FIG. 2.

Soluble factors released from infected apoptotic human macrophages do not induce apoptosis in uninfected bystander macrophages. (A) Macrophages were infected with M. tuberculosis strain H37Ra for 4 h. At 96 h postinfection, the level of apoptosis in infected cells, as indicated by DNA fragmentation, was determined using a cell death detection ELISA. H37Ra infection induced a significant level of apoptosis compared to the uninfected control (P < 0.0001). Treatment with staurosporine was used as a positive control. (B) Supernatants and conditioned medium from infected apoptotic cells were harvested 24, 48, and 96 h after infection and added to a parallel culture of uninfected PMA-differentiated THP-1 cells. The apoptosis levels in uninfected THP-1 cells were determined using the cell death detection ELISA 96 h after the addition of conditioned medium. No significant apoptosis occurred in uninfected cells after exposure to conditioned medium from apoptotic infected cells. The data are expressed as fold increases compared with the uninfected control (means and standard errors of the means) of at least three separate experiments. (C) Transwell culture dishes were seeded with PMA-differentiated THP-1 macrophages (0.5 × 10⁵ cells/ml). Macrophages in the upper chamber were infected with M. tuberculosis strain H37Ra for 4 h and were separated from uninfected macrophages in the lower chamber by a permeable membrane (pore size, 0.1 μm). At 48 h postinfection the uninfected THP-1 macrophages (0.5 × 10⁵ cells) in the lower chamber had not undergone significant levels of apoptosis compared to the uninfected control as determined by TUNEL TMR staining. As a positive control, PMA-differentiated THP-1 macrophages were treated with staurosporine (P < 0.0001). (D) Fluorescent microscopy (magnification, ×20) images of uninfected macrophages from the lower chambers of transwell culture dishes. The left panels show Hoechst and TUNEL staining of uninfected control macrophages. The middle panels show staining of uninfected bystander macrophages that were exposed to culture medium from infected cells for 48 h. The right panels show staining of macrophages that were exposed to staurosporine as a positive control. Statistical significance was determined using Student's t test (one asterisk, P < 0.05; two asterisks, P < 0.001; three asterisks, P < 0.0001). Mtb, M. tuberculosis.