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
2005; Elsevier BV; Volume: 166; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)62256-0
ISSN1525-2191
AutoresVeselina Korcheva, John Wong, Christopher L. Corless, Mihail S. Iordanov, Bruce E. Magun,
Tópico(s)Transgenic Plants and Applications
ResumoRecent 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)-α, interleukin (IL)-1, the transcription factors c-Fos, c-Jun, and EGR1, and the appearance of TNF-α 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-α, IL-1β, 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. 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)-α, interleukin (IL)-1, the transcription factors c-Fos, c-Jun, and EGR1, and the appearance of TNF-α 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-α, IL-1β, 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. In view of its wide availability and ease of purification, ricin has been used as a toxic and lethal agent by totalitarian regimes and, recently, by terrorist groups.1Franz DR Jaax JK Ricin toxin.in: Sidell FR ET Takfuji DR Franz Textbook of Military Medicine, Part 1. Warfare, Weaponry, and the Casualty: Medical Aspects of Chemical and Biological Warfare. Office of the Surgeon General, Department of the Army, Washington1997: 631-642Google Scholar In humans, the estimated lethal dose of ricin is 1 to 10 μg per kg of body weight.2Smallshaw JE Firan A Fulmer JR Ruback SL Ghetie V Vitetta ES A novel recombinant vaccine which protects mice against ricin intoxication.Vaccine. 2002; 20: 3422-3427Crossref PubMed Scopus (81) Google Scholar The majority of described cases of ricin intoxication has resulted from the ingestion of castor beans and is manifested by hemorrhagic diarrhea, liver necrosis, diffuse nephritis, and splenitis.1Franz DR Jaax JK Ricin toxin.in: Sidell FR ET Takfuji DR Franz Textbook of Military Medicine, Part 1. Warfare, Weaponry, and the Casualty: Medical Aspects of Chemical and Biological Warfare. Office of the Surgeon General, Department of the Army, Washington1997: 631-642Google Scholar One of the few described cases of ricin injection was the political assassination of the noted Bulgarian dissident Georgi Markov3Crompton R Gall D Georgi Markov—death in a pellet.Med Leg J. 1980; 48: 51-62PubMed Google Scholar whose body was penetrated by a ricin-containing pellet. Before his death, which occurred 3 days later, he developed fever, lymphadenopathy near the site of inoculation, hypotension with vascular collapse, and shock.3Crompton R Gall D Georgi Markov—death in a pellet.Med Leg J. 1980; 48: 51-62PubMed Google Scholar Although the toxicity of ricin varies according to the route of administration, the clinical symptoms frequently are related to a severe inflammatory response and multiorgan failure. Ricin is a member of a family of protein toxins whose cytosolic target is the 28S rRNA of the 60S ribosomal subunit.4Barbieri L Battelli MG Stirpe F Ribosome-inactivating proteins from plants.Biochim Biophys Acta. 1993; 1154: 237-282Crossref PubMed Scopus (803) Google Scholar The cytotoxicity of ricin results from the depurination of the 28S rRNA at a single adenine nucleotide (A4565 in humans and A4256 in mouse) with consequent inhibition of protein translation. 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These features include extensive thrombotic microangiopathy, hemolytic anemia, thrombocytopenia, and acute renal failure.14Gerber A Karch H Allerberger F Verweyen HM Zimmerhackl LB Clinical course and the role of Shiga toxin-producing Escherichia coli infection in the hemolytic-uremic syndrome in pediatric patients, 1997–2000, in Germany and Austria: a prospective study.J Infect Dis. 2002; 186: 493-500Crossref PubMed Scopus (299) Google Scholar, 15Paton JC Paton AW Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections.Clin Microbiol Rev. 1998; 11: 450-479Crossref PubMed Google Scholar, 16Ruggenenti P Noris M Remuzzi G Thrombotic microangiopathy, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura.Kidney Int. 2001; 60: 831-846Crossref PubMed Scopus (371) Google Scholar, 17Proulx F Seidman EG Karpman D Pathogenesis of Shiga toxin-associated hemolytic uremic syndrome.Pediatr Res. 2001; 50: 163-171Crossref PubMed Scopus (156) Google Scholar Both ricin and Stx act to depurinate the same adenine within the ricin/sarcin loop of eukaryotic mammalian 28S rRNA.18O'Brien AD Holmes RK Shiga and Shiga-like toxins.Microbiol Rev. 1987; 51: 206-220Crossref PubMed Google Scholar Each toxin consists of A and B subunits, of which the B subunits determine the binding to cell surfaces. Whereas ricin binds to galactose residues,19Olsnes S Kozlov JV Ricin.Toxicon. 2001; 39: 1723-1728Crossref PubMed Scopus (147) Google Scholar Stx binds to cell surfaces via a glycosphingolipid receptor, Gb3.20Cohen A Hannigan GE Williams BR Lingwood CA Roles of globotriosyl- and galabiosylceramide in verotoxin binding and high affinity interferon receptor.J Biol Chem. 1987; 262: 17088-17091Abstract Full Text PDF PubMed Google Scholar After endocytosis and retrograde transport through the Golgi apparatus, the A subunits of each toxin enter the cytosol where they depurinate 28S rRNA, thereby inhibiting protein synthesis21Sandvig K Grimmer S Lauvrak SU Torgersen ML Skretting G van Deurs B Iversen TG Pathways followed by ricin and Shiga toxin into cells.Histochem Cell Biol. 2002; 117: 131-141Crossref PubMed Scopus (125) Google Scholar and activating the SAPK cascade.5Iordanov MS Pribnow D Magun JL Dinh TH Pearson JA Chen SL Magun BE Ribotoxic stress response: activation of the stress-activated protein kinase JNK1 by inhibitors of the peptidyl transferase reaction and by sequence-specific RNA damage to the alpha-sarcin/ricin loop in the 28S rRNA.Mol Cell Biol. 1997; 17: 3373-3381Crossref PubMed Google Scholar HUS is a major cause of acute renal failure in children in North America.22Rowe PC Orrbine E Wells GA McLaine PN Epidemiology of hemolytic-uremic syndrome in Canadian children from 1986 to 1988. The Canadian Pediatric Kidney Disease Reference Centre.J Pediatr. 1991; 119: 218-224Abstract Full Text PDF PubMed Scopus (149) Google Scholar, 23Thorpe CM Shiga toxin-producing Escherichia coli infection.Clin Infect Dis. 2004; 38: 1298-1303Crossref PubMed Scopus (116) Google Scholar Abundant evidence supports the conclusion that diarrhea-associated HUS involves an acute inflammatory response, the extent of which is a predictor of the clinical outcome. Patients with HUS display markedly elevated proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and chemokines such as monocyte chemoattractant protein-1 (MCP-1), IL-8, growth related oncogene (Gro)-α and -γ.15Paton JC Paton AW Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections.Clin Microbiol Rev. 1998; 11: 450-479Crossref PubMed Google Scholar, 17Proulx F Seidman EG Karpman D Pathogenesis of Shiga toxin-associated hemolytic uremic syndrome.Pediatr Res. 2001; 50: 163-171Crossref PubMed Scopus (156) Google Scholar, 24Litalien C Proulx F Mariscalco MM Robitaille P Turgeon JP Orrbine E Rowe PC McLaine PN Seidman E Circulating inflammatory cytokine levels in hemolytic uremic syndrome.Pediatr Nephrol. 1999; 13: 840-845Crossref PubMed Scopus (49) Google Scholar, 25Yamamoto T Isokawa S Miyata H Yoshioka K Evaluation of thrombomodulin and tumor necrosis factor-alpha levels in patients with hemolytic uremic syndrome caused by enterohemorrhagic Escherichia coli O157: H7 infection.Nippon Jinzo Gakkai Shi. 1999; 41: 60-64PubMed Google Scholar, 26Lopez EL Contrini MM Devoto S de Rosa MF Grana MG Genero MH Canepa C Gomez HF Cleary TG Tumor necrosis factor concentrations in hemolytic uremic syndrome patients and children with bloody diarrhea in Argentina.Pediatr Infect Dis J. 1995; 14: 594-598Crossref PubMed Scopus (58) Google Scholar The availability of suitable experimental animal models of HUS could provide insight into the molecular mechanisms and sequence of events that occur in HUS. However, the distribution of Gb3 receptors for Stx on cell types varies widely among species, and it has been suggested that these differences may account for the inability of Stx to recapitulate the hallmarks of the human HUS in the available animal models.13Taylor CM Williams JM Lote CJ Howie AJ Thewles A Wood JA Milford DV Raafat F Chant I Rose PE A laboratory model of toxin-induced hemolytic uremic syndrome.Kidney Int. 1999; 55: 1367-1374Crossref PubMed Scopus (42) Google Scholar To bypass the restricted distribution of Stx receptors, Taylor and colleagues13Taylor CM Williams JM Lote CJ Howie AJ Thewles A Wood JA Milford DV Raafat F Chant I Rose PE A laboratory model of toxin-induced hemolytic uremic syndrome.Kidney Int. 1999; 55: 1367-1374Crossref PubMed Scopus (42) Google Scholar administered ricin, which, unlike Stx, was able to recapitulate many of the features of HUS in rats. An additional rationale for elucidating the mechanisms responsible for ricin's toxicity relates to the administration of ricin as an immunotoxin in the therapy of hematological malignancies and solid tumors.27Kreitman RJ Immunotoxins in cancer therapy.Curr Opin Immunol. 1999; 11: 570-578Crossref PubMed Scopus (180) Google Scholar, 28Frankel AE Tagge EP Willingham MC Clinical trials of targeted toxins.Semin Cancer Biol. 1995; 6: 307-317Crossref PubMed Scopus (61) Google Scholar, 29Engert A Sausville EA Vitetta E The emerging role of ricin A-chain immunotoxins in leukemia and lymphoma.Curr Top Microbiol Immunol. 1998; 234: 13-33PubMed Google Scholar The dose-limiting side effect of many of the ricin-containing immunotoxins that have been applied in clinical trials is the appearance of vascular leak syndrome,27Kreitman RJ Immunotoxins in cancer therapy.Curr Opin Immunol. 1999; 11: 570-578Crossref PubMed Scopus (180) Google Scholar whose main feature is the altered integrity of the vascular endothelial cells. Although the cause of endothelial cell damage remains unknown, it has been suggested that the direct uptake of ricin by the endothelial cells and macrophages constitutes the triggering event in vascular leak syndrome pathogenesis.28Frankel AE Tagge EP Willingham MC Clinical trials of targeted toxins.Semin Cancer Biol. 1995; 6: 307-317Crossref PubMed Scopus (61) Google Scholar In this study, we have demonstrated that ricin activates members of the MAPK family, induces the expression of proinflammatory genes in vitro, and causes a severe inflammatory response in vivo. The in vitro activation of a variety of proinflammatory mediators by ricin requires the activation of ERK1/2, JNK, and p38 MAPK. We have shown that after the intravenous administration of ricin, mice developed a syndrome that shared most of the characteristics of human HUS, including thrombocytopenia, hemolytic anemia, renal failure, and microvascular thrombosis. C57BL/6J mice were purchased from The Jackson Laboratory, Bar Harbor, ME. Male mice 8 to 10 weeks of age and weighing 18 to 24 g were used throughout the experiments. Mice were housed under 12-hour light-dark cycle and fed with standard diet ad libitum. To collect 24-hour diuresis, mice were housed for 24 hours in diuresis metabolic cages (model M-D-METAB; Nalgene, Braintree, MA) provided with grounded standard diet ad libitum. Before experimental procedures, mice were anesthetized intraperitoneally with 80 mg/kg of ketamine and 10 mg/kg of xylazine. All of the animal procedures were performed according to protocols that have been approved by the Institutional Animal Care and Use Committee at Oregon Health and Science University, Portland, OR. The murine macrophage cell line RAW 264.7 was purchased from the American Type Culture Collection (Rockville, MD) and maintained in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Inc., Grand Island, NY) supplemented with gentamicin (50 μg/ml) and 10% fetal bovine serum (Cellgro, Herndon, VA) at 37°C in humidified CO2. RAW 264.7 cells were plated into 6- and 10-cm tissue culture plates (Sarstedt, NC) at a concentration of 5.0 × 106 and 8.0 × 106cells, respectively. Before experiments, cells were incubated in serum-free DMEM for 2 hours. The human macrophage cell line THP-1 was obtained from American Type Culture Collection and maintained in RPMI 1640 medium (Cellgro) supplemented with 50 μg/ml of gentamicin and 10% fetal bovine serum. THP-1 cells were plated in 10-cm tissue culture dishes at a concentration of 8.0 × 106cells and were differentiated in the presence of 0.1 μmol/L 12-O-tetradecanoylphorbol 13-acetate (Sigma-Aldrich, St. Louis, MO) for 72 hours. Before experiments, cells were incubated in serum-free RPMI 1640 for 2 hours. Ricin was purchased from Vector Laboratories, Burlingame, CA. Lipopolysaccharide (LPS), derived from Escherichia coli O111:B4, was obtained from Sigma-Aldrich. The MAP kinase inhibitors UO126, SP600125, and SB203580 were obtained from Calbiochem, La Jolla, CA. The mouse TNF-α enzyme-linked immunosorbent assay (ELISA) Ready-SET-Go was purchased from eBioscience (catalog no. 88-7324-76), San Diego, CA; the mouse IL-6 immunoassay kit was obtained from R&D Systems (catalog no. M6000), Minneapolis, MN; the mouse IL-1β ELISA kit was purchased from BD Bioscience (catalog no. 559603), San Diego, CA. Antibodies against phospho-JNK, phospho-p38, and phospho-ERK1/2, were purchased from Cell Signaling Technology, Beverly, MA; antibody against p38 was obtained from Santa Cruz Biotechnology, Santa Cruz, CA; rabbit polyclonal antibody against mouse fibrin(ogen) was purchased from Nordic Immunology, Tilburg, The Netherlands; and horseradish peroxidase-conjugated antibody was obtained from Vector Laboratories. RAW 264.7 cells were grown in 12-well tissue culture dishes in 10% fetal bovine serum-DMEM. Before the treatments with ricin, the cells were serum-deprived for 2 hours. Two and a half hours after the addition of ricin, the cells were pulsed-labeled for 30 minutes with 2 μCi of [3H]-leucine in 0.3 ml of serum-free DMEM. The incorporation of leucine was stopped by the addition of 10% trichloroacetic acid. Cells were washed 3× with 5% trichloroacetic acid, followed by 88% formic acid to solubilize the trichloroacetic acid-insoluble proteins, and the samples were counted in a scintillation counter. To determine a suitable background for the assay, untreated cells were maintained on ice for 15 minutes, before pulse-labeling on ice for 30 minutes with 2 μCi of [3H]-leucine in 0.3 ml of serum-free DMEM. The reaction was stopped by the addition of 10% trichloroacetic acid, after which samples were further processed as described above. RAW 264.7 cells cultured in 6-cm tissue culture dishes were lysed in 250 μl of 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and boiled for 1 minute. The lysates were separated via 10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA), and probed with the corresponding primary antibodies. For the immunoblot analyses performed on mouse tissue, the whole kidneys were homogenized in lysis buffer (20 mmol/L Tris-HCl, pH 7.5, 140 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L Na4P2O7, 1 mmol/L C3H7O6PNa2, 1 mmol/L Na3VO4, 10 mmol/L NaF, 1× Protease inhibitors cocktail; Roche Applied Science, Indianapolis, IN). After incubation for 10 minutes at 4°C, the lysates were centrifuged at 4°C for 10 minutes at 10,000 × g. The protein concentration of the supernatants was determined using the Bradford assay (Bio-Rad protein assay; Bio-Rad Laboratories, Hercules, CA). One hundred fifty μg of each lysate were mixed 1:4 with 4× SDS-PAGE loading buffer, boiled at 95°C for 5 minutes, and separated via 10% SDS-PAGE. After dissection of the kidneys and other organs, the tissues were immediately frozen and ground in liquid nitrogen. RNA was extracted using TRIzol reagent in accordance with the manufacturer's instructions and was further digested with DNase. Both reagents were purchased from Invitrogen Life Technologies, Carlsbad, CA. Cells were lysed directly in TRIzol reagent and processed as above. Integrity of RNA was determined by the appearance of distinct 28S and 18S rRNA bands when analyzed by electrophoresis on 1% agarose gels. The integrity of RNA was confirmed for all samples before microarray and reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis. Gene expression profiling was performed by the Oregon Health and Science University Gene Microarray Shared Resource Affymetrix Microarray Core using MOE-430A chips, which contain 22,690 transcripts. Labeled target cDNA was prepared from purified total kidney RNA (10 μg) from three saline- and three ricin-injected mice. The total RNAs were run on a 1% agarose gel and additionally analyzed on an Agilent Bioanalyzer to determine the integrity of 18S and 28S subunits of RNA. Each sample was hybridized to a test array and a MOE-430A GeneChip array. Image processing and normalization were performed using Affymetrix Microarray suite 5.0 (MAS 5.0) software to obtain the estimate of the fold change for each paired group (saline-injected mice and ricin-injected mice). Signals represent the abundance of each RNA transcript. A two-sample independent t-test was applied to each gene individually to identify differentially expressed genes and to estimate the average magnitude of differential expression. The resulting P values were adjusted using the false discovery rate proposed by Hochberg and Benjamini.30Hochberg Y Benjamini Y More powerful procedures for multiple significance testing.Stat Med. 1990; 9: 811-818Crossref PubMed Scopus (1672) Google Scholar Genes with a false discovery rate P value of 3 or <−3 were selected as potentially significant genes. Genes with fold change <−3 belong to further studies. Two μg of RNA were reverse-transcribed in the presence of SuperScript II and oligo-dT primers (both reagents were purchased from Invitrogen Life Technologies). The amplification of the cDNA was accomplished using the ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA) in the presence of the commercially available SYBR Green PCR Master Mix (Applied Biosystems) and 20 μmol/L of the corresponding sense and anti-sense RT-PCR primers for 120-bp amplicons in a 40-cycle PCR (Table 1). Fold induction in gene expression was measured using absolute quantitation of a standard curve in arbitrary units. The denaturing, annealing, and extension conditions of each PCR cycle were 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, respectively.Table 1Forward and Reverse Primer Sequences Used for Real-Time RT-PCRGeneForward primer sequenceReverse primer sequenceTNF-α5′-AAATGGGCTTTCCGAATTCA-3′5′-CAGGGAAGAATCTGGAAAGGT-3′IL-1α5′-AAAAAAGCCTCGTGCTGTCG-3′5′-TTGTCGTTGCTTGGTTCTCCT-3′IL-1β5′-CAAATCTCGCAGCAGCACA-3′5′-TCATGTCCTCATCCTGGAAGG-3′IL-65′-AGGATACCACTCCCAACAGACCT-3′5′-CAAGTGCATCATCGTTGTTCATAC-3′GRO-α5′-TGTCAGTGCCTGCAGACCAT-3′5′-CGCGACCATTCTTGAGTGTG-3′MCP-15′-AGCAGCAGGTGTCCCAAAGA-3′5′-TCATTTGGTTCCGATCCAGG-3′RANTES5′-CCTGCTGCTTTGCCTACCTC-3′5′-ACTTGGCGGTTCCTTCGAGT-3′c-JUN5′-GAAAACCTTGAAAGCGCAAAA-3′5′-TAGCATGAGTTGGCACCCAC-3′c-FOS5′-TGGTGAAGACCGTGTCAGGA-3′5′-GCAGCCATCTTATTCCGTTCC-3′ATF35′-CGTCAACAACAGACCCCTGG-3′5′-TTGTTTCGACACTTGGCAGC-3′EGR-15′-CCTTCCAGTGTCCAATCTGCA-3′5′-CTGGCAAACTTCCTCCCACA-3′C/EBPβ5′-GGGACTTGATGCAATCCGG-3′5′-AACCCCGCAGGAACATCTTT-3′E-selectin5′-ACTGTGTGCAAGTTCGCCTG-3′5′-TGGACTCAGTGGGAGCTTCAC-3′GAPDH5′-TTGTGGAAGGGCTCATGACC-3′5′-GATGCAGGGATGATGTTCTGG-3′ Open table in a new tab All murine forward and reverse primers (Table 1) were designed by using MacVector or Primer Express software programs and were synthesized by Invitrogen Life Technologies. Reverse transcription of rRNA was performed as described by Iordanov and colleagues.5Iordanov MS Pribnow D Magun JL Dinh TH Pearson JA Chen SL Magun BE Ribotoxic stress response: activation of the stress-activated protein kinase JNK1 by inhibitors of the peptidyl transferase reaction and by sequence-specific RNA damage to the alpha-sarcin/ricin loop in the 28S rRNA.Mol Cell Biol. 1997; 17: 3373-3381Crossref PubMed Google Scholar The oligonucleotide primer 5′-CACATACACCAAATGTC-3′ (Invitrogen Life Technologies) was end-labeled with T4 polynucleotide kinase (Life Technologies, Inc.). A 10-μl mixture of 2 μg total RNA and 1.0 pmol primer in 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl2 was incubated at 90°C for 3 minutes and then placed on ice for 5 minutes, followed by an incubation at room temperature for 5 minutes. The reverse transcription was initiated by the addition of 10 μl of a mixture of 2 mmol/L deoxynucleoside triphosphates and 30 U SuperScript (Life Technologies, Inc.) in 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl2, 10 mmol/L dithiothreitol, followed by an incubation at 48°C for 15 minutes, when the reactions were stopped by the addition of 5 mmol/L EDTA. Reaction products were precipitated in ethanol, resuspended in formamide gel loading buffer, heat denatured, and electrophoresed in 8% acrylamide sequencing gel, which was subsequently dried and exposed to a PhosphorImager screen. For detection of phospho-p38 MAPK, phospho-ERK1/2, and phospho-JNK, mice were anesthetized (see above) and perfused through the heart with 10 ml of 0.9% NaCl (LabChem Inc., Pittsburgh, PA) to flush the circulation, followed by 30 ml of 4% paraformaldehyde. After dissection, the organs were further fixed in 4% paraformaldehyde solution for 48 hours, processed in an automatic tissue processor, embedded in paraffin blocks, and sectioned into 7-μm sections. Before immunohistochemical staining, the tissue sections underwent antigen-retrieval procedures in accordance with the instructions provided with the corresponding primary antibody. For detection of fibrin(ogen), mice were sacrificed by cervical dislocation. The organs were dissected, fixed in Carnoy solution for 2 hours, transferred to 70% ethanol, and further processed in an automatic tissue processor as described above. The tissue sections were incubated for 24 hours with goat polyclonal antibody against mouse fibrin(ogen). All blood analyses were performed by the Core Laboratory, Division of Laboratory Medicine, Oregon Health and Science University, Portland, OR. Blood was obtained by a puncture of the retro-orbital plexus. Blood for cell-count analysis and cell-type determination was collected in hematology tubes with tripotassium ethylenediaminetetraacetic acid (EDTA) (Microtainer; Becton Dickinson, Franklin Lakes, NJ) and analyzed with an automated cell counter (Beckman-Coulter, Inc., Fullerton, CA). Biochemical determinations of blood urea nitrogen and serum creatinine were performed with a Synchron LX Clinical Chemistry System (Diagnostic Chemicals Ltd., Oxford, CT). Total blood, obtained by puncture of the retro-orbital plexus, was left to clot at room temperature for 1 hour, and was then centrifuged at 8000 rpm, 4°C, for 20 minutes to separate serum from blood cells. Ten μl of serum were mixed 1:1 with 2× sample buffer (20% glycerol, 150 mmol/L Tris-HCl, pH 9.2, bromphenol blue) and run on 1.5% agarose gels in the presence of Tris-glycine electrophoresis buffer (130 mmol/L Tris, 95 mmol/L glycine, pH 9.2). Gels were stained with Gelcode Blue staining solution (Pierce, Rockford, IL). Hemoglobin, isolated from lysed erythrocytes in 0.065 mol/L KCl, was used as positive control. Mice were housed in diuresis metabolic cages for 24 hours during which time urine was collected. Urine was further concentrated through 30-kd Micropore concentration tubes. Equal amounts of urine from three control mice and three ricin-injected animals were mixed 1:4 with 4× SDS-PAGE loading buffer, boiled at 95°C for 5 minutes, and separated via 10% SDS-PAGE. Gels were stained with Gelcode Blue staining solution. Increasing concentrations of bovine serum albumin were loaded on the same gel as a positive control. Individual groups were compared using unpaired t-test analysis. To estimate P values, all statistical analyses were interpreted in a two-tailed manner. P values <0.05 were considered to be statistically significant. To examine the ability of ricin to induce inflammatory responses in vitro, we used RAW 264.7 cells, a murine monocyte/macrophage cell line that has been used frequently to examine responses to proinflammatory agents.31Chen CC Wang JK p38 but not p44/42 mitogen-activated protein kinase is required for nitric oxide synthase induction mediated by lipopolysaccharide in RAW 264.7 macrophages.Mol Pharmacol. 1999; 55: 481-488Crossref PubMed Scopus (87) Google Scholar, 32Chakravortty D
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