Brian Keyser

Sulfur Mustard Induces Apoptosis in Cultured Normal Human Airway Epithelial Cells: Evidence of a Dominant Caspase-8-Mediated Pathway and Differential Cellular Responses

We have shown that sulfur mustard (SM; bis-(2-chloroethyl) sulfide), an alkylating, vesicating chemical warfare agent, causes dermal toxicity, including skin microblisters, via the induction of both death receptor (DR) and mitochondrial pathways of apoptosis in human epidermal keratinocytes. While SM is known for its skin-vesicating properties, respiratory tract lesions are the main source of morbidity and mortality after inhalation exposure. We, therefore, investigated whether SM induces apoptotic cell death in normal human bronchial epithelial (NHBE) cells and small airway epithelial cells (SAEC) in vitro. Cells were exposed to various concentrations of SM (0, 50, 100, and 300 μM for 16 h) in the culture medium and then tested for the activation of apoptotic executioner caspase-3 and initiator caspases-8 and -9. Caspases-8 and -3 were activated by SM in both airway cell types, indicating the induction of a DR pathway of apoptosis in these cells; however, the levels of enzyme activation were different, depending on the cell type and the SM concentrations used. Consistent with enzyme activity results, immunoblot analyses revealed the proteolytic processing of the proenzymes to the active forms of caspases-8 and -3 in these cells after SM exposure. Interestingly, NHBE cells were found to be exquisitely sensitive to SM, compared to SAEC, with caspase-3 activities in SM-exposed NHBE cells ∼2-fold higher and caspase-8 activities ∼10-fold higher than in SAEC. Furthermore, SM activated caspase-9 in NHBE cells, but not in SAEC, indicating a possible role of the mitochondrial pathway only in the NHBE cells. The present study shows that both upper airway (NHBE cells) and deep lung (SAEC) epithelial cells undergo SM-induced apoptotic death in vitro, but distinct cell-type specific responses can be elicited, which may be attributed to intrinsic properties that characterize the response of these cells to SM. These findings need to be taken into consideration in the search for modulators of these pathways for the therapeutic intervention to reduce SM injury due to respiratory tract lesions.

Sulfur mustard induces apoptosis in lung epithelial cells via a caspase amplification loop

Sulfur mustard (SM [bis-(2-chloroethyl) sulfide]) is a chemical warfare agent that causes skin blisters presumably due to DNA alkylation and cross-links. We recently showed that SM also induces apoptotic death in cultured normal human bronchial/tracheal epithelial (NHBE) cells and small airway epithelial cells (SAEC) in vitro. In this process, caspases-8 and -3, but not caspase-9, were strongly activated; this suggests a death receptor pathway for apoptosis. We now show that rat lungs were induced to undergo apoptosis in vivo following exposure of rats to SM by inhalation. Further study of the mechanism of apoptosis due to SM was performed with cultured NHBE cells and SAEC using tetrapeptide inhibitors of caspases-3, and -8. Inhibition of caspase-8 drastically reduced the activation of caspase-3 and almost eliminated that of caspase-9. Moreover, caspase-3 inhibition markedly reduced the activation of caspase-8 and also almost completely inhibited activation of caspase-9. These results suggest a death receptor pathway of apoptosis that utilizes a feedback amplification mechanism involving an activated death receptor complex that leads to the activation of caspase-9 via a caspase-3 pathway. These results may be important for the design of inhibitors of these pathways for therapeutic intervention to attenuate SM injury in respiratory tract lesions.

Apoptotic cell death in rat lung following mustard gas inhalation

To investigate apoptosis as a mechanism of sulfur mustard (SM) inhalation injury in animals, we studied different caspases (caspase-8, -9, -3, and -6) in the lungs from a ventilated rat SM aerosol inhalation model. SM activated all four caspases in cells obtained from bronchoalveolar lavage fluid (BALF) as early as 6 h after exposure. Caspase-8, which is known to initiate the extrinsic Fas-mediated pathway of apoptosis, was increased fivefold between 6 and 24 h, decreasing to the unexposed-control level at 48 h. The initiator, caspase-9, in the intrinsic mitochondrial pathway of apoptosis as well as the executioner caspases, caspase-3 and -6, all peaked (P < 0.01) at 24 h; caspase-3 and -6 remained elevated, but caspase-9 decreased to unexposed-control level at 48 h. To study further the Fas pathway, we examined soluble as well as membrane-bound Fas ligand (sFas-L and mFas-L, respectively) and Fas receptor (Fas-R) in both BALF cells and BALF. At 24 h after SM exposure, sFas-L increased significantly in both BALF cells (P < 0.01) and BALF (P < 0.05). However, mFas-L increased only in BALF cells between 24 and 48 h (P < 0.1 and P < 0.001, respectively). Fas-R increased only in BALF cells by 6 h (P < 0.01) after SM exposure. Apoptosis in SM-inhaled rat lung specimens was also confirmed by both immunohistochemical staining using cleaved caspase-3 and -9 antibodies and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining as early as 6 h in the proximal trachea and bronchi, but not before 48 h in distal airways. These findings suggest pathogenic mechanisms at the cellular and molecular levels and logical therapeutic target(s) for SM inhalation injury in animals.