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

Hodges Hodges,Parker Parker,Larocca Larocca,Mccaig Mccaig,Kempen Kempen,Mantis Mantis

doi:10.3390/toxins11080450

Abstract

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.

Highlights

Ricin is a 60–65 kDa glycoprotein toxin derived from the castor bean plant, Ricinus communis [1,2,3]
To test the hypothesis that extrinsic cytokines cause an increase in ricin-induced cell death, A549 cells were treated with increasing doses of ricin in the absence or presence of 100 ng/mL TNF-related apoptosis-inducing ligand (TRAIL), TNF-α, or Fas ligand (FasL) for 24 h at 37 ◦C
Our results indicate that ricin induces distinct cell death modalities in A549 human lung epithelial cells depending upon the TNF family cytokine with which it is paired

Summary

Introduction

Ricin is a 60–65 kDa glycoprotein toxin derived from the castor bean plant, Ricinus communis [1,2,3]. Ricin is a member of the type II family of ribosome-inactivating proteins (RIPs), consisting of a catalytic A subunit (RTA) attached via disulfide bond to a cell-binding B subunit (RTB) [6,7]. RTA inhibits protein synthesis by virtue of its ability to cleave a specific glycosidic bond in the so-called sarcin-ricin loop (SRL) of rRNA in the 60s ribosomal subunit [10,11,12]. Depurination of the SRL leads to the cessation of protein synthesis [11,12]. This activity is so potent that it has been noted that a single RTA can inhibit the function of 1500 ribosomes per minute [1]. Wide-scale damage caused by inhaled ricin leads to acute respiratory distress syndrome (ARDS) which is characterized by a potent proinflammatory response [16,17,18,19]

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