Figure 5

Light microscopic analysis of the effect of Cpn infection on HASMC. HASMC were infected with Cpn-K6 128 IFU/cell (A, B, C, D), Cpn-VR1310 32 IFU/cell (E, F) for 72 h or were inoculated with mock lysate (G) or treated with staurosporin 1 μM for 14 h (H). The cells were subsequently stained for TUNEL (green), NHS-biotin (brown), anti-Cpn-MOMP (red) and DNA (blue). A: HASMC bearing a Cpn-K6 inclusion. DAPI stained chromatin structure is normal and TUNEL as well as NHS-biotin labeling are negative. B: Cpn-K6 infected HASMC with multiple chlamydial spots (arrow heads) and normal nuclear structure. C, E: Cpn-K6 (C) or Cpn-VR1310 (E) infected HASMC with spot like infection (arrow heads). The condensed nucleus is TUNEL positive, the cytoplasm is diffusely labeled with NHS-biotin. D, F: HASMC with Cpn-K6 (D) or Cpn-VR1310 (F) in aggregated (arrow) spots and single spots (arrow heads). Note positive TUNEL labeling of the shrunken nucleus and NHS-biotin staining of the cytoplasm. G: Mock inoculated HASMC. Note the fine granular chromatin structure and lack of TUNEL- and cytoplasmic NHS-biotin staining. H: HASMC incubated with staurosporin. The nucleus exhibits a distinct nuclear fragmentation (DAPI staining) but no TUNEL labeling. The cytoplasm is NHS-biotin negative indicating an intact cell membrane. I: Quantification of TUNEL positive nuclei depending on the chlamydial infectious dose 72 h post infection reveals a dose dependent increase of TUNEL positive cells. Cells in 5 random fields with an area of 62'500 μm² were counted and the percentage of TUNEL positive cells was calculated. -▲-: Cpn-K6; -■-: Cpn-VR1310