Am J Physiol Lung Cell Mol Physiol 293: L1385–L1394, 2007.
First published September 14, 2007; doi:10.1152/ajplung.00207.2007.
Proinflammatory responses of human airway cells to ricin involve
stress-activated protein kinases and NF-␬B
John Wong,1 Veselina Korcheva,1 David B. Jacoby,2 and Bruce E. Magun1
1
Department of Cell and Developmental Biology and 2Division of Pulmonary and Critical Care Medicine, Oregon Health and
Science University, Portland, Oregon
Submitted 22 May 2007; accepted in final form 10 September 2007
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 ⬃5% ricin, which is readily extracted and purified
(53). Because of its toxicity, availability, and ease of production, ricin is included in the U.S. Centers for Disease Control
and Prevention’s select agent list. Ricin has been studied for
development as a bioweapon since the 1940s, was used in an
assassination in 1978, and has been 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 ␮g/100 g body wt),
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 ␮g/100 g body wt), 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).
Many proinflammatory genes have binding sites for activator protein-1 (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 members of the AP-1
family of transcription factors are activated by 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 Refs. 4, 50, and 67). NF-␬B is
normally sequestered in the cytoplasm, where it is bound to
I␬B; on activation, the I␬B kinase (IKK) phosphorylates I␬B,
targeting it for proteasome-mediated proteolysis, thereby releasing NF-␬B to translocate to the nucleus, where it transactivates genes (recent reviews include Refs. 29, 35, and 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 marrow-derived 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 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 respi-
Address for reprint requests and other correspondence: B. Magun, Oregon
Health and Science Univ., 3181 SW Sam Jackson Park Rd., Portland, OR
97239 (e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
lung; airway epithelium; inflammation; tumor necrosis factor-␣; p38
mitogen-activated protein kinase; nuclear factor-␬B
RICIN IS A POTENT TOXIN FOUND
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1040-0605/07 $8.00 Copyright © 2007 the American Physiological Society
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Wong J, Korcheva V, Jacoby DB, Magun BE. Proinflammatory responses of human airway cells to ricin involve stressactivated protein kinases and NF-␬B. Am J Physiol Lung Cell Mol
Physiol 293: L1385–L1394, 2007. First published September 14,
2007; doi:10.1152/ajplung.00207.2007.—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 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 TNF-␣ did 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.
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MATERIALS AND METHODS
Cells. Primary airway cells were isolated from tracheae (3–5 in)
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 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
(no. 9251), phospho-p38 (no. 4631), and active caspase-3 (no. 9662)
were purchased from Cell Signaling Technology (Danvers, MA).
Antibodies against p38 (sc-535), the p65 subunit of NF-␬B (sc-372),
and I␬B-␣ (sc-371) were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Ricin was purchased from Vector Laboratories
(Burlingame, CA). SB-203580 (an inhibitor of p38 MAPK) was
purchased from Calbiochem (San Diego, CA). Etanercept (Enbrel)
was manufactured by Immunex (Thousand Oaks, CA).
Immunocytochemistry. Cells were grown on collagen-coated plastic
dishes and were fixed with methanol. Cells were then blocked with
blocking buffer (PBS ⫹ 1.5% goat serum) and were incubated with
primary antibody against p65 subunit of NF-␬B (sc-372) diluted in
blocking buffer overnight. After incubation in biotinylated goat antirabbit secondary antibody, samples were treated with hydrogen peroxide to block endogenous peroxidase and were further processed by
using the VECTASTAIN Elite ABC kit (Vector Laboratories, Burlingame, CA) with 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 ␮Ci 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 collagencoated 3.5-cm culture dishes and were treated with ricin for 6 h.
AJP-Lung Cell Mol Physiol • VOL
Medium was collected from each well and was concentrated by
ultrafiltration by using Centricon-3 microconcentrators (Millipore,
Billerica, MA) to increase sensitivity. Measurement of TNF-␣ in the
concentrated samples was performed by 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 were
transferred onto polyvinylidene difluoride membranes according to
standard laboratory procedures. Membranes were incubated with the
indicated antibodies and the corresponding horseradish peroxidaseconjugated secondary antibodies; signals were detected by using
enhanced chemiluminescence. Quantification of band intensities was
performed by scanning of the autoradiograph and analysis by the
software IPLab Gel (Signal Analytics, Vienna, VA).
Transfection with siRNA. siRNA was transfected into airway cells
grown on collagen-coated 2.2-cm wells at 50 nM siRNA per well by
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
by using TRIzol (Invitrogen, Carlsbad, CA), following the manufacturer’s instructions. RNA from each sample was treated with DNase
I (Invitrogen) and was reverse transcribed with SuperScript II and
oligo(dT) primer (Invitrogen). Real-time RT-PCR was performed by
using SYBR Green reagents on an ABI Prism 7900HT (Applied
Biosystems, Foster City, CA). Fold induction was calculated for each
sample by absolute quantification by using levels of GAPDH 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 was performed twice with the use of cells from
different donors; in all cases, replicate experiments on cells from
different donors yielded similar results. Confidence levels of P ⬍ 0.05
were considered statistically significant. Further descriptions of statistical measures for each experiment are included in the figure
legends.
Microarray analysis. Gene-expression profiling was performed by
the Gene Microarray Shared Resource Affymetrix Microarray Core at
Oregon Health and Science University. To verify RNA integrity, total
RNA was run on 1% agarose gels and was analyzed on an Agilent
Bioanalyzer (Palo Alto, CA). Following reverse transcription, each
cDNA was hybridized to the HG-U133 Plus 2.0 GeneChip Array
(Affymetrix, Santa Clara, CA), which interrogates 47,000 transcripts.
Data were processed with Affymetrix GCOS v. 1.2 software (GEO
accession no. GSE7845) and were further analyzed by using EASE
(http://david.abcc.ncifcrf.gov/ease/ease.jsp) and EXPANDER (http://
www.cs.tau.ac.il/⬃rshamir/expander/expander.html) software.
RESULTS
TNF-␣ is a cytokine whose increased expression and release
have been tied to the generation of inflammatory cascades in
multiple tissues and organs (2, 47, 68). To determine whether
ricin increases the expression of TNF-␣ mRNA and protein in
airway cells, primary cultures of airway cells were exposed to
varying concentrations of ricin for 5 or 6 h. TNF-␣ mRNA was
measured by real-time RT-PCR, and TNF-␣ content of the
culture medium was analyzed by ELISA. Addition of ricin
resulted in a dose-dependent increase in mRNA encoding
TNF-␣ transcripts (Fig. 1A).
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ratory 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 TNF-␣ in response
to ricin, blocking TNF-␣ 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.
RICIN-INDUCED RESPONSES IN HUMAN AIRWAY CELLS
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In contrast to the progressive increase in expression of
TNF-␣ mRNA that was induced by increasing doses of ricin,
the appearance of TNF-␣ protein in the culture increased
dramatically at low ricin concentrations but decreased thereafter (Fig. 1B). Ricin doses of 1 ng/ml and 10 ng/ml stimulated
a sixfold increase in secreted levels of TNF-␣ protein. Addition
of 100 ng/ml ricin to cells resulted in the appearance of slightly
less TNF-␣ protein than at 1 or 10 ng/ml ricin. Remarkably,
cells exposed to 1,000 ng/ml ricin showed a complete suppression of secreted TNF-␣ protein.
We previously reported that exposure of macrophages to
increasing doses of ricin resulted in a dose-dependent increase
in expression of TNF-␣ mRNA but resulted in a decreased
appearance of TNF-␣ in the culture medium at high concentrations of ricin (39). We demonstrated that the inhibition of
protein translation at the higher doses of ricin could account for
the inability of transcribed TNF-␣ mRNA to be productively
translated into protein. To determine whether a similar inhibition of translation could account for the inability of airway
cells to secrete TNF-␣ into the culture medium at elevated ricin
concentrations, we measured the incorporation of [3H]leucine
into these cells between 2.5 and 3 h after the addition of graded
doses of ricin (Fig. 1C). The results of this experiment revealed
that the incorporation of [3H]leucine decreased to ⬍60% of
control values after addition of 1 ng/ml ricin and ⬍3% of
control values after addition of 10 ng/ml ricin. Together, the
data shown in Fig. 1 demonstrated that primary cultures of
human airway cells responded to ricin by progressively increasing the expression of TNF-␣ mRNA. Furthermore, the
data suggest that the productive translation and subsequent
release of TNF-␣ protein that occur at lower ricin concentrations were progressively impeded at higher concentrations as a
consequence of ricin-mediated inhibition of protein synthesis.
The ability of ricin to modulate a proinflammatory response
has been tied to ricin’s ability to activate the SAPKs in multiple
cell types (22, 27, 33, 39). To determine whether ricin activates
SAPKs in primary airway cells, we exposed airway cell cultures to ricin and examined the phosphorylation of JNK and
p38 MAPK by immunoblotting. Figure 1 demonstrated that
exposure of airway cells to concentrations of ricin ⬍100 ng/ml
resulted in both the accumulation of TNF-␣ mRNA and its
translation into protein.
To determine whether ricin would be effective in causing the
phosphorylation of SAPKs, we exposed airway cells to concentrations of ricin from 1 to 100 ng/ml. The antibody against
phosphorylated JNK detected three bands in Western blotting
(Fig. 2A). The two higher-molecular-weight bands represented
the phosphorylated forms of p55 and p45 isoforms of JNK.
[We employed several methods to determine the identity of the
phosphorylated species that appears below p45 JNK, including
siRNA knockdown of both JNK species, and have concluded
that the band is not a JNK isoform (unpublished data)]. By 2 h
after addition, only 100 ng/ml ricin was capable of increasing
the phosphorylation of JNK and p38 MAPK (Fig. 2A). By 4 h,
ricin induced the phosphorylation of both JNK and p38 MAPK
at all concentrations of ricin that were tested.
Ricin induces apoptosis in a number of cell types (21, 27, 34,
37, 56). As determined by morphological criteria, ricin at
0.1–10 ng/ml failed to induce apoptosis in airway cells at 6 h
(not shown). However, cells exposed to 10 ng/ml became
apoptotic at 24 h, as demonstrated by the appearance of the
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Fig. 1. Effect of ricin on protein translation and secretion of TNF-␣ in airway
cells. Human airway cells were treated with ricin at indicated doses. A: cells
were cultured in 6-cm dishes and were treated with indicated concentrations of
ricin for 5 h, at which time cells were processed for RNA isolation and
RT-PCR by using specific primers for TNF-␣. Means ⫾ SD of triplicate
measurements of each sample are displayed. B: cell cultures in groups of
triplicate 3.5-cm dishes were treated with indicated concentrations of ricin for
6 h, at which time medium was collected for determination of TNF-␣ by
ELISA. Mean ⫾ SD for each treated group is displayed. C: cells cultured in
24-well dishes were treated with indicated concentrations of ricin for 3 h.
Thirty minutes before harvesting, [3H]leucine was added, as described in
MATERIALS AND METHODS. Each value represents a mean ⫾ SD of 3H incorporation from triplicate culture wells. Levels of significance are indicated for
ricin-treated cells compared with untreated cells: *P ⬍ 0.05; **P ⬍ 0.01;
***P ⬍ 0.001.
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RICIN-INDUCED RESPONSES IN HUMAN AIRWAY CELLS
Fig. 2. Effect of ricin on activation of MAPKs in airway cells. Human airway
cells in 6-cm dishes were treated with ricin at indicated doses and times. Cell
lysates were obtained from each dish and were processed for immunoblotting.
Lysates were examined by immunoblotting against antibodies as indicated in
each panel. A: cells were treated with serial dilutions of ricin for 2 and 4 h.
Reactivity with antibodies against phosphorylated p38 MAPK and phosphorylated JNK are indicated. B: cells were treated with varying concentrations of
ricin for 24 h and were reacted with antibodies specific for caspase-3. For both
panels, immunoblotting with anti-p38 MAPK served as loading control.
p17/p19 cleavage products of caspase-3 (Fig. 2B). In cells
exposed to 3 ng/ml, the cleavage of caspase-3 was barely
detectable at 24 h, and concentrations of ricin ⬍3 ng/ml failed
to induce cleavage of caspase-3 by that time. The appearance
of cleavage products of caspase-3 in cells exposed to 10 ng/ml
for 24 h was associated with morphological changes characteristic of apoptotic cells (data not shown).
The binding of NF-␬B to the regulatory regions of many
proinflammatory genes (52, 61) is critical for the development
of proinflammatory responses (5, 44, 65). To determine
whether the exposure of airway cells to ricin would lead to the
activation of NF-␬B, cells were treated with ricin and the
redistribution of the p65 subunit of NF-␬B from cytoplasm to
nuclei was visualized by immunocytochemical localization of
NF-␬B (Fig. 3). In untreated airway cells, p65 was localized
primarily in cytoplasmic regions, with some reaction product
apparent in nuclei (Fig. 3A), suggesting that there may be
partial activation of NF-␬B endogenously in some cells. Cells
that were exposed to varying concentrations of ricin (1 to 1,000
ng/ml) for varying times (0.5 to 24 h) revealed that the optimal
condition for activating NF-␬B was 100 ng/ml ricin for 6 h
(data not shown). Exposure of cells to 100 ng/ml ricin for 6 h
resulted in the accumulation of p65 in the nuclei, although
considerable p65-specific reactivity remained in the cytoplasm
(Fig. 3C).
Although ricin has been shown previously to elevate levels
of SAPKs in a variety of cells by acting on intracellular
transduction pathways (22, 27, 33, 39), the ability of ricin to
activate NF-␬B directly in cell cultures by intracellular mechanisms has not been demonstrated. The activation of NF-␬B
seen in Fig. 3C may have occurred following the ricin-provoked increase in TNF-␣ secretion. To test this possibility,
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Fig. 3. Effect of etanercept on intranuclear migration of NF-␬B following
exposure of human airway cells to TNF-␣ or ricin. Human airway cells in
plastic dishes were treated with 100 ng/ml ricin for 6 h (C and D) or 100 ng/ml
TNF-␣ for 0.5 h (E and F) in presence of saline (A, C, and E) or 10 ␮g/ml
etanercept (B, D, and F). Cells treated with saline or etanercept looked
identical at 0.5 or 6 h, but only those fixed at 6 h were shown (A and B). Cells
were fixed and processed for immunocytochemical detection of p65 subunit of
NF-␬B. Size bar (A) represents 20 ␮m for all panels.
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airway cells were exposed to ricin or TNF-␣ in the presence or
absence of etanercept (Enbrel), an inhibitor of TNF-␣ activity.
Etanercept is a fusion protein that consists of the extracellular
ligand-binding portion of the human 75-kDa TNF receptor
linked to the Fc portion of IgG1 (48). Addition of TNF-␣ to
airway cultures for 30 min led to the nuclear accumulation
of NF-␬B (Fig. 3E), which did not occur in the presence of
etanercept (Fig. 3F). However, the nuclear translocation of
NF-␬B in response to ricin was not diminished in the presence
of etanercept (Fig. 3D). These data suggest that the nuclear
accumulation of NF-␬B in response to ricin did not result from
the ricin-mediated increase in the expression and secretion of
TNF-␣.
The prominent ricin-mediated activation of SAPKs and the
nuclear appearance of NF-␬B suggest that these signaling
pathways participate in the transcriptional activation of genes
in airway cells. One nanogram per milliliter ricin was capable
RICIN-INDUCED RESPONSES IN HUMAN AIRWAY CELLS
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Table 1. Selected microarray data from airway cells treated
with 1 ng/ml ricin for 24 h
Transcription Factors and DNA-Binding Proteins
Fold Increase
Activating transcription factor 3
Basic leucine zipper nuclear factor 1
CREB binding protein
CREB5
Distal-less homeo box 2
Early growth response gene 2
Early growth response gene 3
Early growth response gene 4
E2F transcription factor 7
ETS1
FOSB
Histone 1, H4 h1
Histone 1, H2am
Histone 1, H4d
Histone 1, H3d
Histone 1, H3 h
Histone 2, H2aa
Histone 1, H2bj
Histone 1, H2bg
Histone 1, H3d
Histone 1, H2al
Histone 2a, member J
Histone 3, H2ae
Histone 1, H2ae
Histone deacetylase 9
c-Jun
L-Myc
Microphthalmia-associated transcription factor
Nucleear factor, interleukin 3 regulated
NF-␬B, p105
c-Rel
Suppressor of cytokine signaling 1
Suppressor of cytokine signaling 4
Suppressor of cyto0kine signaling 3
Regulators of inflammatory response
Activator of S phase kinase
CCL5 (RANTES)
CCL20
CXCL2 (Gro-beta)
CXCL3 (Gro-gamma)
CXCL-10 (IP-10)
CXCL-11 (I-TAC)
IL-6 signal transducer, oncostatin M receptor, GP130
Interleukin-1␣
Interleukin 1 receptor-like 1
Interleukin 1 receptor, type II
Interleukin 1 family, member 9
Interleukin-6
Interleukin-8
Interleukin-23, alpha subunit (p19)
Lymphotoxin beta
Receptor-interacting serine-threonine kinase-2
TAK1-binding protein 3
Thrombospondin-1
TRAF-interacting protein
TNF-␣
TNF-␣-induced protein 3
TNF-␣-induced protein 8
TNF receptor superfamily, member 19
TNF receptor-associated factor-1
9.8
9.2
3.5
11.3
13.0
39.4
7.5
59.7
3.5
7.5
24.3
17.1
18.3
16.0
21.1
18.4
19.7
10.6
10.6
10.6
14.9
9.8
8.6
8.6
5.7
3.7
13.0
11.3
6.1
5.6
3.5
4.6
4.6
4.3
4.0
6.5
52.0
16.0
12.1
10.6
17.1
3.7
4.6
5.7
3.7
4.3
9.8
8.0
3.7
9.2
3.0
3.0
6.5
3.0
12.1
12.1
4.3
7.5
3.0
sponse-1 (EGR-1), and activating transcription factor 3, which
are known to be associated with the transcriptional regulation
of proinflammatory genes. Among the proinflammatory transcripts whose expression was increased were the cytokines
TNF-␣, IL-1␤, IL-6, and a number of chemokines. We applied
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of stimulating the phosphorylation of p38 MAPK and JNK by
4 h (Fig. 2A), but the effect of ricin exposure on the levels of
TNF-␣ RNA (Fig. 1A) and other proinflammatory transcripts at
this time point was small. One nanogram per milliliter ricin
failed to induce apoptosis by 24 h (Fig. 2B) and resulted in a
dramatic increase in the expression of many proinflammatory
transcripts at that time. In previous studies (71), we had
determined that maximal increase of proinflammatory transcripts occurred 24 – 48 h following intratracheal instillation of
ricin in mice. For these reasons, we determined the expression
profile of transcripts in airway cells 24 h after exposure of cells
to 1 ng/ml ricin. We employed an unbiased genome-wide
analysis of expressed transcripts by using Affymetrix microarrays on HG-U133 Plus 2.0 chips, which interrogate over
47,000 human transcripts (GEO accession no. GSE7845). We
then used the GCOS software to compare the signals of our
control vs. ricin-treated samples; the statistical-expression algorithm assigned a no-change call if the change P value was
⬍0.0025. Of the set of transcripts analyzed, over 1,660 distinct
transcripts were declared upregulated by threefold or greater
following administration of ricin. Ninety percent of these
upregulated transcripts were found to be significantly different
from the control levels based on the change calls. Selected
genes from this analysis are listed in Table 1.
To identify overrepresented gene-ontology groups and biological pathways associated with the genes upregulated by
ricin, two analytical software tools were applied to analyze the
microarray data: EASE (30) and EXPANDER (60). EASE
automates the process of biological theme determination by
analyzing the overrepresentation of genes that belong to categories that are functionally and structurally defined. Table 2
displays the hierarchical analyses of the annotated genes whose
expression was induced greater than threefold by 1 ng/ml ricin
for 24 h. The most highly overrepresented genes activated
following administration of ricin were associated with transcription, nucleosome assembly, inflammation, and response to
stress and wounding. The molecular functions of these induced
genes were associated with DNA binding, transcriptional regulation, cytokine activity, and chemokine activity; we have
previously reported analysis of microarray data by using RNA
isolated from the lungs of saline- vs. ricin-treated mice, and
similar ontology groups were identified by EASE (71).
We further analyzed our microarray data by using the
PRIMA module of EXPANDER (17), which identifies transcription factors whose binding sites are enriched in the promoters of input genes. EXPANDER identified 12 transcription
factors from airway cells treated with 1 ng/ml ricin for 24 h
(Table 3). Also shown in this table are the P values and the
frequency (i.e., percentage of identified promoters within the
set of input genes). When we reanalyzed our previously published microarray data (71) from lungs of mice treated with 20
␮g ricin/100 g body wt for 48 h, we identified four transcription factors by using the same software. NF-␬B is found at or
near the top on both lists of transcription factors. Several other
transcription factors identified by EXPANDER belong to the
forkhead box proteins and paired box proteins, which have
been implicated in tissue development and cell proliferation
(7, 57).
The microarray analyses (GEO accession no. GSE7845)
revealed ricin-mediated increased expression of a number of
transcription factors such as c-Jun, c-Fos, early growth re-
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RICIN-INDUCED RESPONSES IN HUMAN AIRWAY CELLS
Table 2. Overrepresentation analysis of microarray data
from airway cells treated with 1 ng/ml ricin for 24 h, as
determined by EASE
EASE Score
Transcription
Nucleosome assembly
Chromatin assembly
Establishment and/or maintenance of chromatin architecture
Inflammatory response
Innate immune response
Response to wounding
Response to stress
Programmed cell death
Molecular Function DNA binding
Transcription factor activity
Transcription coactivator activity
Chemoattractant activity
Transcription factor binding
Chemokine activity/receptor binding
1.2 ⫻ 10⫺26
2.6 ⫻ 10⫺12
2.1 ⫻ 10⫺9
1.0 ⫻ 10⫺6
1.2 ⫻ 10⫺3
2.1 ⫻ 10⫺3
5.8 ⫻ 10⫺3
8.4 ⫻ 10⫺3
1.6 ⫻ 10⫺2
1.8 ⫻ 10⫺24
9.5 ⫻ 10⫺10
8.8 ⫻ 10⫺3
8.3 ⫻ 10⫺3
1.3 ⫻ 10⫺2
1.9 ⫻ 10⫺2
real-time RT-PCR to the analysis of transcript abundance for
several proinflammatory genes and transcription factors to
verify their increased expression 24 h after exposure of airway
cells to 1 ng/ml ricin. We previously reported that many of
these genes, which had been chosen because of their known
involvement in the inflammatory process, were upregulated
in vivo in the lungs following intratracheal instillation of ricin
(71). Figure 4 demonstrates, by real-time RT-PCR, the increased expression of a number of selected chemokines
(CXCL1, CCL2, IL-8), cytokines (TNF-␣, IL-1␤, IL-6), and
transcription factors (EGR-1, c-Jun, c-Fos) following exposure
of airway cells to ricin.
Of the SAPKs, p38 MAPK has been shown to occupy a
central role in mediating inflammatory processes (25, 42). To
determine the requirement for activated p38 MAPK in ricinmediated expression of proinflammatory transcripts, airway
cells were exposed to ricin in the presence of SB-203580, an
Table 3. Analysis of transcription factor binding sites as
determined by EXPANDER
Human airway cells treated with 1 ng/ml ricin for 24 h
Transcription Factor
P Value
Frequency
FoxQ1 (HFH-1)
NF-␬B p65
FoxF2 (Freac-2)
SRY
TATA
STAT4
FoxC1 (Freac-3)
Tax/CREB
FoxD3
HMG-1(Y)
Pax-3
FoxO4
3.1 ⫻ 10⫺8
1.8 ⫻ 10⫺6
1.9 ⫻ 10⫺6
7.5 ⫻ 10⫺6
8.1 ⫻ 10⫺6
2.7 ⫻ 10⫺5
3.2 ⫻ 10⫺5
3.3 ⫻ 10⫺5
6.2 ⫻ 10⫺5
6.6 ⫻ 10⫺5
7.4 ⫻ 10⫺5
8.2 ⫻ 10⫺5
18.1
11.8
15.8
31.6
9.7
19.2
15.4
20.0
16.4
20.0
12.0
16.2
Lung from mice treated with 20 ␮g ricin per 100 g body weight
Transcription Factor
NF-␬B p65
ISRE
Pax-4
NF-␬B p50
P-Value
Frequency
3.4 ⫻ 10
1.3 ⫻ 10⫺5
3.8 ⫻ 10⫺5
8.4 ⫻ 10⫺5
15.2
17.1
28.0
14.7
⫺12
AJP-Lung Cell Mol Physiol • VOL
Fig. 4. Effect of SB-203580 on ricin-mediated increase in expression of
proinflammatory RNA transcripts in human airway cells. Cells cultured in
2.2-cm wells were exposed to 1 ng/ml ricin for 24 h in presence (gray bars) or
absence (open bars) of 10 ␮M SB-203580. From each well, RNA was
extracted and real-time RT-PCR was employed to measure relative abundance
of RNA by using primers corresponding to indicated genes, employing
GAPDH as an invariant transcript. EGR, early growth response. Values are
displayed as means ⫾ SD of triplicate measurements of each sample. Level of
significance (***P ⬍ 0.001) is indicated for cells exposed to ricin compared
with cells exposed to ricin plus SB-203580.
inhibitor that has high specificity for p38 MAPK (8, 41). The
efficacy of SB-203580 as an inhibitor of p38 MAPK was
verified by using immunoblotting by employing an antibody
against phospho-MAPKAP kinase-2 (a specific substrate for
p38 MAPK; data not shown). In the presence of SB-203580,
the ricin-mediated expression of transcripts that encode a
variety of cytokines, chemokines, and transcription factors was
reduced significantly (P ⬍ 0.001; Fig. 4), indicating that
activation of p38 MAPK was required for ricin-mediated
increased expression of these mRNAs.
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Biological Process
RICIN-INDUCED RESPONSES IN HUMAN AIRWAY CELLS
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Fig. 6. Effect of reduced expression of p65 subunit NF-␬B on ricin-mediated
expression of proinflammatory RNA transcripts in human airway cells. Cells
cultured in 2.2-cm wells were transfected with control siRNA (open bars), a
single p65 siRNA (gray bars), or a combination of 2 different p65 siRNAs
(solid bars). Two days later, cells were treated with saline (control) or with 1
ng/ml ricin for 24 h, at which time RNA was extracted and processed by
real-time RT-PCR. Experiment shown was performed contemporaneously with
experiment shown in Fig. 5 to verify ability of siRNA to reduce expression of
p65 protein. Values are displayed as means ⫾ SD of triplicate measurements
of each sample. Levels of significance (*P ⬍ 0.05; **P ⬍ 0.01) are indicated
for cells transfected with a single p65 siRNA and exposed to ricin compared
with cells transfected with two different p65 siRNAs and exposed to ricin.
encode transcription factors whose increased expression has
been tied to the activation of cytokines and chemokines.
Fig. 5. Effect of reduced expression of p65 subunit NF-␬B on ricin-mediated
activation of MAPKs in human airway cells. Cells in 2.2-cm wells were
transfected with control siRNA and 1 or a combination of 2 short interfering
RNAs (siRNAs) targeted against p65 subunit of NF-␬B. Two days later, cells
were treated with 1 ng/ml ricin for 24 h, at which time cell lysates were
prepared and processed for immunoblotting against antibodies as shown.
AJP-Lung Cell Mol Physiol • VOL
DISCUSSION
Inflammation of airway epithelium caused by bacterial,
viral, and fungal pathogens is strongly associated with the
activation of pattern-recognition receptors such as the toll-like
receptors, Nod1, Nod2, retinoic acid-inducible protein I (RIG-
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Because NF-␬B is crucially involved in the regulation of a
large number of cytokines, chemokines, and other proinflammatory molecules (52, 61), and because we found NF-␬B
binding sites to be enriched in the promoters of genes that were
upregulated by ricin (Table 3), we determined whether NF-␬B
was required for airway cells to respond to ricin. To this end,
we applied siRNA-mediated knockdown of the p65 subunit
(RelA) of NF-␬B before exposure of airway cells to ricin.
Following exposure of cells to one p65 siRNA or a combination of two different p65 siRNAs, levels of p65 NF-␬B protein
were reduced by ⬃70% when compared with exposure to
control siRNA alone (Fig. 5). Knockdown of p65 NF-␬B did
not interfere with the levels of JNK and p38 MAPK (data not
shown) nor the ability of 1 ng/ml ricin to induce the phosphorylation of these two SAPKs, demonstrating the absence of
nonspecific effects on these kinases. Interestingly, cells exposed to ricin exhibited diminished levels of I␬B-␣ (lanes 2, 4,
and 6), a molecule that is responsible for inhibiting NF-␬B
activation by preventing its nuclear translocation (29, 35, 44).
To determine whether p65 NF-␬B was required for the
ricin-mediated expression of proinflammatory mRNAs, we
exposed control or NF-␬B-deficient airway cells to 1 ng/ml
ricin and determined the expression of transcripts 24 h later
(Fig. 6). Reduction of NF-␬B abundance resulted in the reduced expression of TNF-␣, IL-1␤, CXCL1, CCL2, and IL-8
(P ⬍ 0.01) but not IL-6 or the transcription factors EGR-1,
c-Jun, and c-Fos. A slightly larger decrease in expression was
observed when two p65 siRNAs were used in combination for
TNF-␣, IL-1␤, CXCL1, and CCL2, with P values as shown.
Together, Figs. 4, 5, and 6 demonstrate that the ricin-mediated
expression of a variety of mRNA molecules associated with
inflammation requires the activation of p38 MAPK and that a
subset of these transcripts also requires the activation of NF␬B. Some transcripts that are independent of NF-␬B activation
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RICIN-INDUCED RESPONSES IN HUMAN AIRWAY CELLS
AJP-Lung Cell Mol Physiol • VOL
intoxication produces responses that are similar to those observed in chronic obstructive pulmonary disease, acute lung
injury, and adult respiratory distress syndrome. One of the
genes that has been studied extensively in lung inflammation
and injury encodes the antimicrobial chemokine CCL20/macrophage inflammatory protein (MIP)-3␣, which is secreted by
airway cells and has chemical properties both as a chemoattractant and as a ␤-defensin (64, 73). Expression of CCL20/
MIP-3␣ was increased by ricin (Table 1). In addition to
proinflammatory genes, ricin increased the expression of many
histones (Table 1), a response that also occurs in keratinocytes
following UV irradiation (10). Elevation of histone expression
has been employed as a marker for cell proliferation (26) and
may be a general response to toxic stimuli.
TNF-␣ has been shown to play a major role in many
inflammatory diseases of the lung (reviewed in Ref. 47). In
addition to stimulating its own transcription in different cell
types (49, 51), TNF-␣ upregulates the expression of many
other proinflammatory transcripts (15) and causes the release
of CCL5 and soluble ICAM-1 from airway cells (69). Because
TNF-␣ directly activates both p38 MAPK and NF-␬B (12, 32),
it is reasonable to speculate that TNF-␣ may be responsible for
the activation of NF-␬B by ricin by an autocrine mechanism.
However, blocking the TNF-␣ pathway by use of etanercept
was unable to prevent the nuclear translocation of NF-␬B
induced by ricin (Fig. 3), suggesting that ricin may activate
NF-␬B via another secreted molecule or may directly activate
the NF-␬B pathway in the absence of autocrine signaling by an
unknown mechanism. Translational repression induced by UV
radiation or double-stranded RNA results in the inhibition of
new synthesis of I␬B and in the consequent activation of
NF-␬B (11, 72). Our data demonstrating substantial reduction
in the level of I␬B-␣ protein following administration of ricin
to airway cells (Fig. 5) supports the notion that the direct effect
of ricin on inhibition of protein synthesis may be responsible
for activation of NF-␬B.
ACKNOWLEDGMENTS
We acknowledge excellent technical assistance of David E. Purdy and
Thanh-Hoai Dinh and thank the members of the Pacific Northwest Transplant
Bank for their help in obtaining human tracheas. The Institutional Review
Board at Oregon Health and Science University granted an exemption for this
research.
GRANTS
These studies were supported by grants ES-889456 and AI-1059335,
HL-71795, HL-54659, and HL61013 from the National Institutes of Health.
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