovani and L. amazonensis promastigotes.Duplicate aliquots (50 µl) of promastigotes (107/ml) were mixed with 50 µl 50% NHS and incubated (37°C) for the times indicated. Promastigotes were then washed twice by centrifugation (11,000×g, 1 min) in cold PFS, and promastigote-bound C3 measured with 125I-SIM27–49 (anti-human C3α mAb). C3 binding (mean of duplicate values) from one representative experiment. (A) L. donovani (n = 4), (B) L. amazonensis (n = 5). Insets: plots of L (C3max/C3max−C3ti) against time (ti), from which kapp constants of C3 deposition reactions were obtained.
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pntd-0000743-g002: Kinetics of C3 deposition on L. donovani and L. amazonensis promastigotes.Duplicate aliquots (50 µl) of promastigotes (107/ml) were mixed with 50 µl 50% NHS and incubated (37°C) for the times indicated. Promastigotes were then washed twice by centrifugation (11,000×g, 1 min) in cold PFS, and promastigote-bound C3 measured with 125I-SIM27–49 (anti-human C3α mAb). C3 binding (mean of duplicate values) from one representative experiment. (A) L. donovani (n = 4), (B) L. amazonensis (n = 5). Insets: plots of L (C3max/C3max−C3ti) against time (ti), from which kapp constants of C3 deposition reactions were obtained.

Mentions: To establish how Leishmania circumvents complement effector activity, the kinetics and velocity of complement activation must be understood. We incubated promastigotes with NHS and measured the reaction time course and k+1 of promastigote-C3 binding. Promastigote-C3 binding follows a sigmoidal course, with an initial ∼30-sec lag during which 10 to 20% of total C3 ligands are fixed; C3 molecules that bind during this lag period probably attach to IgM. This amount of C3 is insufficient to activate the alternative pathway and the lytic cascade, but is sufficient to establish multipoint contacts with clustered E-CR1, triggering the IA reaction [40]–[42]. Thereafter, C3 binds at an exponential rate for ∼1 min, during which C3 is bound at an average rate of ∼1,800 molecules/sec for L. donovani (Fig. 2A) and ∼1,200 molecules/sec for L. amazonensis (Fig. 2B). During the C3 lag period, IgM5 binding to Leishmania reaches maximum and the [IgM5] bound is ∼4.1×10−11 M (L. amazonensis) and ∼2.1×10−11 M (L. donovani).


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 C3 deposition on L. donovani and L. amazonensis promastigotes.Duplicate aliquots (50 µl) of promastigotes (107/ml) were mixed with 50 µl 50% NHS and incubated (37°C) for the times indicated. Promastigotes were then washed twice by centrifugation (11,000×g, 1 min) in cold PFS, and promastigote-bound C3 measured with 125I-SIM27–49 (anti-human C3α mAb). C3 binding (mean of duplicate values) from one representative experiment. (A) L. donovani (n = 4), (B) L. amazonensis (n = 5). Insets: plots of L (C3max/C3max−C3ti) against time (ti), from which kapp constants of C3 deposition reactions were obtained.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000743-g002: Kinetics of C3 deposition on L. donovani and L. amazonensis promastigotes.Duplicate aliquots (50 µl) of promastigotes (107/ml) were mixed with 50 µl 50% NHS and incubated (37°C) for the times indicated. Promastigotes were then washed twice by centrifugation (11,000×g, 1 min) in cold PFS, and promastigote-bound C3 measured with 125I-SIM27–49 (anti-human C3α mAb). C3 binding (mean of duplicate values) from one representative experiment. (A) L. donovani (n = 4), (B) L. amazonensis (n = 5). Insets: plots of L (C3max/C3max−C3ti) against time (ti), from which kapp constants of C3 deposition reactions were obtained.
Mentions: To establish how Leishmania circumvents complement effector activity, the kinetics and velocity of complement activation must be understood. We incubated promastigotes with NHS and measured the reaction time course and k+1 of promastigote-C3 binding. Promastigote-C3 binding follows a sigmoidal course, with an initial ∼30-sec lag during which 10 to 20% of total C3 ligands are fixed; C3 molecules that bind during this lag period probably attach to IgM. This amount of C3 is insufficient to activate the alternative pathway and the lytic cascade, but is sufficient to establish multipoint contacts with clustered E-CR1, triggering the IA reaction [40]–[42]. Thereafter, C3 binds at an exponential rate for ∼1 min, during which C3 is bound at an average rate of ∼1,800 molecules/sec for L. donovani (Fig. 2A) and ∼1,200 molecules/sec for L. amazonensis (Fig. 2B). During the C3 lag period, IgM5 binding to Leishmania reaches maximum and the [IgM5] bound is ∼4.1×10−11 M (L. amazonensis) and ∼2.1×10−11 M (L. donovani).

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