Role of Apoptosis in Pseudomonas aeruginosa Pneumonia by Richard S. Hotchkiss, W. Michael Dunne, Paul E. Swanson, Christopher G. Davis, Kevin W. Tinsley, Katherine C. Chang, Timothy G. Buchman, and Irene E. Karl Science Volume 294(5548):1783-1783 November 30, 2001 Published by AAAS

Figure 1 (A) Electron micrograph of ciliated bronchial epithelial cells from saline-treated mouse (sham), demonstrating normal morphology. (A) Electron micrograph of ciliated bronchial epithelial cells from saline-treated mouse (sham), demonstrating normal morphology. Arrows identify nuclei with normal diffuse chromatin pattern. (B) Ciliated respiratory bronchial epithelial cells from P. aeruginosa–treated mouse show normal cell morphology. Nuclei are identified by arrows and have normal diffuse chromatin pattern without chromatin condensation or fragmentation. Bacteria (arrowheads) and inflammatory debris are in bronchial lumen in the right half of the image. Richard S. Hotchkiss et al. Science 2001;294:1783 Published by AAAS

Figure 2 (A) Two capillary endothelial cells (arrows, upper left portion of photomicrograph) are positive for active caspase 3 and have secondary morphologic changes consistent with apoptosis. (A) Two capillary endothelial cells (arrows, upper left portion of photomicrograph) are positive for active caspase 3 and have secondary morphologic changes consistent with apoptosis. Two lymphocytes (remaining arrows) are also positive for active caspase 3. Magnification, ×600. It should be noted that bronchial cells (lower right margin of the image) are negative for active caspase 3. (B) Macrophages (arrows) have ingested apoptotic debris that is positive for active caspase 3. Magnification, ×600. Richard S. Hotchkiss et al. Science 2001;294:1783 Published by AAAS

Figure 3 Lung tissue obtained 24 hours after intratracheal injection and evaluated by TUNEL method. Lung tissue obtained 24 hours after intratracheal injection and evaluated by TUNEL method. TUNEL-positive bronchial epithelial cells have brown staining nuclei and are present in both (A) saline-treated (sham) lung and (B) bacterial-treated (pneumonia) lung. Magnification, ×400. It should be noted that the nuclei of the bronchial epithelial cells have normal morphology, without evidence of contraction or fragmentation. Richard S. Hotchkiss et al. Science 2001;294:1783 Published by AAAS

Figure 4 Thymi obtained 24 hours after intratracheal injection in (A) saline-treated (sham) and (B) bacterial-treated (pneumonia) mice. Thymi obtained 24 hours after intratracheal injection in (A) saline-treated (sham) and (B) bacterial-treated (pneumonia) mice. Magnification, ×600. Cells positive for active caspase 3 (brown stain) show a marked increase in mice with pneumonia; nuclei are also compacted and fragmented. Richard S. Hotchkiss et al. Science 2001;294:1783 Published by AAAS

Figure 1 TEM photomicrographs (14) of lung tissue. TEM photomicrographs (14) of lung tissue. (A) Lung section from uninfected mice show nuclei with homogeneous chromatin (magnification, ×6700). (B) Lung section from mice 12 hours after infection with ATCC 27853 reveals condensation and extensive fragmentation of nuclear chromatin (magnification, ×6700). (C) The presence of cilia indicates apoptosis in epithelial cells (magnification, ×21,000). Similar results were obtained in 5 independent infection experiments. Richard S. Hotchkiss et al. Science 2001;294:1783 Published by AAAS

Figure 2 Monoclonal antibody test for single-stranded DNA (15) in (A) cells from uninfected mice, (B) cells from mice infected with early P. aeruginosa grown to early mid-logarithmic phase, and (C) cells from mice infected with plateau phase–grown bacteria. Monoclonal antibody test for single-stranded DNA (15) in (A) cells from uninfected mice, (B) cells from mice infected with early P. aeruginosa grown to early mid-logarithmic phase, and (C) cells from mice infected with plateau phase–grown bacteria. Single-stranded DNA is detected in (B) but is absent in (A) and (C). Richard S. Hotchkiss et al. Science 2001;294:1783 Published by AAAS

Figure 3 FACS and fluorescence microscopy (20) confirm that only logarithmic-grown P. aeruginosa trigger apoptosis of more than 90% of WI-38 cells after 60 min of infection. FACS and fluorescence microscopy (20) confirm that only logarithmic-grown P. aeruginosa trigger apoptosis of more than 90% of WI-38 cells after 60 min of infection. Apoptosis was detected by FITC-Annexin labeling; simultaneous staining with PI excluded necrosis. Data are representative for five experiments. (A) Histogram of FITC-Annexin fluorescence 1 hour after infection. Blue line, uninfected cells; red line, cells infected with early mid-logarithmic growth bacteria; yellow line, cells infected with plateau phase bacteria; pink line, cells infected with plate-grown bacteria. (B) Dot blots for logarithmic-grown bacteria 1 hour after infection, comparing fluorescence for PI against fluorescence for FITC-Annexin for uninfected and infected cells. (C) Fluorescence microscopy for uninfected cells (top) and for cells 1 hour after infection with logarithmic-grown bacteria. Richard S. Hotchkiss et al. Science 2001;294:1783 Published by AAAS

Figure 4 Internalization of P Figure 4 Internalization of P. aeruginosa by epithelial cells depends on bacterial growth conditions (21). Internalization of P. aeruginosa by epithelial cells depends on bacterial growth conditions (21). Left-hand panels show representative results of crystal violet stainings of WI-38 cells in vitro, for (A) uninfected cells, (B) cells infected with early mid- logarithmic–grown P. aeruginosa ATCC 27853, and (C) cells infected with plateau phase bacteria. (D) Invasion of WI-38 cells by P. aeruginosa in vitro, 15 min and 30 min after infection. Results show mean ± SD for 600 cells. Gray bars, early mid-logarithmic; black bars, plateau phase; white bars, plate growth. (E) Invasion of murine lung cells by P. aeruginosaATCC 27853 in vivo. Results show mean ± SD for five independent infections. Gray bar, early mid-logarithmic; black bar, plateau phase; white bar, plate growth. Richard S. Hotchkiss et al. Science 2001;294:1783 Published by AAAS