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.

Figure 1

Response of RAW 264.7 cells to ricin. a: Immunoblot analysis of cell lysates after exposure to varying doses of ricin. Antibodies used were reactive against phospho-ERK1/2, phospho-p38 MAPK, phospho-JNK, and p38 MAPK, as shown. Cells were harvested 4 hours after the addition of ricin. b: Inhibition of protein synthesis measured by [³H] leucine incorporation 3 hours after the addition of varying concentrations of ricin. Each treatment was performed in triplicates. Data are presented as means ± SEM. c: TNF-α mRNA abundance measured by real-time RT-PCR, 5 hours after exposure to varying concentrations of ricin. Representative data from three different experiments are shown. Each treatment was performed in triplicates. Data are presented as means ± SEM. d: Ricin-induced release of TNF-α protein into the culture medium was measured by ELISA; media were harvested 6 hours after the addition of ricin. Representative data from three different experiments are shown. Each treatment was performed in triplicates. Data are presented as means ± SEM.

Figure 2

Real-time RT-PCR analysis of RNA in RAW 264.7 cells 5 hours after the addition of 1 μg/ml of ricin. Cells were pretreated for 30 minutes with selective inhibitors of the MAPK family members [20 μmol/L SP600125 (SP) for JNK, 10 μmol/L SB203580 (SB) for p38 MAPK, and 10 μmol/L UO126 (UO) for ERK1/2] or with DMSO as a vehicle control. Ricin (R) was then added (0.1 μg/ml) for 5 hours, at which time RNA was isolated. GAPDH was used as a comparator to obtained fold induction, whose value is shown in each panel. Data are expressed as percentage of maximal induction to permit relative comparisons among the six genes whose expression was analyzed. Each treatment was performed in triplicates; error bars represent SEM.

Figure 3

Analysis of blood and urine after administration of ricin in vivo. A–E, G–H: Analyses of blood from three sham-injected mice and six mice injected with 12 μg of ricin/100 g body weight. Data are presented as mean ± SEM; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. A, B: Development of anemia after intravenous ricin administration, determined by a decrease in the erythrocyte count and hemoglobin concentration. C: Thrombocytopenia. D, E: Increased numbers of polymorphonuclear leukocytes and decreased number of lymphocytes. G, H: Evidence of renal failure, as determined by increased concentrations of serum creatinine (G) and blood urea nitrogen (H). F: Decreased urine volume in mice that received 12 μg of ricin/100 g body weight compared to the saline-injected controls. Error bars show SEM; ***, P < 0.001. I: Appearance of free hemoglobin in the serum of mice that received ricin. Equal amounts of serum from three saline (1, 2, and 3) and three mice injected with 12 μg of ricin/100 g body weight (4, 5, and 6) were separated by charge by electrophoresis under nondenaturing conditions. Hemoglobin (Hb) isolated after 0.065 mol/L KCl lysis of erythrocytes was used as a positive control. I: The appearance of free hemoglobin in all of the animals that received ricin. J: Absorption spectrophotometry of sera from I demonstrates absorbance maxima at 431 nm and 555 nm, characteristic of hemoglobin. An increase in the absorbance peaks at these maxima was observed in the sera of mice injected with ricin. Although the peaks of hemoglobin absorbance maxima were lower for each of the control mice, the control sera were pooled in this figure to facilitate graphic representation. K: Albuminuria after ricin administration. Equal amounts of urine from three saline-injected mice (1, 2, and 3) and three mice injected with 12 μg of ricin/100 g body weight (4, 5, and 6) were run on a 10% SDS-PAGE. Representative data are shown from two different experiments, each treatment performed in triplicate. Bovine serum albumin at increasing concentrations (10, 50, and 100 μg) was used as a standard. MW stands for molecular weight of the commercially available protein markers.

Figure 4

Immunohistochemical localization of fibrin(ogen) in kidneys of ricin-treated animals. Mice injected with saline (c, d) or 12 μg/100 g body weight ricin (a, b) were sacrificed 24 hours after the injections. Carnoy-fixed tissues were embedded in paraffin and sections were reacted with primary goat polyclonal antibody against mouse fibrin(ogen). Kidneys of ricin-injected mice display fibrin(ogen) depositions in the glomerular capillary loops (b) and in the peritubular microvasculature (a). Representative pictures from two independent experiments, each performed in triplicate animals, are shown.

Figure 5

Activation of p38 MAPK, JNK, and ERK1/2 in kidneys of mice injected with ricin. Kidney lysates from three sham-injected mice and four mice injected with 12 μg of ricin/100 g body weight were analyzed by immunoblotting with antibodies against phospho-ERK1/2, phospho-p38 MAPK, and phospho-JNK. Results demonstrate activation of all three MAPK family members after ricin administration. Immunoblotting with anti-p38 MAPK antibody was used as a loading control. This experiment was performed three times; each treatment group included six animals. Representative data from one experiment is shown, each sample representing kidney lysate from an individual animal.

Figure 6

Ricin induces the phosphorylation of ERK, JNK, and p38 MAPK in glomerular cells. Immunohistochemical staining with phospho-ERK1/2, phospho-p38 MAPK, and phospho-JNK antibodies performed on kidney tissue sections of control and ricin-injected mice (12 μg/100 g body weight i.v. for 24 hours.) after fixation by perfusion with 4% paraformaldehyde solution. Each image contains one representative glomerulus. Immunohistochemical staining reactions were performed on sections from animals from three different experiments, each performed on groups of six mice.

Figure 7

Real-time RT-PCR analysis of ricin-induced gene expression in mouse kidney. Groups of three mice received intravenously 12 μg of ricin/100 g body weight and were sacrificed at the designated times when kidneys were removed. After RNA extraction, real-time RT-PCR analysis was performed using primers to the corresponding genes. Error bars represent SEM. P values are as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; and NS, nonsignificant.

Figure 8

Primer-extension analysis of ricin-induced lesions in 28S rRNA in mouse kidney. a: THP-1 cells were exposed to vehicle alone (lane1) or 1 μg/ml of ricin for 6 hours (lane 2); kidneys from either sham-injected (lane 3) or ricin-injected (12 μg of ricin/100 g body weight; lane 4) mice were harvested 24 hours after the injection. The circled adenine indicates the site of action of ricin within the sarcin/ricin loop of 28S rRNA. b: Ribotoxic damage of A 4256 in 28S rRNA from kidneys after ricin administration in vivo. Animals were injected with saline (CO) or ricin (12 μg/100 g body weight) and were sacrificed at the times shown. Error bars represent SEM.

Figure 9

Gene expression in mouse kidneys after administration of LPS, ricin, and LPS plus ricin. Mice received 12 μg of ricin/100 g body weight i.v., LPS 10 μg i.p., or both. Each group contains triplicate mice, which were sacrificed at 24 hours for harvesting kidneys. Real-time RT-PCR analyses were performed on RNA extracted from harvested organs. P values are as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; and NS, nonsignificant. Asterisks above bars show comparison between treatments and controls; P values shown below bars of LPS plus ricin treatment show the comparison of this treatment to ricin alone. Error bars represent SEM.