(A) HEp-2 cells infected with C. pneumoniae are resistant to apoptotic chromatin condensation induced by staurosporine and TNF-α. HEp-2 cells 36 h postinfection were treated with 1 μM staurosporine (STS) for 4 h (top), and infected and uninfected HEp-2 cells were treated with 40 ng of TNF-α/ml and 2 μg of cycloheximide (CHX)/ml for 5 h (bottom). The cells were examined at ×400 magnification under a fluorescence microscope equipped with a digital camera. Nuclei were stained with Hoechst 33342 (blue), and the chlamydial inclusions (Cpn) were stained in red. (B) Chlamydia-infected HEp-2 cells are resistant to apoptosis induced by staurosporine and TNF-α HEp-2 cells infected with C. pneumoniae (MOI = 5) were treated with staurosporine (STS) or TNF-α, as described in Materials and Methods. Nuclei were stained with Hoechst 33342, and the apoptotic and nonapoptotic cells from the infected and uninfected specimens in five random fields were counted under a fluorescence microscope (×400 magnification). The percentage of apoptotic cells was calculated based on data obtained in two independent experiments. Error bars, SD of the means.

HEp-2 cells infected with C. pneumoniae are resistant to staurosporine-induced DNA fragmentation. Infected (Cpn) and uninfected HEp-2 cells were treated with staurosporine (STS) and tested for DNA fragmentation using the TUNEL reaction. Shown are phase-contrast images (left) of the corresponding fluorescent fields (right). The TUNEL reaction is revealed by green fluorescence, and the presence of chlamydial inclusions is shown in red (arrows). The staining shows a mutually exclusive pattern: cells carrying inclusions are negative for the TUNEL reaction, and cells without inclusions are positive for the TUNEL reaction. Microscopic fields shown here were photographed from the samples infected at a MOI of 1 and visualized under ×400 magnification.

(A) Cells infected at high MOI detach from the culture plate. HEp-2 cells (5 × 10⁶) grown in six-well plates were infected with C. pneumoniae at MOI of 5, 10, and 50. At 15 h postinfection cells in the supernatants were collected by centrifugation and counted in a hemocytometer. Shown are the absolute numbers of detached cells for every sample. (B) Cells detaching upon C. pneumoniae infection are not apoptotic. Supernatants of HEp-2 cells infected with C. pneumoniae at MOI of 5, 10, and 50 were collected at 15 h postinfection. Cells were harvested, stained with annexin-V–FITC, and PI and analyzed by flow cytometry. The dot plots show the FITC and PI fluorescence intensities of 10,000 events. Note that infection at MOI of 10 and 50 increases the necrotic annexin-V and PI double-positive population but not the apoptotic annexin-V single-positive population. (C) MOI used influences the antiapoptotic effect in host cells treated with staurosporine. HEp-2 cells infected with C. pneumoniae at two different MOI (1 and 5) were treated with staurosporine (STS) at different time points postinfection (12, 24, 36, and 48 h). Nuclei were stained with Hoechst 33342, and the apoptotic and nonapoptotic cells from the infected and uninfected specimens were counted in five random fields (total of more than 400 cells) under a fluorescence microscope (×400 magnification). The plot shows the results of two independent experiments. Error bars, SD of the means.

Caspase-3 activation induced by staurosporine is inhibited in HEp-2 cells infected with C. pneumoniae. Infected (MOI = 1) and uninfected HEp-2 cells were treated with staurosporine (STS) at 36 h postinfection. After 4 to 5 h of treatment with STS, the cells were fixed and stained for chlamydial inclusions (Cpn) and activated caspase-3 as described in Materials and Methods. Activated caspase-3 (green) can only be seen in the uninfected cells or in the individual cells from the infected sample that do not contain chlamydial inclusions. Furthermore, activated caspase-3 is absent in the cells carrying inclusion(s) (red; arrows).

(A) Cytochrome c release is prevented in cells infected with C. pneumoniae. HEp-2 cells infected for 36 h (MOI = 3) were treated with TNF-α in the presence of cycloheximide (CHX). Cytochrome c (green) and chlamydial inclusions (Cpn) (red) were detected using immunofluorescence. Cells carrying inclusions measuring more than 4 μm in diameter were protected from cytochrome c release as shown by double-positive staining. Cells containing chlamydial inclusions measuring less than 4 μm released cytochrome c upon induction of apoptosis. The phase-contrast images show the presence of apoptotic cells. (B, top) Cells of five random fields in two independent experiments were analyzed. Shown is the relative number of cells which did not stain with the anti-cytochrome c antibody. (Bottom) Mitochondria were separated from the cytosol, and the concentration of cytochrome c in the cytosol of cells was determined by ELISA. The data were normalized by the cell number of each sample.