Necroptosis of Lung Epithelial Cells Triggered by Ricin Toxin and Bystander Inflammation.

BACKGROUND/AIMS: The ribosome-inactivating proteins include the biothreat agent, ricin toxin (RT). When inhaled, RT causes near complete destruction of the lung epithelium coincident with a proinflammatory response that includes TNF family cytokines, which are death-inducing ligands. We previously demonstrated that the combination of RT and TNF-related apoptosis inducing ligand (TRAIL) induces caspase-dependent apoptosis, while RT and TNF-α or RT and Fas ligand (FasL) induces cathepsin-dependent cell death in lung epithelial cells. We hypothesize that airway macrophages constitute a major source of cytokines that drive lung epithelial cell death. METHODS: Here, we show that RT-induced apoptosis of the monocytic cell line, U937, leads to the bystander killing of the lung epithelial cell line, A549. U937 cells were treated with ricin. Following this, A549 cells were treated with supernatants from U937 cells and death was measured by WST-1 viability assay. RESULTS: Upon RT-induced U937 cell death, released RT and FasL contributed to A549 cell death. U937 cells also released nuclear protein HMGB1. The release of RT, FasL, and HMGB1 triggered A549 cell necroptosis, rather than cathepsin-dependent killing observed previously with RT and FasL. Reactive oxygen species (ROS) were produced in A549 cells due to HMGB1 ligation of the receptor for advanced glycation end products (RAGE). CONCLUSION: These findings demonstrate the potential for bystander necroptosis of lung epithelial cells during RT toxicosis which may perpetuate or increase the proinflammatory response.

Storage Primes Erythrocytes for Necroptosis and Clearance.

BACKGROUND/AIMS: Like nucleated cells, erythrocytes (red blood cells, RBCs) are capable of executing programmed cell death pathways. RBCs undergo necroptosis in response to CD59-specific pore-forming toxins (PFTs). The relationship between blood bank storage and RBC necroptosis was explored in this study. METHODS: Human RBCs were stored in standard blood bank additive solutions (AS-1, AS-3, or AS-5) for 1 week and hemolysis was evaluated in the context of necroptosis inhibitors and reactive oxygen species (ROS) scavengers. Activation of key factors including RIP1, RIP3, and MLKL was determined using immunoprecipitations and western blot. RBC vesiculation and formation of echinocytes was determined using phase-contrast microscopy. The effect of necroptosis and storage on RBC clearance was determined using a murine transfusion model. RESULTS: Necroptosis is associated with increased RBC clearance post-transfusion. Moreover, storage in AS-1, AS-3, or AS-5 sensitizes RBCs for necroptosis. Importantly, storage-sensitized RBCs undergo necroptosis in response to multiple PFTs, regardless of specificity for CD59. Storage-sensitized RBCs undergo necroptosis via NADPH oxidase-generated ROS. RBC storage led to RIP1 phosphorylation and necrosome formation in an NADPH oxidase-dependent manner suggesting the basis for this sensitization. In addition, storage led to increased RBC clearance post-transfusion. Clearance of these RBCs was due to Syk-dependent echinocyte formation. CONCLUSION: Storage-induced sensitization to RBC necroptosis and clearance is important as it may be relevant to hemolytic transfusion reactions.

Mitochondrial ROS prime the hyperglycemic shift from apoptosis to necroptosis.

We have previously identified a shift from TNF-α-induced apoptosis to necroptosis that occurs under hyperglycemic conditions. This shift involves the downregulation or silencing of caspases and concurrent upregulation of necroptotic proteins leading to activation of the necrosome. In addition, under hyperglycemic conditions in vivo, this shift in cell death mechanisms exacerbates neonatal hypoxia-ischemia (HI) brain injury. Here, we identify two major factors that drive the hyperglycemic shift to necroptosis: (1) reactive oxygen species (ROS) and (2) receptor-interacting protein kinase 1 (RIP1). ROS, including mitochondrial superoxide, led to the oxidation of RIP1, as well as formation and activation of the necrosome. Concurrently, ROS mediate a decrease in the levels and activation of executioner caspases-3, -6, and -7. Importantly, hyperglycemia and mitochondrial ROS result in the oxidation of RIP1 and loss of executioner caspases prior to death receptor engagement by TNF-α. Moreover, RIP1 partially controlled levels of mitochondrial ROS in the context of hyperglycemia. As a result of its regulation of ROS, RIP1 also regulated necrosome activation and caspase loss. Mitochondrial ROS exacerbated neonatal HI-brain injury in hyperglycemic mice, as a result of the shift from apoptosis to necroptosis.

TNF Family Cytokines Induce Distinct Cell Death Modalities in the A549 Human Lung Epithelial Cell Line when Administered in Combination with Ricin Toxin.

Ricin is a member of the ribosome-inactivating protein (RIP) family of toxins and is classified as a biothreat agent by the Centers for Disease Control and Prevention (CDC). Inhalation, the most potent route of toxicity, triggers an acute respiratory distress-like syndrome that coincides with near complete destruction of the lung epithelium. We previously demonstrated that the TNF-related apoptosis-inducing ligand (TRAIL; CD253) sensitizes human lung epithelial cells to ricin-induced death. Here, we report that ricin/TRAIL-mediated cell death occurs via apoptosis and involves caspases -3, -7, -8, and -9, but not caspase-6. In addition, we show that two other TNF family members, TNF-α and Fas ligand (FasL), also sensitize human lung epithelial cells to ricin-induced death. While ricin/TNF-α- and ricin/FasL-mediated killing of A549 cells was inhibited by the pan-caspase inhibitor, zVAD-fmk, evidence suggests that these pathways were not caspase-dependent apoptosis. We also ruled out necroptosis and pyroptosis. Rather, the combination of ricin plus TNF-α or FasL induced cathepsin-dependent cell death, as evidenced by the use of several pharmacologic inhibitors. We postulate that the effects of zVAD-fmk were due to the molecule's known off-target effects on cathepsin activity. This work demonstrates that ricin-induced lung epithelial cell killing occurs by distinct cell death pathways dependent on the presence of different sensitizing cytokines, TRAIL, TNF-α, or FasL.

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation.

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.