Polymorphonuclear leukocyte apoptosis is accelerated by sulfatides or sulfatides-treated Salmonella Typhimurium bacteria

Abstract

Neutrophils die by apoptosis following activation and uptake of microbes or enter apoptosis spontaneously at the end of their lifespan if they do not encounter a pathogen. Here we report that sulfatides or sulfatides-treated Salmonella Typhimurium bacteria accelerated human neutrophil apoptosis. Neutrophil apoptosis was examined by flow cytometry. Sulfatides caused prominent increase in percentage of apoptotic cells after 2.5 hrs of incubation. Salmonella Typhimurium bacteria by themselves did not affect the basal level of apoptosis in neutrophil population. When neutrophils were added to S. Typhimurium "opsonized" by sulfatides, apoptotic index significantly increased, whereas the number of phagocyting cells was not influenced. Sulfatides' proapoptotic effect was strongly dependent on the activity of β-galactosidase; inhibition of this enzyme impaired its potency to accelerate apoptosis. These data support the mechanism of neutrophil apoptosis triggering based on sulfatides' ability to accumulate in intracellular compartments and mediate successive increase in ceramide content resulting from β-galactosidase activity.

Figure 1

Dot plots for phosphatidylserine externalization of the apoptotic cells. We treated PMNLs (1.5 × 10⁶/mL) for 2.5 h at 37°C without (a) or with (b) 25 μg/mL sulfatide (St) and stained them with Annexin V-Alexa Fluor 488 and PI and processed them by flow cytometer as described in Section 2. The percentage of viable cells (region O3), early apoptotic cells (region O4), and late apoptotic and necrotic cells (regions O2 and O1) is indicated. Representative dot plots are shown from four independent experiments.

Figure 2

Evaluation of sulfatide-induced PMNL apoptosis. (a), (c), and (d) PS exposure of PMNL was determined using Annexin V-Alexa Fluor 647. PMNLs (1.5 × 10⁶/mL) were cultured for 2.5 h at 37°C without or with sulfatides (indicated concentration). Then, flow cytometry determined the percentage of Annexin V positive cells. Shown are the mean ± SD (a) and flow cytometric histogram samples of baseline (c) and 25 μg/mL sulfatide (d) groups, from six independent experiments. (b) PMNLs (1.5 × 10⁶ cells/mL) were added or not added with 25 μg/mL sulfatides, and, after 1.5 h at 37°C, the BIOMOL Carboxyfluorescein Multicaspase Activity Kit used flow cytometry to count the cells with activated caspases. Shown is the mean ± SD of three independent experiments.

Figure 3

Assay of PMNL apoptosis developed in probes added with S. Typhimurium bacteria, after 2.5 hours of coincubation. Neutrophils were challenged with plain bacteria (Salm), opsonized with normal serum bacteria (O-Salm), and treated with sulfatides bacteria (St-Salm), as described in Section 2. We also present data of similar experiments in which zymosan—nonopsonized (Z), opsonized by normal serum (OZ) or treated with sulfatides (St-Z)—provided phagocytosable stimulus of PMNLs. Then, PS exposure of PMNL was determined using Annexin V. Shown are the mean ± SD (a) and representative spectra (b) of five independent experiments.

Figure 4

Effect of Cytochalasin D on PMNL apoptosis. We cultured PMNLs (1.5 × 10⁶/mL) for 2.5 h at 37°C without or with 25 μg/mL sulfatides (St) or challenged with sulfatides-treated bacteria (St-Salm), and/or 10 μM Cyto D, as indicated. Shown are the mean ± SD (a) and representative flow cytometric spectra (b) of four independent experiments.

Figure 5

Phagocytosis of S. Typhimurium by PMNLs. We added PMNLs with plain bacteria (Salm), opsonized by normal serum (O-Salm) or treated by sulfatides (St-Salm) at a ratio of 10 bacteria/1 neutrophil. We assessed percentage of phagocyting cells by flow cytometry after 15 min of incubation of PMNLs with bacteria and removal of nonphagocytized bacteria. Shown are the mean ± SD (b) and representative flow cytometric spectra (a) of three independent experiments.

Figure 6

β-galactosidase inhibitor PETG attenuates sulfatide-induced apoptosis. We cultured PMNLs for 2.5 h at 37°C without or with tested compounds (25 μg/mL sulfatides, 3 mM PETG; PETG was added 30 min prior to sulfatides). Then flow cytometry determined the percentage of Annexin V positive cells. Shown are the mean ± SD (a) and representative flow cytometric spectra (b) of four independent experiments.