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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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Time course of normal serum IgM binding to L. donovani and L. amazonensis promastigotes.Aliquots (50 µl) of L. donovani or L. amazonensis promastigotes (107/ml) were mixed with 50 µl 50% NHS and incubated (37°C) for varying times. Promastigotes were then washed by centrifugation (11,000×g, 1 min) with cold PFS. Untreated promastigotes (5×106) were added to the pellet, washed twice in cold PFS, and promastigote-bound IgM measured with 125I-goat anti-μ antibody. Each point (mean of triplicate samples) is expressed as a percentage of the point of maximum IgM binding in one representative experiment of five performed for each species. (A) L. donovani, (B) L. amazonensis. Insets: plots of L (IgM5max/IgM5max−IgM5ti) against reaction time (ti), from which kapp values were derived.
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pntd-0000743-g001: Time course of normal serum IgM binding to L. donovani and L. amazonensis promastigotes.Aliquots (50 µl) of L. donovani or L. amazonensis promastigotes (107/ml) were mixed with 50 µl 50% NHS and incubated (37°C) for varying times. Promastigotes were then washed by centrifugation (11,000×g, 1 min) with cold PFS. Untreated promastigotes (5×106) were added to the pellet, washed twice in cold PFS, and promastigote-bound IgM measured with 125I-goat anti-μ antibody. Each point (mean of triplicate samples) is expressed as a percentage of the point of maximum IgM binding in one representative experiment of five performed for each species. (A) L. donovani, (B) L. amazonensis. Insets: plots of L (IgM5max/IgM5max−IgM5ti) against reaction time (ti), from which kapp values were derived.

Mentions: Non-immune serum from most vertebrates contains natural anti-trypanosomatid antibodies that act as an innate recognition system in the host [28], [39]. To determine the velocity of IgM binding to Leishmania parasites, we measured the association rate constant (k+1) by incubating promastigotes in NHS for varying times. The reaction was terminated by sample dilution with PFS, and promastigote-bound IgM was measured using 125I-goat anti-μ. Pentameric IgM (IgM5) binding reached maximum after 20 sec incubation for both L. donovani (Fig. 1A) and L. amazonensis (Fig. 1B). [IgM5] in adult NHS is ∼1.3 g/L; if the Mr of IgM5 is considered to be 950,000, then serum [IgM5] is ∼1.4×10−6 M. Exhaustive adsorption of 25% NHS with L. amazonensis and L. donovani promastigotes removed ∼15% and ∼30% of IgM5, respectively [28], indicating that [IgM5] anti-L. amazonensis in unadsorbed serum is ∼0.5×10−7 M and that of anti-L. donovani, ∼1×10−7 M. At equilibrium, L. amazonensis and L. donovani promastigotes bind ∼5,000 and ∼2,500 IgM molecules/cell, respectively. Early in the promastigote-IgM5 interaction, antibody binding is assumed to be monovalent [29], which would render [Pmbs] on L. amazonensis promastigotes of ∼4.1×10−11 M and on L. donovani promastigotes of ∼2.1×10−11 M. In a monovalent promastigote-IgM5 interaction, the [IgM5]/[Pmbs] ratio is ∼1,300∶1 in L. amazonensis and ∼5,000∶1 in L. donovani; in the case of a decavalent promastigote-IgM5 interaction, this ratio would be ∼130∶1 (L. amazonensis) and ∼500∶1 (L. donovani). In both cases, the promastigote-IgM5 interaction obeys pseudo-first-order kinetics. The percentage of IgM5 bound, plotted as L (IgM5max/IgM5max−IgM5ti) against reaction time (ti), gives straight lines whose slopes are the kapp values of the reactions, ∼0.23 sec−1 for L. amazonensis and ∼0.18 sec−1 for L. donovani. Second-order rate constants were obtained from k+1 = kapp/[IgM5], with values of ∼4.4×106 M−1 sec−1±0.05×106 M−1 sec−1 for L. amazonensis and ∼1.8×106 M−1 sec−1±0.05×106 M−1 sec−1 for L. donovani. The mean k+1 for natural IgM5 binding to promastigotes of both Leishmania species was ∼3×106 M−1 sec−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)

Time course of normal serum IgM binding to L. donovani and L. amazonensis promastigotes.Aliquots (50 µl) of L. donovani or L. amazonensis promastigotes (107/ml) were mixed with 50 µl 50% NHS and incubated (37°C) for varying times. Promastigotes were then washed by centrifugation (11,000×g, 1 min) with cold PFS. Untreated promastigotes (5×106) were added to the pellet, washed twice in cold PFS, and promastigote-bound IgM measured with 125I-goat anti-μ antibody. Each point (mean of triplicate samples) is expressed as a percentage of the point of maximum IgM binding in one representative experiment of five performed for each species. (A) L. donovani, (B) L. amazonensis. Insets: plots of L (IgM5max/IgM5max−IgM5ti) against reaction time (ti), from which kapp values were derived.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000743-g001: Time course of normal serum IgM binding to L. donovani and L. amazonensis promastigotes.Aliquots (50 µl) of L. donovani or L. amazonensis promastigotes (107/ml) were mixed with 50 µl 50% NHS and incubated (37°C) for varying times. Promastigotes were then washed by centrifugation (11,000×g, 1 min) with cold PFS. Untreated promastigotes (5×106) were added to the pellet, washed twice in cold PFS, and promastigote-bound IgM measured with 125I-goat anti-μ antibody. Each point (mean of triplicate samples) is expressed as a percentage of the point of maximum IgM binding in one representative experiment of five performed for each species. (A) L. donovani, (B) L. amazonensis. Insets: plots of L (IgM5max/IgM5max−IgM5ti) against reaction time (ti), from which kapp values were derived.
Mentions: Non-immune serum from most vertebrates contains natural anti-trypanosomatid antibodies that act as an innate recognition system in the host [28], [39]. To determine the velocity of IgM binding to Leishmania parasites, we measured the association rate constant (k+1) by incubating promastigotes in NHS for varying times. The reaction was terminated by sample dilution with PFS, and promastigote-bound IgM was measured using 125I-goat anti-μ. Pentameric IgM (IgM5) binding reached maximum after 20 sec incubation for both L. donovani (Fig. 1A) and L. amazonensis (Fig. 1B). [IgM5] in adult NHS is ∼1.3 g/L; if the Mr of IgM5 is considered to be 950,000, then serum [IgM5] is ∼1.4×10−6 M. Exhaustive adsorption of 25% NHS with L. amazonensis and L. donovani promastigotes removed ∼15% and ∼30% of IgM5, respectively [28], indicating that [IgM5] anti-L. amazonensis in unadsorbed serum is ∼0.5×10−7 M and that of anti-L. donovani, ∼1×10−7 M. At equilibrium, L. amazonensis and L. donovani promastigotes bind ∼5,000 and ∼2,500 IgM molecules/cell, respectively. Early in the promastigote-IgM5 interaction, antibody binding is assumed to be monovalent [29], which would render [Pmbs] on L. amazonensis promastigotes of ∼4.1×10−11 M and on L. donovani promastigotes of ∼2.1×10−11 M. In a monovalent promastigote-IgM5 interaction, the [IgM5]/[Pmbs] ratio is ∼1,300∶1 in L. amazonensis and ∼5,000∶1 in L. donovani; in the case of a decavalent promastigote-IgM5 interaction, this ratio would be ∼130∶1 (L. amazonensis) and ∼500∶1 (L. donovani). In both cases, the promastigote-IgM5 interaction obeys pseudo-first-order kinetics. The percentage of IgM5 bound, plotted as L (IgM5max/IgM5max−IgM5ti) against reaction time (ti), gives straight lines whose slopes are the kapp values of the reactions, ∼0.23 sec−1 for L. amazonensis and ∼0.18 sec−1 for L. donovani. Second-order rate constants were obtained from k+1 = kapp/[IgM5], with values of ∼4.4×106 M−1 sec−1±0.05×106 M−1 sec−1 for L. amazonensis and ∼1.8×106 M−1 sec−1±0.05×106 M−1 sec−1 for L. donovani. The mean k+1 for natural IgM5 binding to promastigotes of both Leishmania species was ∼3×106 M−1 sec−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