Intracellular survival of Leishmania major in neutrophil granulocytes after uptake in the absence of heat-labile serum factors

Abstract

The role of polymorphonuclear neutrophil granulocytes (PMN) in defense against the intracellular parasite Leishmania is poorly understood. In the present study, the interaction of human PMN with Leishmania major promastigotes was investigated in vitro. In the presence of fresh human serum, about 50% of PMN phagocytosed the parasites within 10 min and the parasite uptake led to PMN activation, resulting in the killing of most ingested parasites. Heat inactivation of the serum markedly reduced the rate of early parasite phagocytosis, suggesting a role of complement components in the early uptake of Leishmania. However, over 50% of PMN were able to ingest parasites in the presence of heat-inactivated serum if the coincubation was extended to 3 h. After 3 h, 10% of the PMN were found to internalize Leishmania even under serum-free conditions. These findings indicate that PMN possess mechanisms for both opsonin/complement-dependent and -independent uptake of Leishmania. Both pathways of uptake could be partially blocked by anti-CR3 antibody. Mannan-binding lectin was found not to be involved in this process. When phagocytosed in the absence of opsonin, the majority of Leishmania parasites survived intracellularly in PMN for at least 1 day. These data suggest a dual role of PMN in the early response to L. major infection. On the one hand, PMN can rapidly eliminate the intracellular parasites, and on the other hand, Leishmania can survive intracellularly in PMN. These data, together with the finding that intact parasites were seen in PMN isolated from the skin of infected mice, suggest that PMN can serve as host cells for the intracellular survival of Leishmania within the first hours or days after infection.

FIG. 1.

Kinetics of phagocytosis of L. major promastigotes by PMN in vitro. PMN were incubated with L. major promastigotes at 37°C at a PMN-to-parasite ratio of 1:5 in RPMI 1640 medium supplemented with either 20% fresh autologous serum, 20% heat-inactivated autologous serum (hi-serum), or 20% heat-inactivated FCS (hi-FCS) or without serum supplementation. The percentage of PMN harboring at least one intracellular parasite was assessed by microscopic evaluation of Giemsa-stained cytocentrifuge preparations at the given time points. The data shown are from one experiment representative of four experiments performed.

FIG. 2.

Effect of anti-CR3 MAb treatment on the uptake of L. major by PMN. PMN were preincubated with anti-CR3 MAb for 20 min at 37°C, and control cultures were incubated with isotype control MAb. PMN were subsequently washed and coincubated with L. major promastigotes at a PMN-to-parasite ratio of 1:5 for 2 h at 37°C in RPMI 1640 medium containing 20% fresh autologous serum (A) or 20% heat-inactivated FCS (B). The uptake of parasites was assessed in two experiments (Exp I and Exp II) by microscopic evaluation of Giemsa-stained cytocentrifuge preparations. Exp I and Exp II were performed with cells and serum from two different donors.

FIG. 3.

Effect of MBL on the uptake of L. major by PMN. (A) PMN were coincubated with L. major promastigotes at a PMN-to-parasite ratio of 1:5 for 10 min at 37°C in medium supplemented with 20% fresh serum from healthy individuals (containing 2.3 μg of MBL per ml ([serum 1] or 2.8 μg of MBL per ml [serum 2]). (B) PMN were coincubated with L. major promastigotes at a PMN-to-parasite ratio of 1:5 for 10 min at 37°C without MBL or in the presence of 3.3 μg of purified MBL in medium without serum supplementation or in medium supplemented with 20% heat-inactivated FCS (hi-FCS). Data are from one representative experiment of two performed.

FIG. 4.

Effect of L. major on the cell surface expression of CD62L and CD66b on PMN. (A) PMN were coincubated with L. major promastigotes at a PMN-to-parasite ratio of 1:5 for 90 min at 37°C in RPMI 1640 medium supplemented with either 20% fresh autologous serum or 20% heat-inactivated FCS (hi-FCS) or without serum supplementation. PMN were then stained with FITC-conjugated MAb to CD66b and PE-labeled MAb to CD62L and analyzed by flow cytometry (A). (B) Mean fluorescence values of CD66b staining.

FIG. 5.

Effect of L. major on induction of the oxidative burst by PMN. PMN were coincubated with L. major promastigotes at a PMN-to-parasite ratio of 1:5 for 10 min at 37°C in RPMI 1640 medium supplemented with either 20% fresh autologous serum or 20% heat-inactivated serum (hi-serum) or without serum supplementation. The oxidative burst in PMN was assessed by using the fluorogenic substrate dihydrorhodamine 123 and measured by flow cytometry (FL-1). The percentage of burst positive cells is shown in the upper left corner.

FIG. 6.

Viability of intracellular Leishmania in PMN. PMN were coincubated with L. major promastigotes at a PMN-to-parasite ratio of 1:5 for 3 h at 37°C in RPMI 1640 medium supplemented with either 20% fresh autologous serum or 20% heat-inactivated FCS (hi-FCS) or without serum supplementation. PMN were then separated from nonphagocytosed L. major promastigotes by fluorescence-activated cell sorting and subsequently incubated for 24 h at 37°C in complete RPMI 1640 medium containing 10% FCS. The viability of L. major in PMN was then assessed by a limiting-dilution Leishmania culture assay. The y axis shows the mean and standard deviation of the three parallel dilutions of PMN.

FIG. 7.

Staining of viable intracellular L. major in PMN with ethidium bromide and acridine orange. PMN were coincubated with L. major promastigotes at a PMN-to-parasite ratio of 1:5 for 3 h at 37°C in RPMI 1640 medium supplemented with 20% heat-inactivated FCS, separated from nonphagocytosed L. major promastigotes, and subsequently incubated for 24 h at 37°C. The viability of L. major in PMN was then assessed by staining with acridine orange to visualize viable cells or parasites (green staining) and ethidium bromide, which stains dead cells. Viable L. major parasites (arrows) are seen in nonapoptotic viable PMN. The morphological appearance of the nuclei and kinetoplasts is typical for intact parasites.

FIG. 8.

Uptake and survival of L. major in PMN in vivo. L. major promastigotes were injected into subcutaneous air pouches in BALB/c mice. At 24 h after infection, the exudate cells were collected from the air pouches. Cytocentrifuge slides of exudate cells were stained with Giemsa. Intracellular Leishmania organisms (arrows) are seen in PMN.