Page 1 of 40 in PresS. Am J Physiol Lung Cell Mol Physiol (September 14, 2007). doi:10.1152/ajplung.00207.2007 Articles
Proinflammatory Responses of Human Airway Cells to Ricin Involve StressActivated Protein Kinases and NF- B.
John Wong1, Veselina Korcheva1, David B. Jacoby2 and Bruce E. Magun1 Department of Cell and Developmental Biology1 and Division of Pulmonary and Critical Care Medicine2, Oregon Health and Science University, Portland, Oregon
Running Title: Ricin-Induced Responses in Human Airway Cells
Corresponding Author: Bruce Magun, Oregon Health and Science University, 3181 SW Sam Jackson Park Rd., Portland, OR 97239 (e-mail:
[email protected])
1 Copyright © 2007 by the American Physiological Society.
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ABSTRACT
Ricin is a potential bioweapon because of its toxicity, availability, and ease of production. When delivered to the lungs, ricin causes severe pulmonary damage with symptoms that are similar to those observed in acute lung injury and adult respiratory distress syndrome. The airway epithelium plays an important role in the pathogenesis of many lung diseases, but its role in ricin intoxication has not been elucidated. Exposure of cultured primary human airway epithelial cells to ricin resulted in the activation of stress-activated protein kinases (SAPKs) and NF- B and in the increased expression of multiple proinflammatory molecules. Among the genes upregulated by ricin and identified by microarray analysis were those associated with transcription, nucleosome assembly, inflammation, and response to stress. Sequence analysis of the promoters of these genes identified NF- B as one of the transcription factors whose binding sites were overrepresented. Although airway cells secrete TNF- in response to ricin, blocking TNFdid not prevent ricin-induced activation of NF- B. Decreased levels of I B- in airway cells exposed to ricin suggest that translational suppression may be responsible for the activation of NF- B. Inhibition of p38 MAPK by a chemical inhibitor or NF- B by short interfering RNA resulted in a marked reduction in the expression of proinflammatory genes, demonstrating the importance of these two pathways in ricin intoxication. Therefore, the p38 MAPK and NF- B pathways are potential therapeutic targets for reducing the inflammatory consequences of ricin poisoning.
Keywords: lung, airway epithelium, inflammation, TNF- , p38 MAPK, NF- B
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INTRODUCTION
Ricin is a potent toxin found in castor beans, the seeds of the castor plant Ricinus communis, which is grown worldwide. From the estimated one million tons of castor beans that are processed every year globally for the production of castor oil and related products, bean mash is produced as waste that contains approximately 5% ricin, which is readily extracted and purified (53). Because of its toxicity, availability, and ease of production, ricin is included in the Centers for Disease Control and Prevention’s select agent list. Ricin has been studied for the development as a bioweapon since the 1940s, used in assassination in 1978, and possessed by suspected terrorist groups in the U.S. and abroad since the early 1990s, according to numerous news reports. Although most experts believe that ricin would be difficult to use as a weapon of mass destruction, it has the potential to be a weapon of terror in small scale attacks.
We have reported that ricin, when delivered intratracheally by instillation to mice at a lethal dose (20 Ag per 100 g body weight), leads to cellular damage that can be detected in the lungs as well as other organs such as kidney and spleen; at a sublethal dose (2 Ag per 100 g body weight), the damage is restricted primarily to the lungs, although some extrapulmonary tissues display increased levels of expression of proinflammatory transcripts (71). Pulmonary symptoms after ricin delivery by inhalation or instillation into the lungs include: pulmonary edema, acute alveolitis, apoptosis, necrosis of the endothelium and epithelium, massive infiltration of inflammatory cells, and hyperplasia of pneumocytes (6, 23, 67, 70, 71).
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Many pro-inflammatory genes have binding sites for AP-1 and NF- B in their promoter regions, and numerous studies using various inhibitory interventions show the importance of these two classes of regulatory proteins in gene expression (1, 5, 38, 44). The AP-1 family of transcription factors are activated by stress-activated protein kinases (SAPKs), such as p38 and JNK, which are in turn activated by a cascade of upstream kinases; this cascade is further regulated by phosphatases (recent reviews include (4, 50, 67)). NF- B is normally sequestered in the cytoplasm where it is bound to I B; upon activation, the I B kinase (IKK) phosphorylates I B, targeting it for proteosome-mediated proteolysis, thereby releasing NF- B to translocate to the nucleus where it transactivates genes (recent reviews include (29, 35, 44)).
It is generally believed that the primary targets of ricin intoxication are endothelial cells (20) and macrophages (6). Treatment of endothelial cells results in apoptosis (6, 31, 43). Exposure of macrophage cell lines (22, 24, 27, 39) and primary alveolar and marrowderived macrophages (40) to ricin results in apoptosis, activation of SAPKs, and secretion of chemokines and cytokines.
The airway epithelium is the initial barrier that separates inhaled substances such as environmental pollutants, toxins, and infectious organisms from the internal milieu and has become increasingly appreciated for its involvement in host defense.
Airway
epithelium is able to synthesize and secrete cytoprotective molecules such as mucins and defensins and to signal to cells of the innate and adaptive immune system by expressing
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adhesion molecules, chemokines, and cytokines (13).
Airway epithelial cells are a
known source of a large number of chemokines and cytokines (9, 13, 35, 62, 63, 66). As a result, it has become apparent that airway epithelial cells actively participate in inflammatory diseases of the lung such as asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, and pneumonia (28, 36, 46, 54). However, the direct involvement of the airway epithelium in the pathogenesis of ricin intoxication is unknown.
In this study, we demonstrate that exposure of cultured primary human airway epithelial cells to ricin resulted in the activation of SAPKs and NF- B and in the increased expression of multiple proinflammatory molecules. Reduction in the expression of many proinflammatory genes was achieved by blocking the p38 MAPK-mediated pathway with a chemical inhibitor. Similarly, experimental inhibition of NF- B by introduction of short interfering RNA (siRNA) resulted in the inhibition of the expression of proinflammatory genes, demonstrating a previously unknown participation of NF- B in ricin intoxication. Although airway cells secrete TNFTNF-
in response to ricin, blocking
did not prevent ricin-induced activation of NF- B. Exposure of cells to ricin
resulted in decreased abundance of I B, suggesting that the inhibition of protein synthesis by ricin may lead to turnover of I B and the consequent activation of NF- B. Therefore, ricin may initiate the inflammatory cascade in airway cells by simultaneously activating SAPKs and NF- B.
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MATERIALS AND METHODS
Cells. Primary airway cells were isolated from tracheae (3-5 inches) obtained at the Pacific Northwest Transplant Bank from anonymous human organ donors. Tracheae were placed in calcium and magnesium containing buffer supplemented with 0.5% pronase, antibiotics, and amphotericin B overnight at 4°C. After incubation, fetal bovine serum was added to a final concentration of 20%, and epithelial cells were detached from the stroma by gentle agitation. The cells were collected by centrifugation at 800 x g for 10 min, washed, and suspended in MEM with 5% fetal bovine serum. After overnight cell attachment on collagen-coated plates, the medium was replaced with LHC-9 (Invitrogen, Carlsbad, CA).
Experiments with human airway cells were conducted
between passages 0 and 4 grown to 70-90% confluence in LHC-9.
Antibodies and other reagents. Antibodies against phospho-JNK (#9251), phosphop38 (#4631), and active caspase 3 (#9662) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against p38 (#sc-535), the p65 subunit of NFB (#sc-372), and I B-
(#sc-371) were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Ricin was purchased from Vector Laboratories (Burlingame, CA). SB203580 (an inhibitor of p38 MAPK) was purchased from Calbiochem (San Diego, CA). Etanercept (EnbrelTM) was manufactured by Immunex (Thousand Oaks, CA).
Immunocytochemistry. Cells were grown on collagen-coated plastic dishes and fixed with methanol. Cells were then blocked with blocking buffer (PBS+1.5% goat serum) and incubated with primary antibody against p65 subunit of NF- B (#sc-372) diluted in 6
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blocking buffer overnight. After incubation in biotinylated goat anti-rabbit secondary antibody, samples were treated with hydrogen peroxide to block endogenous peroxidase and further processed using the VectaStain Elite ABC kit (Vector Laboratories, Burlingame, CA) using 3,3'-diaminobenzidine as substrate.
Incorporation of [3H]-leucine. Cells were grown in collagen-coated 24-well culture dishes. Two and one half hours after the addition of ricin, cells were exposed to 10 ACi of [3H]-leucine for 30 min, at which time 10% trichloroacetic acid was added to terminate incorporation. Culture wells were washed three times with 5% trichloroacetic acid, followed by 88% formic acid to solubilize the trichloroacetic acid-insoluble proteins. The samples were counted in a liquid scintillation counter.
Measurement of TNF- levels. Cells were cultured in collagen-coated 3.5 cm culture dishes and treated with ricin for 6 h.
Medium was collected from each well and
concentrated by ultrafiltration using Centricon-3 microconcentrators (Millipore, Billerica, MA) to increase sensitivity.
Measurement of TNF- in the concentrated samples was
performed using ELISA reagents from eBioscience (San Diego, CA).
Immunoblotting. Equal numbers of cells were plated, treated, and lysed in lysis buffer in preparation for immunoblotting. Equal volumes of the cell lysates were separated on a 10% denaturing polyacrylamide gel in the presence of sodium dodecyl sulfate and transferred onto polyvinylidene difluoride membranes according to standard laboratory procedures. Membranes were incubated with the indicated antibodies and the
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corresponding horseradish peroxidase-conjugated secondary antibodies; signals were detected using enhanced chemiluminescence. Quantification of band intensities was performed by scanning of the autoradiograph and analysis by the software IPLab Gel (Signal Analytics, Vienna, Virginia).
Transfection with siRNA. SiRNA was transfected into airway cells grown on collagencoated 2.2 cm wells at 50 nM siRNA per well using DharmaFECT 4 (Dharmacon, Lafayette, CO) according to the manufacturer’s instructions. A sequence targeting the p65 subunit of NF- B was used singly (74) or in combination with another published sequence targeting the same gene (3). Control siRNA was a published sequence that showed no inhibition of p65 in multiple human cell types (21). We confirmed the inability of this siRNA to reduce expression of p65 in airway epithelial cells.
Real-Time RT-PCR.
Total RNA was harvested from culture dishes using TRIzol
(Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. RNA from each sample was treated with DNase I (Invitrogen) and reverse transcribed with SuperScript II and oligo dT primer (Invitrogen). Real-time RT-PCR was performed using SYBR Green reagents on an ABI Prism 7900HT (Applied Biosystems, Foster City, CA).
Fold
induction was calculated for each sample by absolute quantification using levels of glyceraldehyde phosphate dehydrogenase for normalization. The nucleotide sequences of the primers used in this study have been previously published (19).
Statistical Analyses. Each figure panel displays the results of a single experiment that
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was performed twice using cells from different donors; in all cases, replicate experiments on cells from different donors yielded similar results. Confidence levels of p
