Caspase-8 activation precedes alterations of mitochondrial membrane potential during monocyte apoptosis induced by phagocytosis and killing of Staphylococcus aureus

Abstract

Human peripheral blood monocytes become apoptotic following phagocytosis and killing of Staphylococcus aureus. Although this type of monocyte apoptosis is known to be initiated by Fas-Fas ligand (FasL) interactions, the downstream signaling pathway has not been determined. In this work the involvement of mitochondria and the kinetics of caspase-8 and caspase-3 activation after phagocytosis of S. aureus were studied. Caspase-8 activity was measured in cell lysates by using the fluorogenic substrate Ac-IETD-AFC. Active caspase-3 levels and mitochondrial membrane potential (Deltapsi(m)) were measured in whole cells by flow cytometry using monoclonal antibodies reacting with activated caspase-3 and chloromethyl-X-rosamine, respectively. The results show that caspase-8 was activated shortly after phagocytosis of bacteria. Caspase-8 activation was followed by progressive disruption of Deltapsi(m), which is associated with the production of reactive oxygen intermediates. The irreversible caspase-8 inhibitor zIETD-FMK prevented the disruption of Deltapsi(m) and the release of cytochrome c from S. aureus-exposed monocytes. Caspase-3 activation occurred following disruption of Deltapsi(m). These results strongly suggest that apoptosis of monocytes that have phagocytosed and killed S. aureus is driven by the Fas-FasL-initiated pathway, which is typical for type II cells.

FIG. 1.

Caspase-8 activation in monocytes after phagocytosis of S. aureus. Ac-IETD-AFC was added to monocyte lysates prepared at various times after exposure to bacteria (MO+S.a). Control monocytes without bacteria (MO) were also sampled at the same time points. Ratios of fluorescence signals obtained in samples with S. aureus to those without S. aureus at the indicated time points are shown. Results obtained 15 min after exposure to bacteria are means ± standard deviations of five measurements from independent experiments; results for other time points are means ± standard deviations of measurements from three independent experiments.

FIG. 2.

Evaluation of Δψ_m with the Δψ_m-sensitive probe CMXRos. Control (dotted lines) and S. aureus-exposed (solid lines) monocytes were incubated with CMXRos and analyzed by flow cytometry at the indicated time points. Note the early drop in Δψ_m, followed after 2 h by a dramatic reduction in the signal. Representative results (from one kinetic study out of four performed) are shown.

FIG. 3.

Reductions in Δψ_m are seen predominantly in monocytes that have engulfed bacteria. Monocytes were allowed to phagocytose FITC-labeled S. aureus bacteria, and their Δψ_ms were evaluated by flow cytometry 2 h later. Quadrant statistics are shown to point at the proportions of cells with complete disruption of Δψ_m in both groups. Dot plots present results for control (left) and S. aureus-exposed (right) monocytes from a single experiment out of six performed.

FIG. 4.

Reductions in Δψ_m can be prevented by blocking of NADPH oxidase and by use of antioxidants. Δψ_ms of S. aureus-exposed monocytes without pretreatment (solid lines) or pretreated with DPI or NAC (dotted lines) were evaluated at the indicated time points.

FIG. 5.

Reductions in Δψ_m can be prevented by blocking of NADPH oxidase. Shown is a dot plot analysis of monocytes probed with CMXRos 2 h after exposure to bacteria. (A and C) Monocytes cultured without bacteria; (B and D) monocytes exposed to bacteria. Monocytes for which results are shown in panels C and D were pretreated with apocynin. Quadrant statistics are shown to point at the proportions of cells with complete disruption of Δψ_m.

FIG. 6.

Disruption of Δψ_m and release of cytochrome c by monocytes following phagocytosis of S. aureus does not occur in zIETD-FMK-pretreated cells. Monocytes were either preincubated with the caspase-8 inhibitor (25 μM) for 30 min or left untreated. Then bacteria were added. Δψ_m was measured by use of CMXRos after 60 min of culture. The concentration of cytochrome c in culture (10⁶ monocytes/ml) was measured by enzyme-linked immunosorbent assay in supernatants harvested after 120 min of culture. (A) Overlay histogram shows fluorescence of control and zIETD-FMK-pretreated monocytes exposed to bacteria and of monocytes cultured alone, as indicated. Data from one representative experiment out of three performed are shown. (B) Cytochome c concentrations in culture supernatants. Means and standard deviations from three independent experiments are shown. MO, monocytes.

FIG. 7.

Time course of Δψ_m changes and of the appearance of activated caspase-3. Duplicate sets of samples of monocytes with and without bacteria were cultured and harvested at the indicated time points. In each pair, CMXRos was added to one sample 15 min before harvest while the second sample was stained for the presence of activated caspase-3 (akt. casp-3) at the indicated harvest time.