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Apoptotic Effects of LPS on Fibroblasts are Indirectly Mediated through TNFR1

Department of Periodontology and Oral Biology, Boston University School of Dental Medicine, Boston, MA 02118, USA;

View larger version (62K): [in a new window]   Figure 1. Apoptotic gene expression in wild-type (WT) and TNFR1–/–R2–/– mice measured by the RNase Protection Assay. Total RNA extracted from tissue obtained 24 hrs following inoculation of LPS or vehicle alone was subjected to RPA. (A) The PhosphoImages of gene expression in wild-type (+) and TNFR1–/–R2–/– (-) mice. (B) Densitometric analysis of apoptotic gene expression in wild-type and TNFR1–/–R2–/– mice. The densitometric value of each band was normalized by the value for GAPDH in the same lane. The percentage change values after LPS injection vs. vehicle injection are displayed. Each value represents the mean of 3 RPA ± SEM.

View larger version (26K): [in a new window]   Figure 2. Apoptotic gene expression in TNFR1–/–, TNFR2–/– and matched wild-type mice. The expression of apoptotic genes was assessed by RNase Protection assay 24 hrs after injection of LPS or vehicle alone. The densitometric value of each band was normalized by the value for GAPDH in the same lane. The percentage change values after LPS injection vs. vehicle alone are displayed. Each value represents the mean of 3 RPA ± SEM.

View larger version (38K): [in a new window]   Figure 3. Roles of different caspases in LPS-induced fibroblast apoptosis in vivo. (A) Caspases-3, -8, and -9 activities in wild-type or TNFR1–/–R2–/– mice after LPS injection. Caspase activity was measured by fluorometric assays in lysates from tissues obtained at 6 hrs following inoculation of LPS or vehicle alone. (B) Histologic sections of the site of LPS, or LPS and caspase-3 inhibitor, or LPS and caspase-8 inhibitor injection in the mouse scalp. Upper panel: Apoptotic fibroblasts identified as double-positive in the TUNEL assay and simultaneously for expression of vimentin with the use of a specific antibody as described in MATERIALS & METHODS. Large arrow points to a cell that is TUNEL/vimentin-positive. Small arrows point to vimentin-positive fibroblasts that are TUNEL-negative. Middle panel: Double-staining in mice 24 hrs after injection with LPS and caspase-3 inhibitor. Lower panel: Double-staining in mice 24 hrs after injection with LPS and caspase-8 inhibitor. Bar = 20 µm. (C) Quantitative analysis of fibroblast apoptosis following LPS and different caspase inhibitors’ injection. The number of double-positive TUNEL/vimentin fibroblasts was counted 6 hrs after injection of LPS, or LPS and caspase-3 inhibitor, or LPS and caspase-8 inhibitor, or vehicle alone. Each value represents the mean of 6 specimens ± SEM.