Administration of Ricin Induces a Severe Inflammatory Response via Nonredundant Stimulation of ERK, JNK, and P38 MAPK and Provides a Mouse Model of Hemolytic Uremic Syndrome

Abstract

Recent interest in the health consequences of ricin as a weapon of terrorism has led us to investigate the effects of ricin on cells in vitro and in mice. Our previous studies showed that depurination of the 28S rRNA by ricin results in the inhibition of translation and the coordinate activation of the stress-activated protein kinases JNK and p38 MAPK. In RAW 264.7 macrophages, ricin induced the activation of ERK, JNK, and p38 MAPK, the accumulation of mRNA encoding tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, the transcription factors c-Fos, c-Jun, and EGR1, and the appearance of TNF-alpha protein in the culture medium. Using specific inhibitors of MAPKs, we demonstrated the nonredundant roles of the individual MAPKs in mediating proinflammatory gene activation in response to ricin. Similarly, the intravenous administration of ricin to mice led to the activation of ERK, JNK, and p38 MAPK in the kidneys, and increases in plasma-borne TNF-alpha, IL-1beta, and IL-6. Ricin-injected mice developed the hallmarks of hemolytic uremic syndrome, including thrombotic microangiopathy, hemolytic anemia, thrombocytopenia, and acute renal failure. Microarray analyses demonstrated a massive proinflammatory transcriptional response in the kidneys, coincidental with the symptoms of hemolytic uremic syndrome. Therapeutic management of the inflammatory response may affect the outcome of intoxication by ricin.

Intrapulmonary Delivery of Ricin at High Dosage Triggers a Systemic Inflammatory Response and Glomerular Damage

“…13 The appearance of comparable numbers of lesions in 28S rRNA in lung and kidneys of ricin-instilled mice (Figure 6a) suggested that the kidney may serve as a relevant target of ricin under the experimental conditions that were applied. Histopathological examination of kidney sections after H&E staining revealed mild accumulation of inflammatory cells of mixed origin into the glomerular capillary loops of mice instilled intratracheally with 20 g of ricin/100 g body weight 48 hours before sacrifice (not shown).…”

“…13 Equal amounts of protein of each lysate were separated on a 10% denaturing polyacrylamide gel in the presence of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes according to standard laboratory procedures. Membranes were incubated with the indicated antibodies and the corresponding horseradish peroxidase-conjugated secondary antibodies; signals were detected using enhanced chemiluminescence.…”

“…13 To determine whether instilled ricin would activate these kinases, we subjected lung tissue lysates to analysis by immunoblotting 48 hours after intratracheal instillation of 20 g of ricin/100 g body weight ( Figure 3a). Instillation of ricin led to increased phosphorylation of ERK and JNK, but not p38 MAPK ( Figure 3, lanes 1 to 3 versus lanes 5 to 8), probably attributable to the substantially activated state of the latter kinase in the lungs of control mice.…”

“…Reverse transcription was performed using a primer that binds near the known ricin-mediated depurination site of the 28S rRNA 10,13 to detect the presence of stalled transcripts. Truncated transcripts were detected from all ricintreated tissues except brain.…”

“…15 Previously, we and others have reported that intravenous administration of ricin to mice recapitulates many of the symptoms of hemolytic uremic syndrome, including thrombotic microangiopathy, hemolytic anemia, thrombocytopenia, increases in plasma-borne cytokines, and acute renal failure. 13,16,17 Analysis of proinflammatory gene expression in multiple organs revealed that intravenous ricin induced a robust response, coincident with the symptoms of hemolytic uremic syndrome.…”