Figure 1.
FACS Profile of Immature Human MoDCs
MoDCs were derived from human peripheral blood monocytes. Expression of HLA-DR, CD11c, CD14, CD16, CD80, CD86, and DEC-205 was assessed by flow cytometry. The data were collected from two subjects and are representative of similar experiments. Filled histograms represent isotype-matched controls.
Figure 2.
(A) MoDCs from a representative human subject were treated with LT (500 ng/ml PA and 250 ng/ml LF) or 10 μM camptothecin (Campt.) as a positive control, and annexin V/PI staining was measured by flow cytometry 48 h post-LT exposure.
(B) Percentages of annexin V-positive untreated and LT-treated MoDCs from five different subjects were determined by flow cytometry 48 h post-LT exposure.
(C) MTT assay of LT or camptothecin-treated human MoDCs. Mean + standard deviation from three independent experiments are shown.
(D) LT-treated human MoDCs show signs of apoptotic cell death, as analyzed by electron microscopy 48 h post-LT exposure. Bars: 1 μm.
(E) Human MoDCs were TUNEL-positive 48 h post-LT exposure. Untreated and LT-treated MoDCs were subjected to TUNEL and Hoechst staining.
Figure 3.
(A) BMDCs were derived from a BALB/c mouse and analyzed on day 10. Expression of CD11b, CD11c, CD14, and CD80 was assessed by flow cytometry. The data are representative of four similar experiments in BALB/c and C57BL/6 mice.
(B) BALB/c and C57BL/6-derived BMDCs were stimulated by LPS for 18 h, and CD86 expression was measured by flow cytometry. Filled histograms represent isotype-matched controls.
Figure 4.
BALB/c- and C57BL/6-Derived DCs Differ in Their Response to LT
(A) BALB/c and C57BL/6-derived BMDCs were treated with LT (500 ng/ml PA and 250 ng/ml LF), and cell survival was determined by MTT assay.
(B) Caspase-3 activation of LT-treated BALB/c and C57BL/6 DCs as determined by a colorimetric caspase-3 cleavage assay. A representative experiment is shown.
(C) The caspase inhibitors Z-VAD-FMK (10 μg/ml) and BOC-D-FMK (40 μg/ml) prevent caspase-3 activation in LT-treated BALB/c BMDCs.
(D) The caspase inhibitors Z-VAD-FMK (10 μg/ml) and BOC-D-FMK (40 μg/ml) do not prevent LT killing of BALB/c BMDCs as determined by MTT assay.
(E) C57BL6, but not BALB/c DCs, were TUNEL-positive post-LT exposure. BALB/c and C57BL/6-derived BMDCs were treated with LT (500 ng/ml PA and 250 ng/ml LF), and were stained using a TUNEL reaction and a Hoechst counterstain.
(F) BALB/c and C57BL/6-derived BMDCs were analyzed by electron microscopy 4 and 48 h post-LT exposure, respectively. Bars, 1 μm.
Figure 5.
MAPKK Cleavage Kinetics and LT Susceptibility of BALB/c- and C57BL/6-Derived BMDCs
(A) MAPKK-3 cleavage occurs at similar rates in murine and human DCs treated with LT (500 ng/ml PA and 250 ng/ml LF), as determined by Western blot analysis using anti-MKK3 and anti-actin (control) antibodies.
(B and C) Relative LT susceptibility of BALB/c- (B) and C57BL/6-derived (C) BMDCs. BALB/c and C57BL/6 BMDCs were subjected to PA (500 ng/ml) and varying concentrations of LF. After 4 h (BALB/c DCs) and 72 h (C57BL/6 DCs), cell viability was determined by MTT assays. Representative experiments are shown.
Figure 6.
Proteasome Inhibitors Block LT-Mediated Killing of BALB/c-Derived DCs
Proteasome inhibitors MG132 (10 μM) (A), or Velcade (0.1 μM) (B) were added either simultaneously with LT or 1 or 2 h post-LT exposure, and cell viability was determined by MTT assay. Mean + standard deviation of four independent experiments are shown.
Figure 7.
In Vivo Depletion of DCs and Loss of T Cell Activating Function Following LT Exposure
(A) Murine DCs and macrophages are killed by LT in vivo. Ten-week-old BALB/c mice were injected intraperitoneally with LT, and the percentage of DCs and macrophages in the spleen were determined by flow cytometry. A representative experiment is shown.
(B) Specific depletion of DCs and macrophages in LT-treated BALB/c mice injected intraperitoneally with either PBS or LT (200 μg PA and 200 μg LF). Levels of splenic DCs (CD11c+, MHC class II+), macrophages (CD11b+, MHC class II+), and circulating B cells (B220+) and T cells (CD3+) were determined by flow cytometry. Percent changes are shown by number of cells derived from LT-treated BALB/c mice (three mice per time point), relative to PBS-treated control mice (three mice per control experiment).