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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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Blood leukocyte subpopulations that bind promastigotes in early infection.CMFDA-labeled promastigotes were incubated (5 min) with heparinized blood and the percentage of each Leishmania-binding leukocyte subpopulation was measured by flow cytometry. Leukocyte subpopulations were stained with fluorochrome-labeled mAb and identified in a dot plot as side scatter (SSC) vs. the specific fluorescent label. The percentage of each promastigote-binding subpopulation was analyzed in a secondary plot representing the fluorescence intensity of each gated mAb-labeled population vs. that of cell-bound CMFDA-labeled promastigotes. Dot plots of a representative experiment show cells in the gated populations that bound CMFDA-labeled (A) L. amazonensis or (B) L. donovani promastigotes. (C) Cells of each subpopulation that bound promastigotes expressed as a percentage of total leukocytes in the sample. Results are shown as the percentage (mean ± SEM) of 14 experiments performed with blood of six donors. (▪) L. amazonesis, () L. donovani promastigotes.
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pntd-0000743-g005: Blood leukocyte subpopulations that bind promastigotes in early infection.CMFDA-labeled promastigotes were incubated (5 min) with heparinized blood and the percentage of each Leishmania-binding leukocyte subpopulation was measured by flow cytometry. Leukocyte subpopulations were stained with fluorochrome-labeled mAb and identified in a dot plot as side scatter (SSC) vs. the specific fluorescent label. The percentage of each promastigote-binding subpopulation was analyzed in a secondary plot representing the fluorescence intensity of each gated mAb-labeled population vs. that of cell-bound CMFDA-labeled promastigotes. Dot plots of a representative experiment show cells in the gated populations that bound CMFDA-labeled (A) L. amazonensis or (B) L. donovani promastigotes. (C) Cells of each subpopulation that bound promastigotes expressed as a percentage of total leukocytes in the sample. Results are shown as the percentage (mean ± SEM) of 14 experiments performed with blood of six donors. (▪) L. amazonesis, () L. donovani promastigotes.

Mentions: To measure initial blood leukocyte binding of opsonized leishmanias, we incubated CMFDA-labeled promastigotes with heparin-treated blood and used flow cytometry to determine the percentage of each leukocyte subpopulation that bound parasites after 5 min. Leukocyte subpopulations were identified with fluorochrome-labeled anti-CD15 (for granulocytes), -CD14 (monocytes), -CD3 (T cells), -CD19 (B cells), -CD56+ (NK cells) and -CD209 (monocyte/dendritic cells) mAb. Data from a representative experiment are shown (Fig. 5) in which fluorescence intensity of each gated leukocyte subpopulation is represented in a secondary plot against that of cell-bound CMFDA-labeled promastigotes (FL-1) as the percentage of each subpopulation that bound L. amazonensis (Fig. 5A) or L. donovani (Fig. 5B) parasites. Cells of each subpopulation that bound promastigotes are expressed as a percentage of total leukocytes in the sample (Fig. 5C; mean for eight experiments). After 5 min incubation, 13% of leukocytes bound promastigotes, of which 10.7%±0.15% were CD14+, 76.3%±0.8% CD15+, 2.7%±0.1% CD3+, 8.5%±0.2% CD19+, 1.3%±0.02% CD3− CD56+ and 0.49%±0.03% CD209+ cells. A substantial fraction of B cells (8.5%) bound promastigotes in this early period. Granulocytes are the main subpopulation that bound promastigotes; nevertheless, the percentage of promastigote-binding cells in each subpopulation was nearly identical for CD15+ (19.1±2.4%), CD14+ (17.3%±2.3%) and CD19+ (17.4%±2.5%) (Fig. S6). Other leukocyte subpopulations did not bind promastigotes appreciably.


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)

Blood leukocyte subpopulations that bind promastigotes in early infection.CMFDA-labeled promastigotes were incubated (5 min) with heparinized blood and the percentage of each Leishmania-binding leukocyte subpopulation was measured by flow cytometry. Leukocyte subpopulations were stained with fluorochrome-labeled mAb and identified in a dot plot as side scatter (SSC) vs. the specific fluorescent label. The percentage of each promastigote-binding subpopulation was analyzed in a secondary plot representing the fluorescence intensity of each gated mAb-labeled population vs. that of cell-bound CMFDA-labeled promastigotes. Dot plots of a representative experiment show cells in the gated populations that bound CMFDA-labeled (A) L. amazonensis or (B) L. donovani promastigotes. (C) Cells of each subpopulation that bound promastigotes expressed as a percentage of total leukocytes in the sample. Results are shown as the percentage (mean ± SEM) of 14 experiments performed with blood of six donors. (▪) L. amazonesis, () L. donovani promastigotes.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000743-g005: Blood leukocyte subpopulations that bind promastigotes in early infection.CMFDA-labeled promastigotes were incubated (5 min) with heparinized blood and the percentage of each Leishmania-binding leukocyte subpopulation was measured by flow cytometry. Leukocyte subpopulations were stained with fluorochrome-labeled mAb and identified in a dot plot as side scatter (SSC) vs. the specific fluorescent label. The percentage of each promastigote-binding subpopulation was analyzed in a secondary plot representing the fluorescence intensity of each gated mAb-labeled population vs. that of cell-bound CMFDA-labeled promastigotes. Dot plots of a representative experiment show cells in the gated populations that bound CMFDA-labeled (A) L. amazonensis or (B) L. donovani promastigotes. (C) Cells of each subpopulation that bound promastigotes expressed as a percentage of total leukocytes in the sample. Results are shown as the percentage (mean ± SEM) of 14 experiments performed with blood of six donors. (▪) L. amazonesis, () L. donovani promastigotes.
Mentions: To measure initial blood leukocyte binding of opsonized leishmanias, we incubated CMFDA-labeled promastigotes with heparin-treated blood and used flow cytometry to determine the percentage of each leukocyte subpopulation that bound parasites after 5 min. Leukocyte subpopulations were identified with fluorochrome-labeled anti-CD15 (for granulocytes), -CD14 (monocytes), -CD3 (T cells), -CD19 (B cells), -CD56+ (NK cells) and -CD209 (monocyte/dendritic cells) mAb. Data from a representative experiment are shown (Fig. 5) in which fluorescence intensity of each gated leukocyte subpopulation is represented in a secondary plot against that of cell-bound CMFDA-labeled promastigotes (FL-1) as the percentage of each subpopulation that bound L. amazonensis (Fig. 5A) or L. donovani (Fig. 5B) parasites. Cells of each subpopulation that bound promastigotes are expressed as a percentage of total leukocytes in the sample (Fig. 5C; mean for eight experiments). After 5 min incubation, 13% of leukocytes bound promastigotes, of which 10.7%±0.15% were CD14+, 76.3%±0.8% CD15+, 2.7%±0.1% CD3+, 8.5%±0.2% CD19+, 1.3%±0.02% CD3− CD56+ and 0.49%±0.03% CD209+ cells. A substantial fraction of B cells (8.5%) bound promastigotes in this early period. Granulocytes are the main subpopulation that bound promastigotes; nevertheless, the percentage of promastigote-binding cells in each subpopulation was nearly identical for CD15+ (19.1±2.4%), CD14+ (17.3%±2.3%) and CD19+ (17.4%±2.5%) (Fig. S6). Other leukocyte subpopulations did not bind promastigotes appreciably.

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