Figure 1.

A high dose of L. pneumophila induces necrotic cell death in THP-1 cells. (A) Cytotoxicity of L. pneumophila was evaluated by assay of LDH release from THP-1 cells. (B) LDH release after 6h-stimulation with an MOI 500 of L. pneumophila or 10 μM of the apoptotic inducer MG132. (C) Diff-Quik staining after 6h-stimulation with an MOI 500 of L. pneumophila or MG132. L. pneumophila induced necrotic features such as loss of plasma membrane integrity and irregular and poorly-stained nuclei. The scale bar represents 20 μm. (D) Microscopic findings of the three cell groups identified following 6 h stimulation. L. pneumophila increased the percentage of necrotic cells in a dose-dependent manner. In contrast, MG132 induced mainly apoptotic cells. (E) Flow cytometric analysis of THP-1 cells, infected with an MOI of 500 of L. pneumophila, and stained with Annexin-V and PI. The number at the bottom of each panel indicates the percentage of cells detected in that area. (F) Extracellular HMGB-1 after 6 h incubation following infection with an MOI of 500 of L. pneumophila, or with MG132. HMGB-1 was significantly increased by L. pneumophila but not by MG132. Values are expressed as means ± SD. ** indicates p < 0.01.