Postexposure application of Fas receptor small-interfering RNA to suppress sulfur mustard-induced apoptosis in human airway epithelial cells: implication for a therapeutic approach

• Published in: The Journal of pharmacology and experimental therapeutics 2013-01, Vol.344 (1), p.308-316
• Main Authors: Keyser, Brian M, Andres, Devon K, Nealley, Eric, Holmes, Wesley W, Benton, Betty, Paradiso, Danielle, Appell, Ashley, Carpin, Chris, Anderson, Dana R, Smith, William J, Ray, Radharaman
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis - drug effects; Blotting, Western; Bronchoalveolar Lavage Fluid - chemistry; Burns, Inhalation - drug therapy; Caspase 3 - metabolism; Cell Line; Cells, Cultured; Chemical Warfare Agents - toxicity; Enzyme Activation - physiology; Enzyme-Linked Immunosorbent Assay; Epithelial Cells - drug effects; Fas Ligand Protein - analysis; Fas Ligand Protein - metabolism; fas Receptor - antagonists & inhibitors; fas Receptor - genetics; Flow Cytometry; Humans; Male; Mustard Gas - toxicity; Rats; Rats, Sprague-Dawley; Respiratory System - cytology; Respiratory System - drug effects; RNA, Small Interfering - pharmacology; Transfection
• ISSN: 0022-3565; 1521-0103
• DOI: 10.1124/jpet.112.199935

Sulfur mustard (SM) is a vesicant chemical warfare and terrorism agent. Besides skin and eye injury, respiratory damage has been mainly responsible for morbidity and mortality after SM exposure. Previously, it was shown that suppressing the death receptor (DR) response by the dominant-negative Fas-associated death domain protein prior to SM exposure blocked apoptosis and microvesication in skin. Here, we studied whether antagonizing the Fas receptor (FasR) pathway by small-interfering RNA (siRNA) applied after SM exposure would prevent apoptosis and, thus, airway injury. Normal human bronchial/tracheal epithelial (NHBE) cells were used as an in vitro model with FasR siRNA, FasR agonistic antibody CH11, and FasR antagonistic antibody ZB4 as investigative tools. In NHBE cells, both SM (300 µM) and CH11 (100 ng/ml) induced caspase-3 activation, which was inhibited by FasR siRNA and ZB4, indicating that SM-induced apoptosis was via the Fas response. FasR siRNA inhibited SM-induced caspase-3 activation when added to NHBE cultures up to 8 hours after SM. Results using annexin V/propidium iodide-stained cells showed that both apoptosis and necrosis were involved in cell death due to SM; FasR siRNA decreased both apoptotic and necrotic cell populations. Bronchoalveolar lavage fluid (BALF) of rats exposed to SM (1 mg/kg, 50 minutes) revealed a significant (P < 0.05) increase in soluble Fas ligand and active caspase-3 in BALF cells. These findings suggest an intervention of Fas-mediated apoptosis as a postexposure therapeutic strategy with a therapeutic window for SM inhalation injury and possibly other respiratory diseases involving the Fas response.