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We are interested in the cytotoxic and proinflammatory effects of particulate pollutants in the respiratory tract. We demonstrate that methanol extracts made from diesel exhaust particles (DEP) induce apoptosis and reactive oxygen species (ROS) in pulmonary alveolar macrophages and RAW 264.7 cells. The toxicity of these organic extracts mimics the cytotoxicity of the intact particles and could be suppressed by the synthetic sulfhydryl compounds, N-acetylcysteine and bucillamine. Because DEP-induced apoptosis follows cytochrome c release, we studied the effect of DEP chemicals on mitochondrially regulated death mechanisms. Crude DEP extracts induced ROS production and perturbed mitochondrial function before and at the onset of apoptosis. This mitochondrial perturbation follows an orderly sequence of events, which commence with a change in mitochondrial membrane potential, followed by cytochrome c release, development of membrane asymmetry (annexin V staining), and propidium iodide uptake. Structural damage to the mitochondrial inner membrane, evidenced by a decrease in cardiolipin mass, leads to O⨪2 generation and uncoupling of oxidative phosphorylation (decreased intracellular ATP levels). N-Acetylcysteine reversed these mitochondrial effects and ROS production. Overexpression of the mitochondrial apoptosis regulator, Bcl-2, delayed but did not suppress apoptosis. Taken together, these results suggest that DEP chemicals induce apoptosis in macrophages via a toxic effect on mitochondria.

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Apoptosis is a central host defense mechanism to eliminate virus-infected cells. Activation of NF-κB suppresses apoptosis following some types of stimulation in vitro. To test the physiological importance of this pathway in vivo, we studied murine encephalomyocarditis virus (EMCV) infection in mice and cell lines defective in NF-κB1 (p50) signaling. As previously reported, we find that all p50 knockout (p50 −/−) mice survive an EMCV infection that readily kills normal mice. By introducing the p50 mutation into interferon (IFN) type I receptor knockout (IFNRI −/−) mice, we find that this resistance is not mediated by IFN-β as previously thought. While no IFNRI −/− mice survive, the double-knockout mice survive 60% of the time. The survival is tightly linked to the animals’ ability to clear the virus from the heart in vivo. Using murine embryonic fibroblasts (MEF) derived from wild-type, p50 −/−, and p65 −/− embryos, we found that NF-κB is not required for the replication cycle of EMCV. However, during these experiments we observed that p50 −/− and p65 −/− MEF infected with EMCV undergo enhanced, premature cytotoxicity. Upon examination of this cell death, we found that EMCV infection induced both plasma membrane and nuclear changes typical of apoptosis in all cell lines. These apoptotic processes occurred in an accelerated and pronounced way in the NF-κB-defective cells, as soon as 6 h after infection, when virus is beginning to be released. Previously, only the RelA (p65) subunit of NF-κB has been shown to play a role in suppressing apoptosis. In our studies, we find that p50 is equally important in suppressing apoptosis during EMCV infection. Additionally, we show that suppression of apoptosis by NF-κB1 is required for EMCV virulence in vivo. The attenuation in p50 −/− mice can be explained by rapid apoptosis of infected cells which allows host phagocytes to clear infected cells before the viral burst leading to a reduction of the viral burden and survival of the mice.

There is increasing evidence that particulate air pollutants, such as diesel exhaust particles (DEP), potentiate chronic inflammatory processes as well as acute symptomatic responses in the respiratory tract. The mechanisms of action as well as the cellular targets for DEP remain to be elucidated. We show in this paper that the phagocytosis of DEP by primary alveolar macrophages or macrophage cell lines, RAW 264.7 and THP-1, leads to the induction of apoptosis through generation of reactive oxygen radicals (ROR). This oxidative stress initiates two caspase cascades and a series of cellular events, including loss of surface membrane asymmetry and DNA damage. The apoptotic effect on macrophages is cell specific, because DEP did not induce similar effects in nonphagocytic cells. DEP that had their organic constituents extracted were no longer able to induce apoptosis or generate ROR. The organic extracts were, however, able to induce apoptosis. DEP chemicals also induced the activation of stress-activated protein kinases, which play a role in cellular apoptotic pathways. The injurious effects of native particles or DEP extracts on macrophages could be reversed by the antioxidant, N-acetyl-cysteine. Taken together, these data suggest that organic compounds contained in DEP may exert acute toxic effects via the generation of ROR in macrophages.