Figure 3

HMGB1 release in infected HAEC. HAEC were infected with 2 IFU/cell C. pneumoniae (A, B) or 20 IFU/cell (C, F) and analyzed 48 hpi (F) or 72 hpi (A, B, C). Control cells were additionally treated with chloramphenicol (C), incubated with staurosporin (G), treated with Na-acid (H) or left untreated (I). Mean and SD of three pooled experiments are depicted, differences were considered to be significant if p < 0.05 (D, E). Cells were labelled with anti-MOMP antibody (red), with anti-HMGB1 antibody (green), TUNEL (red), NHS-biotin (yellow), and DAPI (blue). A: Cells containing two inclusions (asterisks) and single C. pneumoniae-spots (arrows) display a regular nuclear morphology associated with a strong HMGB1 signal localized in the nucleus. B: HAEC harbouring C. pneumoniae-spots (arrows) resp. aggregates (arrow head) display condensed nuclei that have released HMGB1. C: Chloramphenicol treated infected HAEC display spots (arrows) together with a strong nuclear HMGB1 signal. D, E: Quantification of HMGB1 negative nuclei revealed a time- and dose-dependent HMGB1 release in the course of the Chlamydia infection. Two representative results such as 48 hpi (D) and 10 IFU/cell (E) are shown. Additionally labelling for TUNEL and NHS-biotin relates HMGB1 to cell death morphology (F, G, H, I). F: Infected HAEC indicated by DAPI positive spots (arrows) contain a TUNEL positive but HMGB1 negative, condensed nucleus as well as NHS-biotin positive cytoplasm. G: Apoptotic cells show a characteristic fragmentation of the nucleus positive for TUNEL and HMGB1. H: Necrotic cells lack TUNEL and HMGB1 labelling in the nucleus. I: Not infected cells display a normal nuclear morphology with a strong nuclear HMGB1 signal. Scale bar; 10 μm; G; 5 μm.