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This is to certify that the

dissertation entitled

DETECTION AND IMMUNOTOXIWLOGIC MECHANISMS
OF TRICHOTEECENE HYCOTOXINS

presented by

Yongjoo Chung

has been accepted towards fulfillment
of the requirements for

Doctoral degree in Food Science and Human
Nutrition and Institute for Environmental Toxicology

 

 

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6/01 c:/ClRC/DateDue.p65.p. 15

DETECTION AND IMMUNOTOXICOLOGIC MECHANISMS OF
TRICHOTHECENE MYCOTOXINS

By

Yongjoo Chung

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Food Science and Human Nutrition
and

Institute for Environmental Toxicology

2002

ABSTRACT

DETECTION AND IMMUNOTOXICOLOGIC MECHANISMS OF
TRICHOTHECENE MYCOTOXIN S

By
Yongjoo Chung

Antibodies for macrocyclic trichothecene, satratoxin G (SG), were produced upon
immunization of rabbits with SG bis-hemisuccinate conjugated to BSA. The antibodies
could detect free SG using a competitive direct-ELISA with a range of detection from 0.1 to
100 ng/ml. The observed antibody cross-reactivity may facilitate simultaneous detection of
other satratoxins and to a lesser extent, other macrocyclic trichothecenes. Methanol content
up to 20% in samples did not affect the immunoassay and this stability expands applicability
of the antibodies. The capacity of representative macrocyclic trichothecenes to alter TNF-a
and IL-6 production and viability was assessed in a murine macrophage model. Macrocyclic
trichothecenes can superinduce the proinflammatory cytokine, TNF-a, at low cytotoxic
concentrations, whereas these compounds are cytotoxic and reduce cytokine production at
higher concentrations. These immunomodulatory effects were observed at relatively low
(ng/ml) concentrations, suggesting that macrocyclic mycotoxins may pose a hazard to
humans exposed to Stachybotrys. In order to expand understanding of how trichothecenes
affect gene regulation, we have isolated vomitoxin (VT)- and SG-responsive genes,
macrophage inflammatory protein-2 (MIP-Z) and complement 3a receptor (C3aR), from a
murine macrophage cell line using differential display-PCR. Both VT and SG up-regulated
expression of MIP-Z, which is a chemokine responsible for chemotaxis of neutrophils to

inflammation site, whereas VT selectively up-regulated C3 aR, which is a receptor involved

in activation by complement. VT up-regulated splenic MIP-Z mRNA and its protein in
serum in a mouse model. To investigate roles of mito gen—activated protein kinase (MAPK)
in cytokine superinduction by VT, the effects of VT on MAPK activation, as well as the
relationship of MAPKs to VT-induced mRNA expression, TNF-a promoter activity, TNF-a
mRNA stability, and TNF-a protein production were assessed in RAW 264.7 murine
macrophage cells. The results indicate that VT may superinduce TNF-a expression by
increasing its transcripts and mRN A stability through activation of MAPK. Trichothecene-
induced activation of MAPK may play a critical role in superinduction of proinflammatory

cytokine, INF-0t, and succeeding pathologic events.

To my heavenly Father, my wife, parents, and three children

iv

ACKNOWLEDGMENTS

I am greatly grateful to Dr. James J. Pestka, my major advisor, for his guidance,
assistance, encouragement, and understanding throughout this study. I wish to express my
gratitude to Dr. John E. Linz, Dr. L. Patrick Hart, and Dr. Kate Claycombe for their
accessibility and advise and for being members of my graduate guidance committee.

I wish to thank Dr. Joseph J. Schroeder, Dr. Hui-Ren Zhou, Dr. Zahid Islam, Dr.
Rebecca Uzarski, Mr. Matthew Rarick, and Mr. Michael Miller for their suggestions,
practical help, and friendship.

I would like to thank my wife, Cindy Y. Chung and my children, Ashley, Joshua, and
Eunice for their love, patience, understanding, and support during this study in my life. I am
also grateful to my father, Woohong Chung, my mother, Kyungsoon Song, and mother-in
law, J unghee Lim, and my twin brother Dr. Yongsoo Chung for their unconditional love and

support in my life.

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................... ix
LIST OF FIGURES ......................................................................................................... x
Page
INTRODUCTION ........................................................................................................... 1
CHAPTER 1. LITERATURE REVIEW ....................................................................... 6
A. TRICHOTHECENES .................................................................................. 7
Al Introduction ..................................................................................... 7
A2 Vomitoxin ....................................................................................... 9
A3 Macrocyclic Trichothecenes ........................................................... 10
A4 Detection of macrocyclic trichothecenes ........................................ 12
B. EFFECT OF TRICHOTHECENES ON IMMUNE MODULATION ........ 13
B.1 Introduction .................................................................................... 13
B.2 IgA Nephropathy ............................................................................ 14
3.3 Role of cytokines in IgA Nephropathy .......................................... 14
B.4 VT-induced IgA Nephropathy ....................................................... 15
C. PROINFLAMMATORY MEDIATORS AND TRICHOTHECENES ...... 16
Cl Introduction ..................................................................................... 16
C2 Effect of VT on production of proinflammatory cytokines ............. 17
C3 Macrophage inflammatory protein-2 ............................................... 17
C4 Complement 3a Receptor ................................................................ 19
D. MAPK AND THEIR EFFECT ON CYTOKINES
MESSENGER RNA REGULATION ..................................................... 20
D.1 Introduction ..................................................................................... 20
D2 Transcriptional control ..................................................................... 22
D3 Post-transcriptional control .............................................................. 22
D4 Superinduction and involvement of MAPKs ................................... 24
E. RATIONALE FOR THIS RESEARCH ....................................................... 26

CHAPTER 1]. DEVELOPMENT OF ANTIBODIES TO SATRATOXINS
AND APPLICATION TO DIRECT COMPETITIVE ENZYME-LINKED
IMMUNOSORBENT ASSAY ...................................................................................... 28

ABSTRACT ...................................................................................................... 29

vi

INTRODUCTION ............................................................................................. 29

MATERIAL AND METHODS .......................................................................... 32
Materials and chemicals ......................................................................... 32
Synthesis of the bis-hemisuccinate of satratoxin G ................................ 32
Preparation of satratoxin G conjugates ................................................... 32
Rabbit immunization ............................................................................... 34
Antibody titration by direct ELISA ........................................................ 34
Competitive direct (CD) ELISA ............................................................. 35
ComparatiVe analysis of S. chartarum cultures by HPLC and ELISA ..... 36

RESULTS AND DISCUSSION .......................................................................... 36

CHAPTER III. MODULATION OF LIPOPOLYSACCHARIDE-INDUCED
PROINFLAMMATORY CYTOKINE PRODUCTION BY SATRATOXIN S
AND OTHER MACROCYCLIC TRICHOTHECENES IN THE MURINE

MACROPHAGE ............................................................................................................. 48
ABSTRACT ....................................................................................................... 49
INTRODUCTION ............................................................................................. 50
MATERIAL AND METHODS ......................................................................... 53

Chemicals ............................................................................................... 53
Cell culture and ELISA .......................................................................... 53
MTT assay .............................................................................................. 54
Real time PCR ........................................................................................ 55
Statistical analysis ................................................................................... 55
RESULTS ............................................................................................................ 56
DISCUSSION ...................................................................................................... 59

CHAPTER IV. UP-REGULATION OF MACROPHAGE
INFLAMMATORY PROTEIN-2 AND COMPLEMENT 3A RECEPTOR

BY THE TRICHOTHECENES DEOXYNIVALENOL AND SATRATOXIN G .......... 67
ABSTRACT ..................................................................................................... 68
INTRODUCTION ............................................................................................. 69
MATERIAL AND METHODS ......................................................................... 71

Reagents and cell culture ........................................................................ 71

vii

RNA preparation ..................................................................................... 72

Differential display analysis ................................................................... 72
Cloning and sequence analysis of differential display bands ................... 73
RT-PCR for mRNA expression in in vitro study .................................... 74
MIP-2 protein quantitation ...................................................................... 75
MlP-2 expression in mouse ..................................................................... 76
Statistics .................................................................................................. 77
RESULTS ............................................................................................................ 77
DISCUSSION ...................................................................................................... 87

CHAPTER V. ROLE OF MITOGEN-ACTIVATED PROTEIN KINASES
IN VOMITOXIN-INDUCED TNF-a MESSENGER RNA EXPRESSION

IN MURINE MACROPHAGE RAW 264.7 CELLS ......................................................... 96
ABSTRACT ..................................................................................................... 97
INTRODUCTION ............................................................................................. 98
MATERIAL AND METHODS ...................................................................... 101

Reagents and plasmids ...................................................................... 101

Cell culture ............................................................................................ 101

MAPK phosphorylation by Western blot analysis ............................... 103

RNA extraction ..................................................................................... 103

Ribonuclease protection assay .............................................................. 103

Real time-PCR ..................................................................................... 104

Cell transfection ..................................................................................... 105

Luciferase and B-galactosidase assay ...................................................... 106

TNF-a production .................................................................................. 106

Statistics ................................................................................................ 106

RESULTS ......................................................................................................... 107
DISCUSSION ................................................................................................... 121
CHAPTER VI. SUMMARY AND CONCLUSIONS ................................................... 128
BIBLIOGRAPHY ......................................................................................................... 131

viii

LIST OF TABLES

Table Page

 

Table 2.1. Comparison of HPLC and CD-ELISA analysis of S. chartarum
rice culture .......................................................................................................... 46

ix

LIST OF FIGURES

F_igur_e figs
Figure 1.1. Major groups of trichothecenes .................................................................... 8
Figure 1.2. Activation of MAPKs and their cellular response ........................................ 21
Figure 2.1. Structures of macrocyclic and selected other trichothecenes ...................... 33

Figure 2.2. Direct ELISA titer determination of rabbit SG antibodies.
Serially diluted antisera were coated onto microtiter wells and
specific binding determined by incubation with HRG-SG ................................. 38

Figure 2.3. Standard curve of SG CD ELISA. Microtiter were coated
with SG antibodies from rabbits (#148, #151, and #155) and then
simultaneously incubated with Standard SG and HRP-SG. Each
point represents the mean i SD for three replicates ............................................. 40

Figure 2.4. Cross-reactivity of SG Rabbit 2 antiserum toward macrocyclic
trichothecenes, verrucarol, and deoxynivalenol. Wells were
coated with SG antibody and incubated with HRP-SG in the
presence of different toxins ................................................................................. 41

Figure 2.5. Effect of methanol on binding of HRP-SG to SG antiserum.
SG antiserum was coated onto microwells and incubated with HRP-SG
in the presence of methanol at concentrations ranging from 0 to 40% ............... 44

Figure 3.1. Structures of macrocyclic trichothecenes .............................................. 51

Figure 3.2. Effect of macrocyclic trichothecenes on TNF-a production

in RAW 264.7 cells. ....................................................................................... 57
Figure 3.3. Effect of satratoxin G (SG) on TNF-ot mRNA level in LPS

stimulated RAW 264.7 cells. ............................................................................ 58
Figure 3.4. Effect of macrocyclic trichothecenes on IL-6 production in

RAW 264.7 cells. ............................................................................................. 60
Figure 3.5. Effect of macrocyclic trichothecene on viability of RAW

264.7 cells by MTT assay. ................................................................................. 61
Figure 4.1. Sequencing gel section from mRNA DD-PCR ............................................. 79

Figure 4.2. Dose-dependent induction of MIP-Z and C3aR mRNAs expression
by VT in RAW 264.7 cells. ................................................................................ 80

Figure 4.3. Dose-dependent induction of MIP-2 mRNAs expression
by SG in RAW 264.7 cells. ............................................................................... 81

Figure 4.4. Time-dependent induction of MIP-2 and C3aR mRNA expression
by VT in RAW 264.7 cells. .............................................................................. 83

Figure 4.5. Time-dependent induction of MIP-2 mRNA expression by
SG in RAW 264.7 cells. .................................................................................... 84

Figure 4.6. Effect of VT or SG on MIP-2 protein production by RAW 264.7
cells for dose-dependent study. ........................................................................... 85

Figure 4.7. Effect of VT or SG on MIP-2 protein production by RAW 264.7
cells for time-course study. ................................................................................. 86

Figure 4.8. Effect of VT on MIP-2 expression in vivo. ................................................ 88

Figure 5.1. Time-dependent activation of p38, ERK1/2, and JNKl/Z by
LPS, VT and LPS+VT in RAW 264.7 macrophage cells ............................... 108

Figure 5.2. Dose-dependent activation of p38, ERK1/2, and JNK1/2 by
VT and LPS+VT in RAW264.7 macrophage cells ........................................ 109

Figure 5.3.A. Cytokine mRNA expression in LPS-, VT-, and LSP+VT
treated RAW264.7 cells ................................................................................. 111

Figure 5.3.B. The bands of TNF-0t and GAPDH mRNA were
quantified using Phospholrnager and are shown as a relative
intensity of control sample at 2 hr .................................................................. 1 12

Figure 5.4. Effect of MAPK inhibitors on LPS-, VT-, and LPS+VT-
induced TNF-0t mRNA expression in RAW 264.7 macrophage cells ............. 1 14

Figure 5.5. Effect of MAPK inhibitors on LPS-, VT-,and LPS+VT-
induced TNF reporter gene expression in RAW 264.7 macrophage cells .......... 1 15

Figure 5.6. Effect of SB2035 80 on VT-induced TNF-0t mRNA expression
in LPS-treated RAW264.7 cells ..................................................................... 117

xi

Figure 5.7. Effect of PD98059 on VT-induced TNF—or mRNA expression
in LPS-treated RAW264.7 cells ..................................................................... 119

Figure 5.8. Effect of SP600125 on VT-induced TNF-0t mRNA expression
in LPS-treated RAW264.7 cells ..................................................................... 120

Figure 5.9. Effect of MAPK inhibitors on LPS-, VT-, and LPS+VT-
induced TNF-0t production in RAW 264.7 macrophage cells ....................... 122

xii

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INTRODUCTION

Trichothecene mycotoxins are a group of secondary metabolites produced by
F usarium, Stachybotrys, and other molds growing on food or in the environment. Over 180
trichothecenes are identified and categorized into four groups (type A-D) based on the
functional groups attached to the trichothecene ring structure. From a public health
standpoint, type D (e. g. satratoxins, verrucarin A, and roridin A), type A (e. g. T-2 toxin and
diacetoxyscirpenol), and type B (e. g. vomitoxin [VT or deoxynivalenol] and nivalenol) are
of particular interest since these toxins are present in the environment and in food. These
mycotoxins bind eukaryotic ribosomes and inhibit protein synthesis. Exposures of farm
animals and humans to trichothecenes have been reported worldwide and have been related
to outbreaks of alimentary toxic aleukia, vomiting, gastroenteritis, leukocytosis, and
circulatory shock.

Satratoxins, a group of macrocyclic trichothecenes, are mainly produced by
Stachybotrys atra, which grows on cellulose materials in water-damaged buildings.
Satratoxins are 10 to 100 times more toxic than are other groups of trichothecenes in mouse
LD50 studies, HeLa cell cytotoxicity tests, and rabbit reticulocyte protein synthesis inhibition
assays. These toxins have been etiologically associated with stachybotryotoxicosis in
animals in Central Europe. Clinical signs of this disease include leukopenia,
thrombocytopenia, hemorrhage, arrhythmic heartbeat, and death. Outbreaks of Stach ybottys-
associated diseases in water-damaged homes have been reported (Etzel et al., 1998;
Johanning et al., 1996). Recently, the toxigenic S. atra was reported to be involved in

pulmonary hemorrhage and hemosiderosis in infants living in S. atra-contaminated homes

(Etzel et al., 1998).

Given the potential harmful effects of macrocyclic trichothecenes on human health,
it is important to monitor their presence in the environment. Current methods for detection
and quantitation of the toxins include thin-layer chromatography, high performance liquid
chromatography, and mass spectrometry, which require time-consuming extraction, sample
clean-up, and/or expensive instrumentation. Enzyme-linked immunosorbent assay (ELISA)
has been widely applied for the detection of mycotoxins because of its high specificity and
sensitivity. It is desirable and feasible to develop a specific ELISA for the satratoxins.

Leukocytes are particularly sensitive to trichothecenes. Depending on dose and
duration of exposure, these toxins can be immunostimulatory as well as immunosuppressive.
Trichothecene-induced immunostimulatory effects include increased cytokine production and
upregulation of immunoglobulin (Ig) production. Immunosuppressive effects of
trichothecenes include apoptosis and impairment of humoral immunity (Bondy and Pestka,
2000). To date, most work on the immunostimulatory effects of trichothecenes has focused
on the type A and type B groups. It is important to better understand effects of macrocyclic
trichothecenes on immune fimction for their potential effects on human health.

The trichothecene VT causes a wide range of toxicolo gical and immunological effects
in mice. Experimentally, high dose oral exposure to VT causes impairment to immune cells
whereas low dose can result in vomiting, diarrhea, and gastroenteritis. Dietary VT in mice
affects mitogen-induced proliferation of lymphocytes, host resistance, Ig production,
especially IgA, and cytokine expression. One of the most prominent effects of dietary VT

is dysregulation of IgA production, which is highly analogous to human IgA nephropathy.

Relevant effects of VT include increases in serum IgA, circulating IgA immune complexes,
kidney mesangial IgA, and hematuria as well as polyclonal activation of IgA secreting cells.
Overproduction of IgA by VT is mediated through alteration of cytokine production by
macrophages and helper T cells.

Cytokine superinduction by VT is well documented in vivo and in vitro. Acute, oral
exposure of mice to VT increased Th1 cytokine, IL-2 and IFN-y mRNA expression, Th2
cytokines IL-4, -5, -6 and their mRNAs, and proinflammatory cytokines IL-6, IL-18, and
tumor necrosis factor (TNF)-a. Superinduction of IL—2 and TNF-0L and IL-6 expression by
VT were observed in a murine T-cell culture (EL-4 cell line) model and in RAW 264.7
macrophage cells, respectively. In order to expand understanding of how trichothecenes
affect gene regulation, murine RAW 264.7 macrophage cells were treated with VT or
satratoxin G (SG) and expressed genes were assessed by differential display PCR (DD-PCR).
The results indicate that both toxins up-regulated expression of macrophage inflammatory
protein-2 (MIP-2), which is a chemokine responsible for chemotaxis of neutrophils to
inflammation sites, whereas VT selectively up-regulated complement 3 a receptor (C3aR),
which is a receptor involved in activation through complement. Induction of these two genes
might contribute to trichothecene-induced immunotoxicity.

Mitogen-activated protein kinase (MAPK) cascades are major signaling systems in
which cells transduce extracellular stimuli into intracellular responses in leukocytes and other
cells. MAPKs have been associated with a wide array of cellular responses including
cytokine production, cell growth, differentiation, and apoptosis. Three distinct MAPKs have

been characterized in the mammalian system; the extracellular signal-regulated kinase

 

(ERK1/2 or p42/p44), the c-Jun amino-terminal kinases (JNK1/2, also known as stress-
activated protein kinases, SAPK), and p38 MAPK. Activation of these MAPKs by stimuli
turns to signal-specific transcriptional and posttranscriptional activation, and determine the
cellular responses.

MAPKs may contribute to gene expression at a transcriptional level by
phosphorylating transcription factors and at post-transcriptional level by mediating
phosphorylation of RNA binding proteins. Activated transcription factors bind their binding
sites in DNA which they subsequently transcribe to mRN A. Phosphorylated RNA binding
protein may regulate binding affinity to its target site and result in stabilization of mRNA.

MAPKs may be critical mediators of VT-induced cytokine production. VT is known
to superinduce TNF-0t and IL-6 in RAW 264.7 cells and IL-2 in EL-4 cells. This
superinduction is found to be due to both increased transcription and increased mRNA
stability. The increased transcripts for TNF-0t and IL-6 were also explained by an increase
of binding activity of NF-KB, AP-l and NF-IL6 transcription factors to their promoter
regions. The increased TNF-O. and IL-6 mRNA stability was explained by the observation
that LPS-induced TNF-0t and IL-6 mRNAs were stabilized by VT in the presence of
transcription inhibitor, 5,6-dichloro-beta-D-ribofuranosyl-benzimidazole (DRB). Recently,
VT was reported to induce p38 kinase, ERK1/2, and JNKl/2. The involvement of MAPKs
in increased TNF-on mRNA expression by VT has yet to be explained.

The aforementioned issues were addressed in this dissertation, which is comprised
of five parts. Chapter I reviews the literature on trichothecenes, effect of trichothecenes on

immune modulation, proinflammatory mediators and trichothecenes, and MAPKs and their

4

effect on cytokine mRNA regulation. Chapter H describes development of antibodies to
satratoxins and application to direct competitive enzyme-linked immunosorbent assay.
Chapter 111 describes modulation of lipopolysaccharide-induced proinflammatory cytokine
production by satratoxins and other macrocyclic trichothecenes in the murine macrophage.
Chapter IV describes up-regulation of macrophage inflammatory protein-2 and complement
3a receptor by the trichothecenes deoxynivalenol and satratoxin G. Chapter V describes
MAPK effects on TNF-or mRNA expression by deoxynivalenol. Chapter VI includes

summary and conclusion of these studies.

 

 

CHAPTER I

LITERATURE REVIEW

A. Trichothecenes
A.I. Introduction

Trichothecenes are a group of mycotoxins, which are secondary metabolites produced
by various strains of fungi including Fusarium and Stachybotrys. Fungal infection,
colonization, and toxin production on grain crops can occur in the field, at harvest, and
during storage. Favorable environmental conditions such as high moisture and cool
temperature support mold colonization with subsequent trichothecene biosynthesis (Ishii,
1983). More than 1 8O trichothecenes have been identified and are categorized into four types
(A-D) depending on the functional groups attached to the trichothecene ring structure (Figure
1.1) (Grove, 1988; Grove, 1993; Grove, 1996). Deoxynivalenol (vomitoxin, VT) and
nivalenol (type B trichothecenes) and T-2 toxin and diacetoxyscirpenol (type A
trichothecenes), are among the most common contaminants in cereal grains (Agriculture,
1989). Satratoxins, the macrocyclic trichothecenes, are type D trichothecenes and are often
found in feeds or water-damaged structures (Bata et al., 1985; Harrach et al., 1983;
Johanning et al., 1996). Because these food-bome mycotoxins are resistant to processing
(Jackson and Bullerman, 1999; Scott et al., 1984; Wolf-Hall et al. , 1999), they are potential
contaminants of human and animal food supplies. Consequently, trichothecene-
contaminated grains have been a concern with food industries and animal feed manufacturers
due to their possible health risks (Biabani and Dowling, 1996).

Trichothecenes have been associated with acute toxicoses of farm animals and
humans (Bhat et al., 1989; Ueno, 1983a; Yagen and Joffe, 1976). With humans, exposure

to trichothecenes has led to outbreaks of alimentary toxic aleukia, vomiting, and

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Figure 1.1 Major groups of trichothecenes

gastroenteritis (Bhat et al., 1989; Li et al., 1999; Luo etal., 1990). Acute effects of VT in
animals are diarrhea, vomiting, leukocytosis, circulatory shock, and death while chronic
effects are feed refusal, reduced rate of weight gain, and immunotoxicity (Arnold et al.,
1986; Bondy and Pestka, 2000; Forsyth et al., 1977; Friend et al., 1982).

In addition to health concerns, contamination of grains by trichothecenes has been
a major economic concern. A VT outbreak occurred across many Mid-Western states
including Michigan in 1996 (Biabani and Dowling, 1996). Kellogg's, one of the biggest
cereal producing companies in the world refused to purchase wheat from producers in the
state of Michigan due to the possible human health risks fi'om VT. Since 1996, American
barley has become less competitive, partly due to contamination with VT (Service, 2000).
Grains contaminated with VT also has reduced value as animal feed due to feed refusal and
reduced weight gain in the animals (Biabani and Dowling, 1996; Kalish, 1995). A current
FDA guideline in US. is 1 ppm for bran, flour, and germ intended for human consumption
while 5 and 10 ppm are the levels for feed ingredients intended for swine diet and cattle diet,

respectively (Trucksess, 1995).

A.2. Vomitoxin (Deoxynivalenol)

VT is one of the major trichothecenes produced by F usarium graminearum and F.
culmarum, which is common contaminant of several grains including corn, wheat, and barley
(Perkowski, 1998; Trigo-Stockli et al. , 1998; Wetter et al. , 1999; Yoshizawa and J in, 1995).
Wheat and barley from the US. Midwest were widely contaminated with VT following the

heavy rain and cool weather of 1993-1996 (Trigo-Stockli et al., 1998; Trucksess et al.,

199

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1995). A recent report indicates that 71% of the 237 commercially available cereal-based
foods including infant foods were positive for VT (Schollenberger et al., 1999).

VT induces a wide range of toxicological and immunological effects. A high and
acute dose (2100 mg/kg) of VT causes necrosis of the gastrointestinal tract, bone marrow,
and lymphoid tissues as well as lesions in heart and kidney (Forsell et al. , 1987). Subchronic
dietary exposure to VT impairs the functions of the intestinal tracks in mice (Hunder et al.,
1991). VT induces anorexia at low doses and emesis at higher acute doses in animals
(Forsyth et al. , 1977; Pestka et al. , 1987; Prelusky and Trenhohn, 1993). Dietary VT affects
mitogen-induced proliferation of lymphocytes, host resistance, immunoglobulin (Ig)
production, especially IgA production, and cytokine expression (Rotter et al., 1996).

The capacity of VT to alter immune function has been extensively studied (Bondy
and Pestka, 2000). Depending on dose and frequency of exposure, VT can be both
immunosuppressive and immunostimulatory. Irnmunosuppression by VT is manifested by
rapid onset of leukocyte apoptosis (Islam et al., 2002; Pestka et al. , 1994; Yang et al., 2000;
Zhou et al. , 2000; Zhou et al. , 1999). On the other hand, low doses of VT exposure enhance
expression of various cytokines in vitro and in vivo (Azcona-Olivera et al. , 1995a; Li et al. ,
1997; Wong et al., 1998; Yan et al., 1997, 1998b; Zhou et al., 1997). These VT-induced
immunomodulatory effects are further related to activation of mitogen-activated protein

kinases (MAPKs) (Moon and Pestka, 2002; Yang et al., 2000).

A.3. Macrocyclic T richothecenes

Macrocyclic trichothecenes are characterized by a cyclic diester or triester ring which

10

connects O4 to C-15 of the trichothecene structure (Figure 1.1). These trichothecenes are
10 to 100 times more toxic than Type A and Type B trichothecenes in mouse LD50 studies,
HeLa cell cytotoxicity tests, and rabbit reticulocyte protein synthesis inhibition assays (U eno,
1983b). Several potentially important macrocyclic trichothecenes are produced by
Stachybotrys atra (also known as S. chartarum ). This mold grows on cellulose substrates
with a high moisture content such as wet hay and straw (Bata et al., 1985; Harrach et al.,
1981). Macrocyclic trichothecenes have been etiologically associated with
stachybotryotoxicosis in horse and sheep in Central Europe (Bata et al. , 1985; Harrach et al. ,
1983; Harrach et al., 1981; Hintikka, 1978). Clinical 'signs of this disease include
leukopenia, thrombocytopeni a, hemorrhage, arrhythmic heartbeat, and death (Forgacs, 1972).

Humans can also be exposed to Stachybotrys and macrocyclic trichothecenes via the
environment. Outbreaks of Stachybotrys-associated diseases in water-damaged structures
have been reported (Etzel et al., 1998; Johanning et al., 1996). The characteristics of the
diseases include recurrent cough, imitation of the eyes and skin, mucous membrane disorder,
headache, and fatigue. Recently, a toxigenic strain of S. atra was reported to be involved in
pulmonary hemorrhage and hemosiderosis in infants living in S. atra-contaminated homes
(Etzel et al., 1998). Furthermore, this fungus was isolated from the lungs of a child with
pulmonary hemorrhage (Elidemir et al. , 1999). This was the first report of the organism in
human tissue, supporting the concept that Stachybotrys is involved in the lung disease. The
involvement of macrocyclic trichothecenes, however, was not determined.

The ability of macrocyclic trichothecenes to modulate immune effect has partially

been characterized. For example, macrocyclic trichothecenes have been shown to

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superinduce H.—2 production in EL-4 thymoma cells as well as induce and impair
blastogenesis of lymphocytes in the presence of mitogen (Hughes et al., 1990; Pestka and
Forsell, 1988). These toxins have been observed to induce apoptosis through activation of
MAPK in Jurkat T cells or RAW 264.7 murine macrophage cells (Shifi'in and Anderson,
1999; Yang et al., 2000). Spores from S. atra induce production of proinflammatory
cytokines, TNF-0L and IL-6, in RAW 264.7 cells (Ruotsalainen et al., 1998). However, the

capacity of macrocyclic trichothecenes to induce those cytokines has not been studied.

A.4 Detection of macrocyclic trichothecenes

Given the potential harmful effects of macrocyclic trichothecenes on human health,
it is important to monitor their presence in the environment. Current methods for detection
and quantitation of the toxins include thin-layer chromatography, high performance liquid
chromatography, and mass spectrometry (Krishnamurthy et al., 1989; Stack and Eppley,
1980; Tuomi et al., 1998). The methods used for macrocyclic trichothecenes, however,
necessitate time-consuming extraction, sample clean-up and/or expensive instrumentation
(Hinkley and Jarvis, 2000). A protein translation assay employing luciferase mRNA was
developed for the purpose of detection of trichothecenes in the spores of toxigenic fungi
(Yike et al. , 1999). This assay is sensitive due to the effect of protein synthesis inhibition by
all trichothecenes present in spores and this method can not distinguish the effects of a single
trichothecene from other trichothecenes or other chemicals in a sample. A further limitation
of this assay is its expense and requirement for special training and equipment.

Enzyme-linked immunosorbent assays (ELISA) have been widely applied to

12

detection of mycotoxins because of their high specificity and sensitiVity (Abouzied etal.,
1993; Azcona-Olivera et al., 1992; Monti et al., 1999; Thirumala—Devi et al., 2000; Yuan
et al., 1999). Although the development of ELISA for a variety of food-borne non-
macrocyclic trichothecenes has been accomplished (Abouzied et al., 1993; Barna-Vetro et
al., 1994; Lee et al., 1989; Park and Chu, 1996; Yuan et al., 1999), relatively little effort to
date has been placed on the environmentally important macrocyclic trichothecenes. In one
exception, an ELISA for the macrocyclic trichothecene, roridin A, was developed using
rabbit polyclonal antibodies (Martlbauer et al., 1988). The antibodies were highly specific
for roridin A and showed low cross-reactivity against satratoxin H (15%) and SG (6.5%),
which are two of the major toxins in Stachybotrys spores(Jarvis et al., 1998). Therefore, it

was desirable and feasible to develop a specific ELISA for the satratoxins.

B. Effect of trichothecenes on immune modulation
B. 1 Introduction

VT and other trichothecenes act in a number of cellular systems as protein synthesis
inhibitors by binding to eukaryotic ribosomes (F einberg and MacLaughlin, 1989; Liao et a1. ,
1976; Middlebrook and Leatherman, 1989; Ueno et al., 1968). VT targets actively dividing
cells in immune organs and intestinal mucosa (Azcona-Olivera et al., 1995a; Yan et al.,
1998a; Zhou et al., 2000) and consequently modulates immune function. One of the most
prominent effects of dietary VT in a mouse model is dysregulation of IgA production, which
is highly analogous to human IgA nephropathy (D'Amico, 1987; Dong et al. , 1991;

Emancipator and Lamm, 1989).

13

 

3.2

glor
the
me:
rais

pic

B.2 IgA Nephropath y

IgA nephropathy is recognized worldwide as the most common form of
glomerulonephritis (D'Amico, 1987; Emancipator and Lamm, 1989). It is characterized by
the predominant deposition of IgA immune complexes (IgA-1C) in the glomerular
mesangium. Common features in IgAN patients are an increased circulating IgA-1C and
raised serum IgA concentration (Czerkinsky et al., 1986; Lesavre et al., 1982). Cytokines
produced by infiltrating monocyte/macrophages, resident glomerular cells, and neutrophils
are suspected as mediators for inflammatory damage in human glomerulonephritis including

IgAN (Kincaid-Smith et al., 1989; Lai et al., 1994b; Yoshioka et al., 1993).

8.3 Role of cytokines in IgA Nephropath y

Cytokines are key mediators of proliferation and differentiation in lymphoid cells (de
Vries et al., 1993; Esser and Radbruch, 1990). They are also involved in immunoglobulin
class-switching to different isotypes (Lebman and Edmiston, 1999). Changes in cytokine
profile may influence the increased IgA production observed in IgAN. An increase of
circulating “type 1" T helper (Th 1) and “type 2" T helper (Th2) lymphocytes subset has been
observed in IgAN patients (Lai et al., 1994a). Cytokines IL-4, IL-5 and IL-6 produced by
Th2 cells are of particular importance in differentiation of B cells to IgA production
(McGhee et al., 1990; McGhee et al., 1989; Murray et al., 1990). Other cytokines such as
H.-2 and IFN-y, which are produced by Th1 cells, can stimulate other cytokine secretion and
activate macrophage cells, respectively. Transforming growth factor-B (TGF-B) produced

by Th1 cells can induce IgA class switch recombination (Defrance et al. , 1992). IL-2, -4, and

14

IFN-y levels were higher in mitogen-stimulated peripheral blood mononuclear cells (PBMC)
from IgAN patients compared with controls (Lai et al. , 1994b; Lai et al. , 1991 ; Schena et a1. ,
1989). TGF-B expression was increased in CD4+ cells in IgAN patients (Lai et al., 1994c).

Overall, different cytokine profiles are found in IgAN patients compared to healthy controls.

8.4 VT-induced IgA Nephropath y

VT can induce IgAN in a mouse model in which alteration of cytokine production
may influence induction of the disease. The level of serum IgA increased while IgG and IgM
levels decreased when dietary VT was fed to mice (Forsell et al., 1986). Chronic dietary
exposure of mice to VT resulted in an elevation of serum IgA and led to IgA nephropathy
(Bondy and Pestka, 1991; Dong and Pestka, 1993; Pestka et al., 1989). Accumulation of
mesangial IgA and increased IgA immune complex in serum were observed in VT-fed mice
(Dong et al., 1991). Dysregulation of IgA production is suspected to result from an
alteration of cytokine profile caused by VT.

VT modulates T helper cytokine profile as well as proinflammatory cytokine profile
in vivo and in vitro. Acute, oral exposure of mice to VT increased Th1 cytokine, IL-2 and
IFN-y mRNA expression, Th2 cytokines, IL-4, -5, -6, and their mRNAs (Azcona-Olivera et
al., 1995b; Ouyang et al., 1996), and proinflammatory cytokines, IL-6, IL-1 [3, and tumor
necrosis factor (TNF)-a (Azcona-Olivera et al., 1995a). With in vitro studies,
superinduction of IL-2 mRN A and its protein by VT was observed in a murine T-cell culture
(EL-4 cell line) model (Li et al., 1997). TNF-0t and IL-6 mRNA and their proteins were

superinduced in RAW 264.7 macrophage cells when VT and lipopolysaccharide (LPS) were

15

cotreated (Wong et al. , 1998). The changes in these cytokine profile are generally suspected
for upregulation of IgA production, leading to IgAN (Dong et al. , 1994; Warner et al. , 1994;
Yan et al., 1997). From a study with IL-6-“knockout” mice, IL-6 was shown to be an
essential cytokine in VT-induced IgA dysregulation (Pestka and Zhou, 2000). However,
from a study with TNF-or receptor “knockout” mice, TNF-or was not involved in VT-

mediated dysregulation of IgA production (Pestka and Zhou, 2002).

C. Proinflammatory mediators and trichothecenes
C.I Introduction

Inflammatory mediators can be categorized into four groups: cytokines, chemokines,
plasma enzyme mediators including complement, and lipid inflammatory mediators. The
major proinflarnmatory cytokines are TNF-or, IL-6, and IL-1. These cytokines generally
function in increasing fever, synthesis of acute phase protein by liver, increased vascular
permeability, and activation of B- and T-cells. Chemokines such as IL-8 and macrophage
inflammatory protein-2 (MIP-2), are small polypeptides that attract other leukocytes and
regulate the expression of adhesion molecules in leukocyte membranes. Plasma enzyme
mediators include the kinin system, clotting system, fibrinolytic system, and complement
system. Binding of complement split products to receptors on cell membranes induces
various immune responses. Lipid inflammatory mediators are prostaglandins, leukotrienes,
and platelet-activating factors, which function in increased vascular permeability, neutrophil

chemotaxis, and platelet aggregation (Kuby, 1997).

16

C.2 ;

macr

deat‘.

Huf:

to V"

199

few

B-c

SEC!

oral.

ofl

C3

C.2 Effect of VT on production of proinflammatory cytokines

Proinflammatory cytokines such as TNF-0t and IL-6 are mainly produced by
macrophage upon stimulation. TNF-or is known to mediate biological responses such as cell
death, cytokine and chemokine induction, antiviral activity, and sepsis (Beutler and van
Huffel, 1994; Sedgwick et al. , 2000; Smith et al. , 1990; Van Damme et al. , 1987). Exposure
to VT increases TNF-0L mRN A and protein level in vivo and in vitro (Azcona-Olivera et al.,
1995a; Wong et al., 1998). IL-6 is involved in the inflammatory response by increasing
fever, synthesis of acute-phase proteins by liver, increased vascular permeability, and T- and
B-cell activation(Kuby, 1997). In addition, IL-6 promotes B cell differentiation to IgA
secreting cells in the Peyer’s patch (Beagley et al. , 1991; Beagley and Elson, 1992). Acute,
oral exposure to VT increases IL-6 production as well as IgA production and the elevation

of IL-6 is mediated mainly by macrophage cells (Yan et al., 1998b).

C3 Macrophage inflammatory protein-2

MIPS are chemokines, a member of structurally related proteins that can induce
migration of a specific subset of leucocytes. Three MIPS (MIP- 1 a, MIP-lb and MIP-2), that
are structurally and functionally similar, have been identified (Sherry et al. , 1988; Wolpe et
al., 1988; Wolpe et al., 1989). MIPS are produced in response to various stimuli such as
LPS, proinflarnmatory cytokines, and oxidative stress in a wide range of cells including
alveolar macrophages, mast cells, peritoneal macrophages, epithelial cells, and fibroblasts

(Burd et al. , 1989; Driscoll et al. , 1993; Kopydlowski et al. , 1999; Shi et al., 1999). MIP-l 0L

and MIP-IB possess proinflammatory properties and can activate both neutrophils and

17

mononuclear cells (Driscoll, 1994). MIP-2 is a potent neutrophil chemoattractant and
epithelial cell mitogen and it is involved in acute pulmonary inflammation (Driscoll et al.,
1995; Huang et al., 1992; Schmal et al., 1996; Yoshidome et al., 1999).

Several in vivo studies demonstrate that MIP-2 is involved in tissue injury and
inflammation. Expression of MIP-2 mRN A and accumulation of neutrophils in lung tissue
were associated with development of lung edema during hepatic ischemia/reperfusion in
mice (Yoshidome et al., 1999). In that study, passive immunization against MIP-2 with
neutralizing antibody reduced lung edema indicating that MIP-2 is involved in tissue injury.
The role of MIP-2 in LPS-induced lung injury in rats was studied demonstrating that LPS
instillation into rat lung up-regulates MIP-Z mRNA in a time dependent manner and MIP-2
is responsible for neutrophil accumulation in lungs (Schmal et al., 1996). These studies
demonstrate that increased MIP-2 expression followed by neutrophil accumulation results
in tissue damage and inflammation.

MIP-2 has been considered a functional analog of human IL-8 (Sherry et al., 1992).
IL-8 mediated neutrophil recruitment to the site of inflammation can occur in patients with
adult respiratory stress syndrome, idiopathic pulmonary fibrosis, rheumatoid arthritis, and
hepatic ischemia-reperfusion injury (Colletti et al. , 1995; Donnelly et al. , 1994; Koch et al. ,
1994; Lynch et al., 1992). A significant increase of IL-8 in sera of IgAN patients was
detected compared to healthy controls. The free IL-8 autoantibodies of the IgA were more
frequently detected in IgAN patients (Lai et al., 1996). Recently, VT induced IL-8

production in human macrophage cells (Sugita-Konishi and Pestka, 2001 ).

18

8C

lys

6’!

prc
in

mi
wa
Pro
IL-
Ten
of I

81in

C4 Complement 3a Receptor
Complement 3a receptor (C3aR) is a seven-transmembrane receptor that is
functionally coupled to G proteins (Roglic et al. , 1996). C3aR is distributed in both myeloid
cells (monocytes/macrophage, neutrophils and eosinophils) and nonmyeloid cells (mast cells,
glial cells, astrocytes, and smooth muscle cells) throughout various tissues including lung,
heart, kidney, liver, testis, and brain (Davoust et al. , 1999; Gasque et al. , 1998; Legler et al. ,
1996; Martin et al., 1997; Sayah et al., 1999). C3aR is activated on binding C3a ligand,
which is one of the anaphylatoxins produced by proteolytic cleavage during complement
activation (Hugli, 1990). C3a ligand stimulates numerous biological responses including
lysosomal enzyme secretion from neutrophils, histamine release from mast cells, smooth
muscle contraction, and modulation of the humoral and cellular immune responses (Glovsky
et al., 1979; Morgan, 1986; Sayah et al., 1999; Showell et al., 1982; Stimler et al., 1983).
C3a can modulate the immune response by stimulating or suppressing cytokine
production. The effects of C 3a on IL-6 gene expression and protein production were studied
in the presence of LPS in human PBMC (Fischer et al., 1999). In this study, both IL-6
mRNA and protein were increased with a co-treatment of C3a and LPS by PBMC while IL-6
was not enhanced with C3a alone. Pretreatment of PBMCs with pertussis toxin inhibited the
production of IL-6. In another study with human astrocytomas, stimulation by C3a enhanced
IL-6 mRNA level and protein level while IL-lB, TNF-cc and TGF-B mRNA expression
remained unchanged (Sayah et al. , 1999). IL-6 mRN A expression was blocked by treatment
of pertussis toxin or C3aR antibody, which indicates that the IL-6 response was specific to

stimulation of C3a in astrocytomas. Regulation of B cell functions by C3a was studied by

19

0r

Ca

measuring IgG, TNF-0t, and IL-6 production in Staphylococcus aureus Cowan strain I
(SAC)/IL-2-activated B cells (Fischer and Hugli, 1997). Incubation with C3a in SAC/IL-2-
activated B cells suppressed polyclonal immune response, IL-6, and TNF-ct production.

Taken together, C3a has immunomodulatory effects upon binding C3aR in various cell types.

D. MAPK and their effect on mRNA regulation of cytokines
0.1 Introduction

MAPK cascades are major signaling systems in which cells transduce extracellular
stimuli into intracellular responses. The essential cascades in MAPK pathways consist of
three protein kinases (Figure 1.2). The stimulated MAPK kinase kinase (MAPKKK or
MEKK), which is the most upstream of the MAPK cascades, phosphorylates and activates
the MAPK kinase (MAPK or MEK). The activated MAPKK, in turn, phosphorylates
closely conserved threonine and tyrosine residues in MAP kinase (MAPK). These highly
sequenced phosphorylation events turn the extracellular stimuli to signal-specific
transcriptional and posttranscriptional activation, and determine the cellular responses
(Tibbles and Woodgett, 1999).

Three distinct, but partially “cross-talking”, MAPKs have been characterized in the
mammalian system; the extracellular signal-regulated kinase (ERKS or p42/p44), the c-Jun
amino-terminal kinase (INKS, also known as stress-activated protein kinases, SAPK), and
p38 MAPK. The ERK pathway is stimulated at the cellular membrane by either a mitogen
or grth factor. ERK causes cell proliferation, differentiation, and mitosis. JNK and p38

cascades are activated by multiple environmental insults such as LPS, heat stress, UV

20

 

 
    
 
    

 
       

Stress, differentiation factor, growth factor
Growth Factors

Mitogens

Stress or
Environmental

 

 

Insults

     

    

\

 

      

     
    
 
  
    
   
   
   

“Raf MEKK1,2, 3 PAK, MEKK4 "
MEK / MKK4,7 MKK3.6
l l j
Cytoplasm ERK “K p38

   

  

Elk I cJun ATF_2
N cleus cJun

/S‘Jlm\/\

AP-l site

 

 

 

 

 

Growth
Prollferatlon leferentlatlon Cytoklno production
Dflferentlatlon Survival Apoptosls
Mltosls Apoptosls

 

 

 

 

 

 

 

Figure 1.2 Activation of MAPKS and their cellular response

21

radiation, and osmotic stress. Activation of JNK and/or p38 mediates cellular processes such
as apoptosis, immune activation, inflammation and adaptation to stress (Tibbles and

Woodgett, 1999).

0.2 Transcriptional control

MAPKS contribute to gene expression at a transcriptional level by phosphorylating
transcription factors. Activation of p38 leads to phosphorylation of activating transcription
factor-2 (ATF-2). The phosphorylated ATP-2 functions as a transcriptional activator via
binding to the CAMP-response element (CRE) (Brinkrnan et al., 1999). Activated JNK1/2
can also phosphorylate c-Jun and ATF-2 (Hazzalin et al. , 1996). Phosphorylated c-Jun binds
to the activating protein—1 (AP-1) binding site resulting in transcriptional activation.
Activation of ERK1/2 can phosphorylate Elk-1, which is a c-fos regulatory transcription
factor (Babu et al., 2000). In addition, all three MAPK pathways are involved in activation
of nuclear factor-kB (NF -kB) by phosphorylating IkBa (Lee et al., 1997; Schwenger et al.,
1998; Zhao and Lee, 1999). CRE, AP-l, and NF-kB cis-acting sites are known to play key

roles in proinflammatory cytokine gene expression, including TNF-a.

D.3 Post-transcriptional control

MAPKS also contribute to gene expression at a posttranscriptional level.
Stabilization of cytokine mRNAs can be achieved by different signaling events in all three
MAPK pathways. For example, IL-6, IL-8, and TNF-on mRNAs are stabilized by activation

of the p38 kinase pathway by stimuli such as lipopolysaccharide (LPS) and IL-1 (Brook et

22

al., 2000; Miyazawa et al., 1998; Winzen et al., 1999). The INK pathway also participates
in stabilization of IL-2 and IL-3 mRNAs by stimulation of ionomycin (Chen et al., 1998;
Ming et al., 1998). The ERK pathway is responsible for granulocyte-macrophage colony-
stimulating factor (GM-CSF) and IL-8 mRN A stabilization induced by TNF-or (Esnault and
Malter, 2002; Jijon et al., 2002).

The mechanisms by which MAPK contributes to cytokine mRNA stabilization are
not clearly understood. However, phosphorylation of RNA binding proteins by MAPK
might be one of the important factors in regulating RNA stability (Sirenko et al. , 1997). AU
rich element (ARE) in 3'-untranslated region (U TR) of mRNA is found in many short-lived
cytokine and proto-oncogene mRNAs and determines stabilization of their transcripts (Chen
and Shyu, 1995; Gillis and Malter, 1991; Sachs, 1993; Winstall et al. , 1995). Several distinct
proteins such as AUFl (Sirenko et al., 1997), HuR (Myer et al., 1997), and tristetraproline
(TTP) (Lai et al., 1999), are known to bind to ARES and regulate mRNA turnover.

Depending on types of protein and state of phosphorylation, binding of these proteins
on ARE contributes to either stabilization or destabilization of mRNA. For example, AUFl
selectively recognizes ARE and facilitates mRNA degradation (DeMaria and Brewer, 1996;
Pende et al., 1996; Sirenko et al. , 1997). Adhesion-dependent stabilization of IL-IB
transcripts in monocytes was abolished when SK&F 86002, p38 kinase inhibitor, was treated
(Sirenko et al., 1997), which indicates that phosphorylation events are necessary for AUFl
to bind to ARE of the transcripts. HuR, a member of the ELAV-like protein family, is
important in translocation and protection of ARE-containing RNA (Fan and Steitz, 1998).

It is a 'I'NF ARE binding protein which stabilizes mRN A containing INF-0t mRNA (Dean

23

et al., 2001). In the study of Dean et al (2001), HuR-mediated stabilization was found to be
independent on activation of p38 kinase. TTP is a zinc finger RNA binding protein that is
responsible for destabilization of TNF-a mRNA (Lai et al. , 1999). Unphosphorylated form
of TTP has high binding activity to ARE RNA probe and facilitates mRNA degradation
while phosphorylated TTP has low binding activity and consequently increases mRNA
stability (Carballo et al., 2001). This protein is inducible by LPS in macrophages and
phosphorylated by MAPK-activated protein kinase-2 (MAPKAPK2 or MK-Z) through

activated p38 kinase (Mahtani et al., 2001).

0.4 Superinduction and involvement of MAPKs

Contribution of MAPKS to gene expression at the transcriptional and
posttranscriptional levels may be associated with the superinduction of cytokines by VT
because both cytokine gene transcription and mRN A half life are increased by VT in cell line
models (Li et al., 1997; Wong et al., 2001; Wong et al., 1998). Superinduction is defined
as the capacity of protein synthesis inhibitors such as VT to prolong stimulation of transiently
induced cytokine genes in response to stimulants such as grth factors, cytokines,
hormones, and interferons (Mahadevan and Edwards, 1991). Superinduction is observed
with an increase of specific transcripts and /or their mRNA stabilization.

The causes of superinduction due to protein synthesis inhibitors are unknown, but
there are several theories to explain it. For example, c-Fos is a transcription factor that
activates expression of other genes. A labile molecule on the c-fos gene in an inactivated

state may be depleted by protein synthesis inhibitors, causing superinduction (Morello et al. ,

24

 

1990

anisc

min

199

pm
the
syr

531

3C

El

bi

01

3L

31

1990; Subrarnaniam et al., 1989). Alternatively, protein synthesis inhibitors such as
anisomycin can act as second-messenger agonists on intracellular kinases leading to
activation of signal transduction cascades (Cano et al., 1994; Mahadevan and Edwards,
1991). Activated kinases are strongly involved in Signaling to c-fos and c-jun (Cano et al.,
1994). Further, superinduction can be modulated by controlling the repressive system that
mediates degradation of mRNA (Roger et al., 1998). ARES are widely accepted as mRN A
unstability determinants (Chen and Shyu, 1995; Gillis and Malter, 1991). Specific binding
proteins to ARE bind to various early response gene mRNAs and regulate degradation of
those mRNAs (Nakamaki et al., 1995; Sirenko et al., 1997; Zhang et al., 1993). Protein
synthesis inhibitors may contribute to repress mRN A degradation in two ways. First, protein
synthesis inhibitors may deplete the specific 3'-UTR binding proteins leading to increased
mRNA half life and superinduction. Second, activation of MAPK by protein synthesis
inhibitors may lead to phosphorylation of RNA binding protein, resulting in low binding
activity to ARE and consequently increase mRNA half life (Carballo et al., 2001; Mahtani
et al. , 2001). VT may contribute to activation of MAPK following phosphorylation of RN A
binding protein, especially, TTP and lead to increased mRN A half life and superinduction
of TNF-a and IL-6.

VT can superinduce TNF—0t and IL-6 in RAW 264.7 cells and IL-2 in EL-4 cells as
indicated by both mRNA and protein levels (Li et al., 1997; Wong et al., 1998). This
superinduction is due to both increased corresponding transcripts and increased mRNA
stability (Li et al., 1997; Wong et al., 2001). The increased transcripts for TNF-0t and IL—6

were explained by an increase of binding activity of NF-KB, AP-l and NF-IL6 transcription

25

factors to their promoter regions (Wong et al. , 2002). The increased TNF-or and IL—6 mRN A
stability was explained that LPS-induced TNF-0L and IL—6 mRNAs were stabilized by VT in
the presence of transcription inhibitor, DRB (Wong et al., 2001). The increased binding
activity of those transcription factors and increased mRN A stability of TNF-0t and IL-6 imply
activation of MAPKS (Brook et al. , 2000; De Cesaris et al. , 1998; Diaz and Lopez—Berestein,
2000; Liu et al., 2000; Miyazawa et al., 1998; Swantek et al., 1997). Recently, VT was
reported to induce p38 kinase, ERK1/2, and JNK1/2 (Moon and Pestka, 2002; Yang et al.,
2000). The involvement of MAPKS in increased mRNA expression during superinduction

of ‘l'NF-a has yet to be explained.

E. Rationale for this research

VT and SG, protein synthesis inhibitors, are often found in foods and in the
environment, respectively. They have immunemodulatory effects in vivo and in vitro.
Furthermore, VT has been suspected as an etiological factor in human IgAN based on a
mouse experimental model. From experiments of dietary exposure to VT, proinflammatory
cytokines, especially IL—6 and TNF-0t, are found to be important to the progression of IgAN.
The ability of SG or other macrocyclic trichothecenes to induce cytokine expression has been
incompletely studied. Study on cytokine production by macrocyclic trichothecenes in RAW
264.7 macrophage cells will provide information about how the toxins can modulate immune
function. Recently, from differential display experiments, two genes, MIP-2 and C3 aR, were
found to be up-regulated by VT and/or SG in RAW 264.7 cells. Expression of these gene

products might play an important role in the progression of IgAN since autoimmune

26

complexes against IL-8 (a functional analog of MIP-2 in human) is higher in mesangium in
IgAN patients than healthy controls. Kinetic studies on the expression of MIP-Z will provide
information about their possible involvement in the autoimmune disease. Finally, the
induction of proinflarnmatory cytokines by VT in RAW 264.7 cells is due both to increased
transcription and to increased mRNA half life. Study of MAPK involvement in mRNA
expression will provide information about the mechanism by which mRNA expression is
increased by VT. Overall, these studies will contribute to understanding how trichothecenes
can modulate immune firnction. In the long run, results of these studies will be used to

reduce health risks due to exposure to trichothecenes.

27

CHAPTER II

DEVELOPMENT OF ANTIBODIES TO SATRATOXINS AND APPLICATION

TO DIRECT COMPETITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY'

‘ This work has been submitted for publication in a similar form with authors Chung,
Y.J., Jarvis, B.B., Tak, H., and Pestka, J .J . in Mycopathologia.

28

ABSTRACT

Although macrocyclic trichothecenes produced by Stachybotrys chartarum have
been associated with numerous outbreaks of indoor illness, no simple method exists for the
detection of these toxins in environmental samples. The goal of this study was to develop an
enzyme-linked immunosorbent assay (ELISA) to the major S. chartarum macrocyclic
trichothecene mycotoxin, satratoxin G (SG). SG was derivatized to its hemisuccinate and
coupled to bovine serum albumin for use as an immunogen to produce antibodies in rabbits.
A direct competitive ELISA employing SG antibodies and SG-hemisuccinate coupled to
horseradish peroxidase (HRP-SG) was used to detect between 0.1 to 100 ng/ml of SG. The
concentrations of SG, satratoxin H, isosatratoxin F, roridin A, and verrucarin A causing 50%
inhibition of HRP-SG conjugate binding were 1.3, 5.4, 3.2, 25.1, and 52.5 ng/ml,
respectively, indicating that the antibodies were most Specific for SG and closely related
satratoxins. Sensitivity was largely unaffected by methanol content up to 20% in assay
diluents. The ELISA was applicable to detection of satratoxins in S. chartarum spore extracts
and cultures. SG antibodies should be useful for identifying toxigenic S. chartarum isolates
as well as the rapid detection of satratoxins in environmental samples from buildings

contaminated with this fungus.

INTRODUCTION
Macrocyclic trichothecenes are a group of mycotoxins produced by Stachybotrys
chartarum (S. atra ) and other fungi (Bata et al., 1985; Harrach et al., 1981; Jarvis et al.,

1988; Jarvis and Wang, 1999; Namikoshi et al., 2001) that have potential human health

29

implications. These trichothecenes are 10 to 100 times more toxic than non-macrocyclic
trichothecenes in mouse LD50 studies, HeLa cell cytotoxicity tests, and rabbit reticulocyte
protein synthesis inhibition assays (Ueno, 1983). Stachybotrys grows well on cellulose
substrates that have a high moisture content such as wet hay and straw and this fungus’s
metabolites contribute etiologically to stachybotryotoxicosis in horse and sheep in Central
Europe (Bata eta1., 1985; Habennehl et al. , 1985; Harrach et al. , 1983; Harrach et al. , 1981 ;
Hintikka, 1978). Outbreaks of Stachybotrys-associated human disease have been
epidemiologically associated with water-damaged buildings (Dearbom, 1997; Etzel et al.,
1998; Johanning et al., 1996). Pulmonary hemorrhage and hemosiderosis in infants have
been associated with toxigenic S. chartarum contaminated homes (Etzel et al., 1998) and
notably, S. chartarum strains isolated from these homes produce several macrocyclic
trichothecenes including satratoxin G (SG), satratoxin H (SH), isosatratoxin F (iSF), roridin
L-2, and trichoverrol B (Jarvis et al., 1998). Recently, spores of this fimgus were isolated
from the lungs of a child with pulmonary hemorrhage (Elidemir et al. , 1999). Although this
was the first report of the organism in human tissue, the specific involvement of macrocyclic
trichothecenes in the disease was not determined.

Given the potential harmful effects of macrocyclic trichothecenes on human health,
it is important to monitor their presence in the environment. Current methods for detection
and quantitation of the toxins include thin-layer chromatography, high performance liquid
chromatography, and mass spectrometry (Krishnamurthy et al., 1989; Stack and Eppley,
1980; Tuomi et al., 1998). These methods used for macrocyclic trichothecenes, however,

necessitate time-consuming extraction, sample clean—up, and/or expensive instrumentation.

30

A protein translation assay employing luciferase mRNA has been developed to detect
trichothecenes in the spores of toxigenic fungi (Yike et al., 1999). This assay is quite
sensitive as all trichothecenes in spores inhibit protein synthesis, but this method is not
specific for any single trichothecene, and may give false positives in response to other
metabolites in a sample. A further limitation of this assay is its expense and requirement for
special training and equipment.

Enzyme-linked immunosorbent assay (ELISA) has been widely applied for the
detection of mycotoxins because of its high specificity and sensitivity (Abouzied et al. , 1993;
Azcona-Olivera et al. , 1992; Thirumala-Devi et al. , 2000). ELISAS have been developed for
a variety of food-borne non-macrocyclic trichothecenes (Abouzied et al. , 1993; Barna-Vetro
et al., 1994; Park and Chu, 1996), however, relatively little effort to date has been placed
on the environmentally important macrocyclic trichothecenes. In one exception, an ELISA
for the macrocyclic trichothecene, roridin A, was developed using rabbit polyclonal
antibodies (Martlbauer et al., 1988). The antibodies were highly specific for roridin A but
showed low cross-reactivity against SH (15%) and SG (6.5%), which are two of the major
toxins in Stachybotrys spores. Therefore, it was desirable and seemed feasible to develop
a specific ELISA for the satratoxins. The purpose of this study was (i) to device a chemical
strategy for linking SG to the protein carrier bovine serum albumin (BSA), (Q) to produce
specific antibodies against the BSA-SG in rabbits, and (m) to apply these antibodies to the
development of a competitive direct enzyme-linked immunosorbent assay (CD-ELISA) for
SG as well as other satratoxins. The results suggested that high titer SG antisera could be

produced and applied to a CD-ELISA for the rapid detection of SG and other satratoxins.

31

MATERIALS AND METHODS

Materials and Chemicals. Bovine serum albumin (BSA, fatty acid free grade), 1,3-
dicyclohexylcarbodiimide (DCCD), N-hydroxysuccinimide, N,N-dimethylformamide
(DMF), complete and incomplete Freund's adjuvants, Tween-20, deoxynivalenol (DON),
roridin A (RA), verrucarin A (VA), and verrucarol were purchased from Sigma Chemical Co.
(St. Louis, MO). Horseradish peroxidase (HRP, ImmunoPure® grade) was obtained from
Pierce (Rockford, IL). SG, satratoxin H (SH), and isosatratoxin F (iSF) (Figure 2.1) were
prepared from cultures as described by Jarvis et al. (Jarvis et al., 1995).

Synthesis of the bis-hemisuccinate of satratoxin G. A solution of 4.0 mg (7.3
mmol) of SG, 3.7 mg (36.5 mmol) of succinic anhydride, and 1 mg of N,N-
dimethylaminopyridine (DMAP) in 5 mL of chloroform (that had first been passed through
a short column of alumina) was refluxed for 1.5 hr. The reaction mixture was cooled, diluted
with 5 mL of dichloromethane, and the solution washed successively with 10 mL of 5%(v/v)
HCl and 5%(w/v) NaHCO3 to yield, after removal of organic solvent, 5 mg of an amorphous
solid that resulted in a single spot on thin layer chromatography (TLC, 1 :9 dichloromethane-
ethyl acetate) analysis. The product was analyzed by NMR at the University of Maryland.
Department of Chemistry and Biochemistry.

Preparation of satratoxin G conjugates. SG hemisuccinate was coupled to BSA
and HRP. Briefly, 1 mg of SG hemisuccinate was dissolved in 50 ul of DMF and was
activated with 1.14 mg of N-hydroxysuccinimide and 4.08 mg of DCCD in 100 pl of dry
DMF for 18 h at room temperature. The reaction mixture (150 pl) was added dr0pwise to

5 mg of BSA or 5 mg of HRP dissolved in 1.0 ml of NaHCO3 (130 mM) and slowly stirred

32

   

 

Satratoxin G Isosatratoxin F

HO

 

H
Verrucarol Deoxynivalenol

Roridln A Verrucarin A

Figure. 2.1. Structures of macrocyclic and selected other trichothecenes

33

for

bu

CO

of

\l

for 2 h at room temperature. The conjugates were dialyzed against 4 liters of phosphate
buffered (pH 7.2, 10 mM) saline (PBS) at 4°C with four changes for 3 days. BSA-SG
conjugate was stored frozen at - 20°C. HRP-SG conjugate was diluted in an equal volume
of glycerol and stored at - 20°C.

Rabbit immunization. Three rabbits (Harlan, 6 weeks, female) were injected
intraderrnally with BSA-SG conjugate (375 pg) in 1 m1 of PBS-F reund's complete adj uvant
(1:1) at 20 to 30 Sites on a shaved back area. A booster consisting of BSA-SG conjugate
(250 pg) in 1 ml of PBS—Freund's incomplete adjuvant (l :1) was injected subcutaneously at
week 18. Blood samples were drawn via marginal ear vein at intervals. Sera were obtained
after overnight incubation of blood at 4°C and centrifiigation at 3,000 x g for 30 min. For
long term storage, immunoglobulins were purified by 50% saturation with ammonium
sulfate, lyophilized and stored at - 20°C.

Antibody titration by direct ELISA. Wells of polystyrene microtiter plates
(Immulon 4 Removawells, Dynex Technologies Inc. Chantilly, VA) were coated with 100
pl of serially diluted serum in PBS (pH 7.2, 10 mM) overnight in a forced air drying oven
at 42°C. Plates were washed three times with PBS-Tween 20 (0.02%, v/v). Wells were
blocked with 300 pl of 3% (w/v) non-fat dried milk in PBS (NFDM-PBS), covered with
parafilrn, and incubated for 30-60 min at 37°C. Plates were washed four times with PBS-
Tween. Then, 100 pl of HRP-SG conjugate (1 :4,000 in NFDM-PBS) was added to each well
and incubated at 37°C for 60 min. After seven washes with PBS-Tween, bound peroxidase
was determined with 3,3',5,5'-tetramethylbenzidine (TMB) _(Fluka Chemika, Buchs,

Switzerland) substrate as described previously (Wong et al., 1998). The reaction was

34

terminated after 10 min incubation at room temperature by adding 100 pl of 6 N sulfuric acid
(HZSO4). The absorbance at 450 nm was measured using Vmax Kinetic Microplate Reader
(Molecular Devices, Menlo Park, CA).

Competitive direct (CD) ELISA. Microtiter plates were coated with antiserum
diluted 15,000-fold in PBS at 4°C overnight and blocked as described above. Then, 50 pl
of standard SG or sample in PBS was simultaneously incubated with 50 pl of HRP-SG
(1 :2000 in NFDM-PBS) for 1 h at 37°C. Bound peroxidase was determined by adding TMB
substrate as described above. The reaction was stopped after 30 min incubation at room
temperature and absorbance read.

Cross-reactivity was measured by CD-ELISA as described above. Briefly, microtiter
plates were coated with antiserum diluted 15,000-fold in PBS at 4°C ovemight. After
blocking with NFDM-PBS, 50 pl of SH, iSF, RA, VA, DON, or verrucarol serially diluted
in PBS, was simultaneously incubated with HRP-SG conjugate (1 :2000 in NFDM-PBS) and
analyzed as described above.

Methanol sensitivity was measured by CD-ELISA as described above with some
modification. Fifty microliters of standard SG dissolved in PBS containing 0 to 80%
methanol was simultaneously incubated with 50 pl of HRP-SG conjugate in antibody coated
wells for 1 h at 37°C and analyzed as described above.

Analysis of spore extracts to detect satratoxins in Stachybotrys spore, spore extracts
from a toxigenic and non-toxigenic isolate, prepared as previously described (Yike et al.,
1999) were kindly provided by DrS. I Yike and D. Dearbom (Case Western Reserve

University, Cleveland, OH) were dissolved in PBS. Briefly, 50 pl of serially diluted spore

35

extracts in PBS were simultaneously incubated with 50 p1 of HRP-SG conjugate in antibody
coated wells for 1 h at 37°C and analyzed as described above.
Comparative analysis of S. chartarum cultures by HPLC and ELISA.

S. chartarum cultures for this study were kindly provided by Ms. Janet Simpson
(NIOSH) DRDS, Morgantown, WV). These cultures were previously collected in Cleveland,
OH (Jarvis et al., 1998). The general procedure for growth, extraction and HPLC analysis
of rice cultures of S. chartarum was altered slightly from a recently published method
(Hinkley et al., 2000). Briefly, after 3 wk of growth, 25 ml of 85% (w/v) methanol in water
was added to 5g of rice culture and the mixture sonicated for 30 min. The extraction was
repeated and the extracts combined and filtered. This procedure tends to preferentially extract
the trichothecenes from the culture (Hinkley et al., 2000) and reduce problems with
interfering analytes during HPLC analyses. Efficiency of recovery by this extraction protocol
(approximately 50%) was assessed using standards and used to correct final estimates in
HPLC analyses. For ELISA, 1.5 ml of 80% (w/v) methanol in water was added to 0.5 g of
S. chartarum rice culture and sonicated for 30 min. Samples were filterd through a 0.45 pm
filter; the extraction repeated and the filtrates combined. Extracts were diluted to attain 20%
(w/v) methanol in water. Extracts were further diluted serially in 20% (w/v) methanol and

subjected to ELISA in which standards were in the same diluent.

RESULTS AND DISCUSSION
Derivatization of SC to its bis-hemisuccinate. Succinic anyhydride was used to

convert SG its bis-hemisuccinate. NMR was used to verify the identity of the product as

36

4.21
H-l
10.f
dd,

774

ton
dirt
BS
sub
ser

$31?

the

inj

31

satratoxin G hemisuccinate {1H NMR (400 MHz) (CDCl3) d: 0.86 (l H, s, H-l4), 1.00 (3
H, d, J = 7 Hz, H-14'), 1.71 (3 H, s, H-16), 2.0 (4 H, m, H-7 and H-8), 2.0 (1 H, m, H—3b),
2.5 (1 H, m, H-3a), 2.5 (2 H, m, H-4'), 2.97 (2 H, center ofAB system, J = 4 Hz, H-13), 3.58
(1H, d, J = 5 Hz, H-1 1), 3.48 (1 H, s, H-2'), 3.84 (1 H, d, J = 5 Hz, H-2), 3.9 (2 H, m, H-5'),
4.21 (2 H, center ofAB system, J =12 Hz, H-15), 4.83 (l H, s, H-12'), 5.32 (1 H, q, I = 7 Hz,
H-13'), 5.42 (1 H, br d, J = 5 Hz, H-10), 5.84 (1 H, d, J= 16.5 Hz, H-7'), 5.96 (1 H, d, J =
10.5 Hz, H-lO'), 6.0 (1 H, m, H-4), 6.68 (1 H, dd, J = 8.0 and 10.5 Hz, H-9'), and 6.97 (l H,
dd, J = 8.0 and 16.5 Hz, H-8'), CH2CH2'S of succinate: 2-7 (8 H, m); HREI-MS m/z
774.2640 M+ (C37H36016 req. 7742629)}.

Production of antibodies against satratoxin G. SG bis-hemisuccinate was
conjugated to BSA for use as an immunogen and to HRP as an ELISA marker ligand. A
direct ELISA was applied to monitor titers of SG antiserum whereby sera from three SG-
BSA immunized rabbits was coated onto a microtiter plate and bound antibodies
subsequently detected with HRP-SG conjugate. The titer was arbitrarily designated as the
serum dilution that showed visually distinct color from pre-immune serum control at the
same dilution. Titration curves for the antisera at week 6 are shown in Figure 2.2. Titers of
the antiserum was 31250 whereas pre-immune serum control had negligible absorbance.

The titers from the three rabbits over 42 weeks are measured: the highest titer was
31250 at week 6 through week 10 and the titers dropped to 6250 at week 17. A booster
injection to two rabbits at week 18 increased the titer of SG antiserum to 31250 from 27 to
31 weeks and the titers decreased slightly to 6250 for the remaining weeks. One rabbit (#2)

was not given a booster injection due to granulomas on its back. The granuloma was self-

37

1 (#148) 2 (#1 51) 3 (#155)

 

 

 

 

 

 

 

 

 

 

 

 

3.0 . 3.0 3.0
+ pre-immune + pro-immune + pie-immune
E 2.5 ~ + week 5 2.5 « —I— week6 2,5 -. —l— week6
C
8 2.0 .4 2.0 r 2.0 r
v
:6 1.5 ~ 1.5 — 1.5 —
§
§ 1.0 a 1.0 — 1.0 ~
a: 0.5 -‘ 0.5 — 0.5 -‘
2
0.0 - 0.0 - 0.0 '-
‘05 01010101010101; '05 iiiiiffii -0.5 iiiiriii
\bbggggo ooooooow ooooooox~
(It (1' (L (L (I! Q N o) (O ‘0 (O (O ‘3 0 N (0 <0 (0 (O ('3 9) Q
\ e ,9 be a "L (L 61,639. 6° 'L .0. ””5065" go
Dilution Dilution Dilution

Figure 2.2. Direct ELISA titer determination of rabbit SG antibodies. Serially diluted
antisera were coated onto microtiter wells and specific binding determined by incubation
with HRG-SG. Three rabbits (#148, #151, and #155) were immunized with SG-BSA.
Collected sera were subjected to direct ELISA for titer determination.

38

resolving but titers remained at 6250.

Competitive direct ELISA. A CD-ELISA was performed by simultaneously
incubating standard SG and HRP-SG conjugate over antibodies coated on microtiter plates.
Binding of HRP-SG conjugate could be inhibited by free SG and the resultant ELISA was
highly sensitive for SG (Figure 2.3). The detection limit for the curve employing rabbit 2
serum was 0.1 ng/ml whereas it was 0.5 ng/ml for the other two rabbits. Ammonium sulfate
precipitated immunoglobulin from rabbit 2 was therefore chosen to further characterize the
properties of the antibodies.

Specificity of satratoxin G antibody. The specificity of the rabbit SG antiserum
was tested by using other macrocyclic trichothecenes, verrucarol, and DON, a non-
macrocyclic type B trichothecene, as competitors in the CD-ELISA (Figure 2.4). Different
macrocyclic trichothecenes exhibited cross-reactivity while verrucarol and DON did not
cross react with the antiserum. Concentrations at 50% inhibition (H350) of HRP-SG conjugate
binding to antibodies on microtiter plates were 1.3, 5.4, 3.2, 25.1, and 52.5 ng/ml for SG, SH,
iSF, RA, and VA, respectively. More than 1 pg/ml of verrucarol were required to inhibit
50% of HRP-SG conjugate binding. DON at 1 pg/ml did not inhibit binding of the SG-HRP.

The side chains in macrocyclic trichothecenes might play an important role in
determining specificity of antibodies produced. Macrocyclic trichothecenes are characterized
by a cyclic diester or triester ring connecting O4 to C-15 of trichothecene structure (Jarvis
et al., 1995). The bis—hemisuccinate of SG facilitated conjugation of SG through the
hydroxyls associated with the six membered ring component (cyclopentanone) in the cyclic

structure (Figure 2.1). Thus, the resultant immunodominant epitopes for SG would be those

39

1 (#148) 2 (#151) 3 (#1 55)

 

 

 

 

 

 

 

 

 

 

 

 

2.0 - 2.0 ~ 2.0 .4
i
E 1.5 "‘ 1.5 ‘1 15 -1
C
o I
10
V 1.0 — 1.0 a 1.0 — I
a I
O I
O 0.5 ~ 0.5 — 0.5 ~
0.0 4 ° 00 4 0.0 a
I LLAULILI Llllull IUIMI llliw 1” I 111411ng 11111111] ruruuI Arum:I rrunuI’iu m1 Anni:I Arum:I Ann-III Allllfll Allllflfi
0 0.1 1 10 100 1000 o 0.1 1 10 100 1000 o 0.1 1 10 100 1000
Satratoxin G (ng/ml) satratoxin G (ng/ml) Satratoxin G (ng/ml)

Figure 2.3. Standard curve of SG CD ELISA. Microtiter were coated with SG antibodies
from rabbits (#148, #151, and #155) and then simultaneously incubated with Standard SG
and I-IRP-SG. Each point represents the mean i SD for three replicates.

40

 

 

    

 

 

 

120
110 —
100 —
90 -
80 -
.5 70 ~
E 60 -
E
32 50 — O Satratoxin G
40 - O Satratoxin H
A lsosatratoxin F
30 r ‘ Roridin A
V Verrucarin A .
20 " V Verrucarol . v
10 T O Deoxynivalenol ‘\\ 3
0 111 1 1111111 1 1111111 1 1111111 1 1111111 1 1111111
0 0'1 1 10 100 1000

Concentration (ng/ml)

Figure 2.4. Cross-reactivity of SG Rabbit 2 antiserum toward macrocyclic trichothecenes,
verrucarol, and deoxynivalenol. Wells were coated with SG antibody and incubated with
I-IRP-SG in the presence of different toxins.

41

moieties projecting distally from these conjugation sites. The presence of the cyclopentanone
in satratoxin H and isosatratoxin F may account for the relatively high cross-reactivity,
approximately 24 and 41 %, respectively (based on the 113505), relative to SG. Roridin A
and verrucarin A, which do not possess the six member ring structure, exhibited much less
cross-reactivity, 5.2 and 2.5 %, respectively, compared to SG. The inability of verrucarol,
a trichothecene that can be generated by base hydrolysis of macrocyclic trichothecenes
(Figure 2.1), or deoxynivalenol, a non macrocyclic trichothecene, to compete in the ELISA
indicated that there is an absolute requirement for a macrocyclic ring structure for antibody
binding.

The observed cross reactivity raises the issue that absolute quantification of SG from
samples might not be achievable. Nevertheless, the capacity of these antibodies to bind to
other satratoxins or other macrocyclic trichothecenes could be beneficial in screening
environmental samples for satratoxins and other macrocyclic trichothecenes as well as in
toxigenicity determinations for Stachybotrys or Myrothecz‘um isolates which potentially
produce different macrocyclic trichothecenes (Bata et al. , 1985; Harrach et al. , 1981; Jarvis
et al., 1988; Jarvis and Wang, 1999; Namikoshi et al., 2001). In such determinations,
macrocyclic trichothecenes can be estimated semiquantitatively as “SG equivalents” when
SG is used as the standard. When desired, the antibodies can be used to identify specific
macrocyclic trichothecenes profiles using a chromatographic step such as immunoaffinity
chromatography in conjunction with HPLC or using the HPTLC ELISAGRAM described
previously by our laboratory (Pestka, 1991).

Effect of methanol in CD-ELISA. Methanol extraction of toxic components from

42

S. chartarum has been applied for the detection of macrocyclic trichothecenes (Sorenson et
al., 1987; Stack and Eppley, 1980) and for the removal of toxic components to study their
effects on pulmonary inflammation (Rao et al., 2000). Methanol might also be used in
procedures for extracting environmental particulate or swab samples for satratoxins.
Therefore, the effects of using different methanol contents in the assay diluent on the CD-
ELISA was assessed. The presence of 20% methanol in the final CD-ELISA slightly affect
the sensitivity of the method (Figure 2.5); however, higher methanol concentrations either
depressed assay sensitivity (30%) or completely suppressed the ELISA. The stability of these
antibodies in 20% methanol should facilitate the detection in extracts of fungal isolates and
in environmental samples

Application of CD ELISA to detect satratoxins from Stachybottys spore extracts.
Two S. chartarum spore extracts from isolates obtained in a case control study (Yike et al. ,
1999) were tested for satratoxin content. Previously, these extracts were estimated by
protein translation inhibition assay to contain 670 fg SG equivalents/ spore (Strain 58-17) and
80 fg SG equivalents/spore (strain 58-06) (I.Yike, personal comunication). Using ELISA,
the 58—17 extract was found to contain 980 fg SG equivalent per spore. The other extract,
58-06, had 0.05 fg SG equivalent per spore. The reason for the differences between the two
assays may relate to the presence of other trichothecenes in the extract that interfere in the
translational assay of SG or enhance it. Nevertheless, these qualitative results suggest that
the ELISA might be readily used to discriminate toxigenic from weakly toxigenic or non-

toxigenic strains of S. chartarum.

43

 

1.0

OD at 450 nm

 

 

 

 

O 0.1 1 10 100 1000
Satratoxin G (ng/ml)

Figure 2.5. Effect of methanol on binding of HRP-SG to SG antiserum. SG antiserum was
coated onto microwells and incubated with HRP-SG in the presence of methanol at

concentrations ranging from 0 to 40%.

44

Comparison of HPLC to CD ELISA in analysis of satratoxins in S. chartarum
rice cultures. Three toxigenic and three atoxigenic S. chartarum cultures isolated from an
epidemiologic study (Jarvis et al., 1998) were grown of rice for 3 wk and subjected to
analysis by HPLC and CD ELISA (Table 2.1). The CD-ELISA correlated qualitatively with
HPLC recovery and identified the three highly toxigenic cultures from the atoxigenic
cultures. ELISA estimates compared to HPLC estimates were approximately identical for
one sample (22-11), one half for a second sample (22-03) and two times higher for a third
sample (22-08). The reason for these differences might be attributed to subsample variability
arising from non-uniformity of culture growth and toxin production. Differences might also
be associated with the fact the ELISA measured satratoxin equivalents which are affected by
differential antibody cross reactivity for various macrocyclics cover, whereas HPLC
measured total macrocylic trichothecenes. The reason for the Slight positive ELISA
responses (1-9 pg/ g) in the three cultures that do not produce macrocyclics as detectable by
HPLC may relate to the cultures capacity to produce the non-macrocyclic verrucarol which
reacts weakly in the ELISA. Taken together, these data suggest that culture or environmental
samples can be analyzed for satratoxins by ELISA after only a methanol-water extraction
without any of the laborious clean-up steps or instrumental requirements associated with

HPLC.

CONCLUSIONS
In summary, high antibody titers to SG were readily achieved upon immunization of

rabbits with SG bis-hemisuccinate conjugated to BSA. The antibodies produced could detect

45

Table 2.1. Comparison of HPLC and CD-ELISA analyses of S. chartanum rice

cultures'.

 

 

 

 

 

 

 

 

 

 

 

HPLCb
Total macrocyclic CD-ELISAc
trichothecenes Satratoxin equivalents
Samvle (rig/g) (118/8)
22-03 680 303 3:140
22-04 0 0.8 :1: 0.3
22-07 0 9.0 :t 0.2
22-08 180 374 i 91
22-10 0 1.20i0.1
22-11 174 177i26

 

a. Cultures were isolated from homes in Cleveland during an epidemiologic study of S.
chartarum-associated pulmonary hemorrhage.

b. Standard error for this method was typically 10 to 20% (12).

c. Mean of two separate 0.5 g subsamples.

46

free SG using a CD-ELISA with a range of detection from 0.1 to 100 ng/ml. The observed
antibody cross-reactivity may facilitate Simultaneous detection of other satratoxins and to a
lesser extent, other macrocyclic trichothecenes. Methanol content up to 20% in samples did
not affect the immunoassay and this stability expands applicability of the antibodies. As has
been implemented on a widescale for foodbome mycotoxins (Abouzied et al. , 1993; Barna-
Vetro et al., 1994; Park and Chu, 1996), potential applications of these CD-ELISA would
be on-site rapid detection of satratoxins in culture, wallboard, floor and air samples from
Stachybotrys-contaminated buildings. Additional analytical studies on Stachybotiys isolates
and environmental samples in which assessment of satratoxins by ELISA are compared with
existing chemical methods such as HPLC would be helpful in this regard. Finally, since
Stachybotrys spores can be inhaled in lungs, these antibodies might also be applied to the

localization of toxigenic spores and released toxin in lung tissue.

ACKNOWLEDGMENT
This work was supported by EPA Contract 00-5674 NAEX and NIH grant ES-0335 8.
We thank Drs. I. Yike and D. Dearbom (Case Western Reserve University, Cleveland, OH)

for providing spore extracts and related analytical data.

47

CHAPTER III
MODULATION OF LIPOPOLYSACCHARIDE-INDUCED
PROINFLAMMATORY CYTOKINE PRODUCTION BY SATRATOXINS AND
OTHER MACROCYCLIC TRICHOTHECENES IN THE MURINE

MACROPHAGE '

' This work has been accepted for publication in a similar form with authors Chung, Y.J .,
Jarvis, 3.3., and Pestka, J .J . in Journal of Toxicology and Environmental Health.

48

ABSTRACT

The satratoxins and other macrocyclic trichothecene mycotoxins, are produced by
Stachybotrys, a mold that is often found in water-damaged dwellings and office buildings.
To test the potential immunomodulatory effects of these mycotoxins, RAW 264.7 murine
macrophage cells were treated with various concentrations of satratoxin G (SG),
isosatratoxin F (iSF), satratoxin H (SH), roridin A (RA), and verrucarin A (VA) for 48 hr in
the presence or absence of suboptimal concentration of lipopolysaccharide (LPS, 50 ng/ml).
Tumor necrosis factor-a (TNF-0t) and interleukin-6 (IL-6) production were assayed by
ELISA. In LPS-stimulated cultures, TNF-ct supernatant concentrations were significantly
increased in the presence of 2.5, 2.5, and 1 ng/ml of SG, SH, and RA, respectively, whereas
IL-6 concentrations were not affected by these macrocyclic trichothecenes. When cells
treated with LPS and SG were evaluated by real time PCR, SG at 2.5 ng/ml increased TNF-0t
mRNA at 24, 36, and 48 hr compared to control cells. At higher concentrations, cytokine
production and cell viability were markedly impaired in LPS-stimulated cells. Without LPS
stimulation, neither TNF-0'. nor IL-6 was induced. These results indicate that low
concentrations of macrocyclic trichothecenes superinduce expression of TNF-ct whereas
higher concentrations of these toxins are cytotoxic and concurrently reduce cytokine
production. The capacity of satratoxins and other macrocyclic trichothecenes to alter
cytokine production may play an etiologic role in outbreaks of Stachybotrys-associated

human illnesses.

49

INTRODUCTION

The trichothecenes are a family of sesquiterpenoid mycotoxins which include some
of the most potent protein synthesis inhibitors known (U eno, 1983). More than 180
trichothecenes have been identified and are categorized into four types based on functional
groups attached to the trichothecene ring (Grove 1993;1996). Mechanisms of toxicity of
foodbome Type A (e.g., T-2 toxin) and Type B (e. g., vomitoxin or deoxynivalenol)
trichothecenes have been studied extensively (Bondy and Pestka, 2000). On the other hand,
toxicological studies on environmentally encountered Type D (e. g., satratoxins, verrucarin,
and roridin) macrocyclic trichothecenes are limited.

Macrocyclic trichothecenes are characterized by a cyclic diester or triester ring which
connects C-4 to C-15 of the trichothecene structure (Figure 3.1). These trichothecenes are
10 to 100 times more toxic than Type A and Type B trichothecenes in mouse LD50 studies,
HeLa cell cytotoxicity tests, and rabbit reticulocyte protein synthesis inhibition assays (U eno,
1983). Several potentially important macrocyclic trichothecenes are produced by
Stachybotrys atra (or also known as S. chartarum ). This mold grows well on cellulose
substrates with a high moisture content such as wet hay and straw (Bata et al. , 1985 ; Harrach
et al. , 1981). Macrocyclic trichothecenes have been etiologically associated with
stachybotryotoxicosis in horse and sheep in Central Europe (Bata et al. 1985; Harrach et al. ,
1981; 1983; Hintikka 1978). Clinical signs of this disease include leukopenia,
thrombocytopenia, hemorrhage, arrhythmic heartbeat, and death (Forgacs, 1972).

Humans can also be exposed to Stachybotrys and macrocyclic trichothecenes via the

environment. Outbreaks of Stachybotrys-associated human disease have been

50

 

   

Satratoxin H Satratoxin G lsosatratoxin F
O O
O H
Q 34' HO H
HOI n H HO? Verrucarol Deoxynivalenol
H
H
Roridin A Verrucarin A
Figure 3.1. Structures of macrocyclic trichothecenes

51

epidemiologically associated with water-damaged buildings (Dearbom, 1997; Etzel et
al.,1998; Johanning et al., 1996). Symptoms reported by exposed individuals include
recurrent cough, irritation of the eyes and skin, mucous membrane disorders, headache, and
fatigue, which are also characteristics of indoor air illnesses. Notably, pulmonary
hemorrhage and hemosiderosis in infants have been associated with toxigenic S. atra
contaminated homes (Etzel et al. , 1998). Recently, spores of this fungus were isolated from
the lungs of a child with pulmonary hemorrhage (Elidemir et al. , 1999). Although this was
the first report of the fungal organism in human tissue, the specific involvement of
macrocyclic trichothecenes was not determined.

Leukocytes are particularly sensitive to trichothecenes (Bondy and Pestka 2000).
Depending on dose and duration of exposure, these toxins can be immunostimulatory as well
as immunosuppressive. Trichothecene-induced immunostimulatory effects include increased
cytokine production and upregulation of immunoglobulin (lg) production.
Immunosuppressive effects of trichothecenes include apoptosis and impairment of humoral
immunity. To date, most work on the immunostimulatory effects of trichothecenes has
focused on the Type A and Type B subgroups. However, macrocyclic trichothecenes have
been shown to superinduce IL-2 production in EL-4 thymoma cells as well as induce and
impair blastogenesis of lymphocytes in the presence of mito gen (Hughes et al. , 1990; Pestka
and F orsell, 1988). Given the potential importance of macrocyclic trichothecenes to human
health, it is important to better understand their effects on immune fimction.

Tumor necrosis factor-a (TNF-0t) and interleukin-6 (Ho-6) are proinflammatory

cytokines which are secreted by activated macrophages. Although these cytokines are

52

important in host defense, overproduction of the cytokines may be important in the
pathogenesis of several inflammatory diseases such as septicemia and fatal circulatory shock
(Chantry and Feldman, 1990; Vogel and Hogan, 1990). These cytokines are also highly
expressed by inflammatory cells in the airways (Noah et al. , 1995) and can potentiate allergic
inflammation (Gosset et al. , 1993). The purpose of this investigation was to test a hypothesis
that macrocyclic trichothecenes can alter TNF-0t and IL-6 production and viability in a

murine macrophage model.

MATERIALS AND METHODS

Chemicals. All cell culture reagents and chemicals were purchased from Sigma
Chemical Co. (St. Louis, MO) unless otherwise noted. Satratoxin G (SG), satratoxin H (SH),
and isosatratoxin F (iSF) were prepared from cultures as described by Jarvis et al. (1995).
All toxins were dissolved in ethanol, dried at a stock concentration of 10 pg, and stored at
4°C for long term storage. Stock solutions were dissolved in ethanol and further diluted to
200 ng/ml in Dulbecco’s Modified Eagles Medium (DMEM, Sigma) with a final ethanol
concentration of < 0.1% of the diluent. Lipopolysaccharide (LPS) from Salmonella
typhimurium was dissolved in DMEM at 250 ng/pl and stored at -20°C.

Cell Culture and ELISA. The murine macrophage RAW 264.7 cell line was
obtained from American Type Culture Collection (Rockville, MD). Cells were maintained
at 37°C in a 7% CO2 humidified incubator in DMEM supplemented with 10% (v/v) fetal calf
serum (F CS; Gibco BRL, Gaithersburg, MD), 1% (v/v) National Cancer Institute Medium

NCTC 135 supplement, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 pg/ml

53

streptomycin. For toxin treatment, RAW 264.7 cells (2.5 X105 cells/ml) were cultured for
24 hr in flat-bottomed 48-well tissue culture plates (Fisher Scientific Co., Corning, NV) with
each well containing 500 pl of cell suspension. Then, supematants were removed and fresh
medium containing various concentrations (0 to 10 ng/ml) of macrocyclic trichothecenes and
LPS (50 ng/ml) was added to culture. Supematants were collected at various time points and
assayed for TNF-a and IL-6 by enzyme-linked immunosorbent assay (ELISA) (Wong et al. ,
1998)

MTT Assay. The MTT assay was performed as described by Marin et al. (1996).
Briefly, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5,-diphenyltetrazolium bromide) (Sigma) was
dissolved in 0.01 M phosphate-buffer saline (PBS) at pH 7.4 with a concentration of 5
mg/ml. Filter sterilized MTT reagent was stored in the dark at 4°C no longer than 1 wk.
RAW 264.7 cells (2.5 x105 cells/ml) were cultured for 24 hr in flat-bottomed 96-well tissue
culture plates (Fisher Scientific) with each well containing 200 pl of cell suspension. Then,
supematants were removed and fresh medium containing macrocyclic trichothecenes and
LPS was added to the culture. After 48 hr incubation, MTT reagent (20 pl/well) was added
to the 96 well plate and incubated for an additional 3 hr in a C02 incubator at 37°C. Then,
plates were centrifuged at 450 x g for 15 min and supematants were carefully removed from
wells by slow aspiration through a 28-gauge needle. Dimethyl sulfoxide (DMSO) (150
pl/well) was added to dissolve purple formazan crystals. Alter complete solubilization of
the crystals by Shaking, absorbance was measured on a microplate reader (Molecular
Devices, Menlo Park, CA) using 570 nm as the test wavelength and 690 nm as the reference

wave length.

54

Real Time PCR. Total RNA was extracted from RAW 264.7 cells by the method
of Chomczynski and Sacchi (1987) using TrizolTM reagent (Gibco BRL, Gaithersburg, MD)
according to the manufacturer’s instructions. Total RNA content was determined
spectrophotometrically at 260 nm.

Total RNA was analyzed to detect TNF-0t mRNA level using an ABI PrismTM 7700
Sequence Detection System (PE Applied Biosystems, Foster City, CA). The levels of 18S
rRNA were also measured as an indigenous control. Specific primers and probes for TNF-0t
and 188 were obtained from PE Appllied Biosystems. Concentrations used for primers and
probes were 200 and 100 nM, respectively, in each reaction. Sixteen pl of master mix
comprising 20 x TNF-0t primers and probe, 20 x 188 primers and probe, predeveloped assay
reagents for PCR and RT (PE Applied Biosystems) was added to 9 pl of samples containing
100 ng total RNA. Reverse transcriptase and polymerase reactions were performed in the
same tubes according to manufacturer’s recommendations. Relative quantitatiies of TNF-0t
mRNA and 18S rRNA were calculated using the comparative threshold cycle number for
each sample fitted to standard curves for TNF-0t mRNA and 188 rRNA, respectively,
generated according to guideline (ABI Prism 7700 Use Bulletin #2, PE Applied Biosystems).
Expression levels for TNF-0t mRNA in each sample were normalized to 18S rRNA.

Statistical Analysis. One-way analysis of variance (AN OVA) using Dunnett’s test
for parametric data or Kruskal-Wallis one-way ANOVA for non parametric data were
performed with Sigma Statistical Analysis System (J andel Scientific, San Rafael, CA). A

p value of less than 0.05 was considered statistically significant.

55

RESULTS

Effects of macrocyclic trichothecenes on TNF-0t and IL-6 production. RAW
264.7 cells were exposed to macrocyclic trichothecenes in the presence of a sub-optimal
concentration of LPS (50 ng/ml). After 48 hr, TNF-0t levels in supernatant were significantly
increased by SG, SH, and roridin A (RA) at 2.5, 2.5, and 1 ng/ml, respectively, while iSF and
verrucarin A (VA) did not significantly induce TNF-0L production at similar concentrations
(Figure 3 2). At 12 and 24 hr with the same concentrations or lower of each macrocyclic
trichothecene, TNF-0t production in toxin-treated cells was not different from LPS-treated
cells (data not Shown) although TNF-0t level gradually increased over time. At 10 ng/ml, all
macrocyclic trichothecenes were cytotoxic, resulting in significant decrease of TNF-0t
production in LPS-treated cells. At 10 ng/ml, all macrocyclic trichothecenes significantly
suppressed TNF-0t production in LPS-treated cells. In the absence of LPS stimulation, TNF-
a production was not induced in macrophages by macrocyclic trichothecenes at 12, 24, and
48 hr (data not shown).

The effect of macrocyclic trichothecenes on TNF-0t production was further related
to TNF-0t gene expression. SG was chosen as a representative of macrocyclic trichothecenes
for mRNA expression study. RAW 264.7 cells were exposed to 2.5 ng/ml of SG in the
presence of LPS (50 ng/ml) and levels of TNF-0t mRNA and 18S rRNA were assayed by real
time-PCR. After 24, 36, and 48 hr incubation, relative expression of TNF-or mRNA in SG
(2.5 ng/ml)-treated cells were higher than that of LPS-treated cells (Figure 3 .3). These results

suggest that the increased TNF-0t production was due, in part, to increased TNF-0t mRNA

56

 

8
1:1noLPS
7— . . IZZJOng
"0.1m
6e 1 ' C:10.25ng
I -1ng
1:12.5ng
E5“ -1Ong
v4_
‘7’
u.
E
3‘ I
2-1
1—

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SH
Macrocyclic Trichothecene

Figure 3.2. Effect of macrocyclic trichothecenes on TNF-0t production in RAW 264.7 cells.
Cells (2.5 x 105 cells/ml) were cultured for 24 hr and then medium was replaced with
medium containing various concentrations of macrocyclic trichothecene in the presence of
LPS (50 ng/ml) and incubated for 48 hr. Supematants from cell cultures were subjected to
TNF-0t assay by ELISA. Data are mean :1: SE of triplicate cultures. Bars marked with an
asterisk differ significantly from values of no toxin (0 ng/ml) treatment (* p < 0.05). Results
are representative of three separate experiments. iSF stands for isosatratoxin F, SG
satratoxin G, SH satratoxin H, RA roridin A, and VA verrucarin A.

57

 

 

 

 

 

120
<Z£100~ —o—LPS
hag —O—LPS+SG
0
<0 80-
529 .
*5\
a; 60-
En:
UE
- 40-
.2?
LL
E 20-
0<I I
0 24 36 48

Hours

Figure 3.3. Effect of satratoxin G (SG) on TNF-0t mRNA level in LPS stimulated RAW
264.7 cells. RAW 264.7 cells were incubated with LPS (50 ng/ml) and LPS +SG (2.5 ng/ml)
for the indicated time periods. After isolation of total RNA, real-time PCR was performed
for TNF-0t mRNA and 18S rRN A. The results were expressed in relative fold induction to
non-stimulated cells after normalization against 188 rRNA. Values are given as mean :E
S.E.M. of duplicate samples. Results are representative of two separate experiments.

58

expression in toxin-treated cells. Another important cytokine involved in regulation of
inflammation, IL-6, was assayed in this system. In LPS- treated cells, IL-6 production was

significantly impaired by 10 ng/ml iSF, SG, and SH, and by 2.5 ng/ml RA and VA at 48 hr

(Figure 3.4). At 12 and 24 hr, the same concentrations of each macrocyclic trichothecene

inhibited IL-6 production in LPS-treated cells (data not shown). Exposure to lower doses of
macrocyclic trichothecenes did not alter IL-6 production in LPS-stimulated cells (Figure 3 .4).

In the absence of LPS stimulation, IL-6 production was not induced in the macrophage cells

by macrocyclic trichothecenes at 12, 24, and 48 hr (data not Shown).

Effect of macrocyclic trichothecenes on MTT response. The MTT assay was also
performed because cytotoxicity might also affect cytokine production. At 48 hr, viability of
RAW 264.7 cells treated with macrocyclic trichothecenes with stimulation by LPS was
significantly reduced at 10 ng/ml iSF, SG, and SH, at 2.5 rig/ml RA, and 1 ng/ml VA (Figure
3.5) whereas viability was not affected by the toxin concentrations that induced TNF-0t
production. These data suggest that proliferation and viability of the cells were impaired at

the same toxin concentrations that impaired l NF-a production.

DISCUSSION
Proinflammatory cytokines are key mediators in regulating immune and inflammation
responses to microbes, toxins, trauma, and ischemia (Dinarello, 2000). Cytokine production
in cloned cell lines has been used as an endpoint to assess key modulatory effects of toxicants
on immune function. The results presented herein indicate that low levels of macrocyclic

trichothecenes superinduced TNF-0t production in a murine macrophage model whereas high

59

 

8
1:1noLPS
[:30ng
7‘ -01ng
[:1025ng
6.. -199
[2:125ng
5 -10ng
E
24-
«p .
d
3—
2‘ O
1..
S
0 T

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SH
Macrocyclic Trichothecene

Figure 3.4. Effect of macrocyclic trichothecenes on IL-6 production in RAW 264.7 cells.
Cells (2.5 x 105 cells/ml) were cultured for 24 hr and medium was replaced with medium
containing various concentrations of macrocyclic trichothecene in the presence of LPS (50
nyml) and incubated for 48 hr. Supematants from cell cultures were subjected to IL-6 assay
by ELISA. Data are mean :E SE of triplicate cultures. Bars marked with an asterisk differ
significantly from values of no toxin (0 ng/ml) treatment (* p < 0.05). ND; not detectable.
Results are representative of three separate experiments. Abbreviations are identified in
Fig.3.2 legend.

60

 

1 60
1:] no LPS

.1 - 0 ng/ml
140 - 0.1 nglml
E: 0.25 ng/ml
- 1 ng/ml

120 ~
I: 2.5 nglml

% Control Response
on
o
1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

T
SH

Macrocyclic Trichothecene

 

Figure 3.5. Effect of macrocyclic trichothecene on viability of RAW 264.7 cells by MTT
assay. Cells (2.5 x105 cells/ml) were cultured for 24 hr in 96-well tissue culture plates
containing 200 pl of cell suspension. Then, supematants were removed and fresh medium
containing macrocyclic trichothecenes and LPS was added to the culture and incubated for
48 hr. Then, MTT assays were performed as written in Materials and Methods. Data are
mean :1: SD of triplicate cultures. Bars marked with an asterisk differ significantly from
values of no toxin (0 ng/ml) treatment (* p < 0.05). Results are representative of two
separate experiments. Abbreviations are identified in Fig.3.2 legend.

61

concentrations decreased both TNF-0L and IL-6 production due to their cytotoxicity.

The concentrations of macrocyclic trichothecenes required for superinduction of
TNF-0t were approximately 100 times less than that found for deoxynivalenol(vomitoxin)
in the same cell line (Wong et al., 1998). Molar concentrations of macrocyclic
trichothecenes required to significantly superinduce TNF-0t were 1.8 - 4.6 nM (1-2.5 ng/ml)
whereas 338 - 845 nM (100-250 ng/ml) of deoxynivalenol is required for the same effect.
Similar macrocyclic trichothecene concentrations superinduce IL-2 secretion in EL-4
thymoma cells (Lee et al., 1999). Remarkable sensitivity of lymphocytes to macrocyclic
trichothecenes, roridin A, and verrucarin A, has been previously observed in mitogen-
induced blastogenesis (Hughes et al., 1990; Pestka and Forsell, 1988). The potency of
macrocyclic trichothecenes might be due to their extremely high ribosome binding affinity
as compared to affinities of other trichothecenes (Cannon et al., 1976).

In this study, sub-optimal concentration of LPS (50 ng/ml) was chosen to investigate
maximal effect of macrocyclic trichothecenes for cytokine production in less stimulated cells.
With less activated cells, effects of the toxins on cytokine production can be more readily
discerned (Sugita-Konishi and Pestka, 2001). The observation that TNF-0t and IL-6
production was impaired by high concentrations of the macrocyclic trichothecenes may relate
to cytotoxicity. Macrocyclic trichothecenes (22.5 ng/ml) significantly decreased MTT
response in LPS-stimulated RAW 264.7 cells. Similar results have been reported in EL-4
T cells (Lee et al. , 1999). Impaired proliferation and cytotoxicity by these toxins may result
from their capacity to induce apoptosis (Yang et al., 2000). Interestingly, toxin

concentrations that superinduced cytokine production produced slight (10~20%) inhibition

62

of MTT response, suggesting that the capacity of TNF-0t production per cell might be
increased. This observation is supported by similar findings in CD4+ T cells that partially
cytotoxic concentrations of deoxynivalenol can increase IL-2 secretion per cell (Ouyang et
al., 1996a).

Although specific mechanisms for increased TNF-0t transcripts upon exposure to
macrocyclic trichothecenes are unknown, it may relate to the capacity of the toxins to inhibit
translation. Superinduction is modulated in part via a repressive system that mediates
degradation of mRNA (Roger et al., 1998). Degradation of mRNA including TNF-0t
transcripts is facilitated in genes containing AU rich elements (ARE) in the 3' untranslated
region (UTR). ARES are known to be mRNA instability determinants (Chen and Shyu,
1995; Gillis and Malter, 1991). Specific binding proteins to ARE, such as AUFl , bind with
high affinity to c-myc, c-fos, GM-CSF, and other early response gene mRNAs, and regulate
degradation of those mRNAs (Nakamaki et al., 1995; Sirenko et al., 1997; Zhang et al.,
1993). Protein synthesis inhibitors may repress mRN A degradation by depleting the specific
3'-UTR binding proteins leading to increased mRNA half life and superinduction (Ohh and
Takei, 1995; Pages et a1. , 2000; Roger et al. , 1998). Notably, TNF-0t, IL-6, and IL-2 mRNAs
and resultant proteins are superinduced by deoxynivalenol and these observations are partly
explained by increased mRNA stability (Li et al., 1997; Wong et al., 2001).

Cytokine mRNA levels might also be elevated through binding of transcription
factors on a promoter region of these genes, resulting in increased transcriptional rates. For
example, superinduction of TNF-0t and IL-6 mRNAs by deoxynivalenol is partly explained

by an increase of NF-KB, AP-l, and NF-IL6 (C/EBPB) binding to the promoter regions in

63

RAW 264.7 cells (Wong, 2000) and EL-4 cells (Li et al., 2000; Ouyang et al., 1996b).
Increased binding activity of these transcription factors may result from activation of mito gen
activated protein kinases (MAPKS) (Guha and Mackman, 2001). For example, activation
of MAPK families (p38, ERKl/2, and JNKl/Z) is required for LPS-induced TNF-0t gene
transcription and TNF-0t mRNA translation (Zhu et aI. , 2000). All three MAPK families are
known to be important for TNF-0t mRNA induction and, furthermore, JNKl/Z is required
for translation of TNF-0t mRNA (Rutault et al., 2001; Swantek et al., 1997). Macrocyclic
trichothecenes have previously been observed to activate all three MAPK families (Shifrin
and Anderson, 1999; Yang et al., 2000). The precise roles of MAPK activation in
controlling cytokine transcript levels during superinduction by macrocyclic trichothecenes
require further study.

Interestingly, the level of IL-6 was not increased despite a significant elevation in
TNF-0t production following stimulation by macrocyclic trichothecenes in RAW 264.7 cells.
This observation was unexpected because exposure to deoxynivalenol up-regulates both
TNF-0t and IL-6 expression in vivo and in vitro (Wong et al. 2001; Zhou et al. 1998;1999).
The differential effect might result from inherent differences in structures of these toxins.
Deoxynivalenol, a type B trichothecene, does not possess a ring structure connected to C4
and C15 positions in the trichothecene nucleus that is found in macrocyclic trichothecenes.
This additional ring structure might interfere with IL-6 induction.

The results found in this study may have relevance to Stachybotrys-induced lung
disease. Exposure to Stachybotrys spores can cause inflammation in the lungs (Nikulin et

al., 1997; Rao et al., 2000). When the spores are introduced to the lung, the number of

64

alveolar macrophages, which can not only phagocytose the spores but also secrete various
immune mediators, are increased (Rao et al., 2000). Relatedly, Rao et al. (2000a)
demonstrated that removal of toxins from Stachybotrys spores reduces inflammatory effects
in the lungs of mice. These studies imply that toxins from the spores may enhance the
inflammatory response in this organ. The present study also supports the possibility that
macrocyclic trichothecenes in Stachybotrys spores may induce inflammation in the lungs
through TNF-0. production.

An in vitro study by Ruotsalainen et al., (1998) examined the effects of exposure to
Stachybotrys spores in production of proinflammatory cytokines, TNF-0t and IL-6, in a RAW
264.7 cell model. In that study, cytotoxicity of spores from 21 different strains was
determined by MTT assay or Trypan blue exclusion test. With a concentration of 10° spores
per 10° cells, all the tested strains of Stachybotrys sp. were cytotoxic producing a 31 to 97
% decrease in cell viability at 24 hr. Interestingly, Stachybotrys sp. strains with low
cytotoxicity induce the production of TNF-0t and IL-6 at the concentrations of 10° spores/10°
cells, whereas other strains that are highly cytotoxic do not induce the cytokine production
in the macrophages. The results from these experiments are consistent with our findings
because exposure to highly cytotoxic spores, presumably containing high concentrations of
macrocyclic trichothecenes, would reduce cytokine production due to overt cytotoxicity. The
ability of weakly cytotoxic strains of Stachybotrys Sp. to induce cytokine production may be
due to phagocytosis by macrophages or the presence of other metabolites in Stachybotrys
spores. Other fungal metabolites such as atranones (Hinkley et al., 2000),

stachybotrylactones, and stachybotrylactams (Jarvis et al., 1995) are found in Stachybotrys

65

sp. and might be responsible for the production of proinflammatory cytokines by
Stachybotrys spores. Further study is necessary to determine the effect of other fungal
metabolites as well as the combination of macrocyclic trichothecenes and metabolites on the
immunomodulatory effects.

Taken together, the results show that at low cytotoxic concentrations, macrocyclic
trichothecenes can superinduce the proinflammatory cytokine, TNF-0t, in murine
macrophages, whereas at higher concentrations, these compounds are cytotoxic and reduce
cytokine production. These immunomodulatory effects were observed at relatively low
(ng/ml) concentrations, suggesting that macrocyclic mycotoxins may pose a hazard to
humans exposed to Stachybotrys. Further studies on immunomodulatory effects of
macrocyclic trichothecenes should improve understanding of possible mechanisms for

Stachybotrys spore-induced lung injury.

ACKNOWLEDGMENTS
This work was supported by Public Health Service grants ES-09521 (J JP) and ES-
03358 (JJP) from the National Institute for Environmental Health Sciences, and by the

Michigan State University Agricultural Experimental Station.

66

CHAPTER IV
UP-REGULATION OF MACROPHAGE INFLAMMATORY PROTEIN-2
AND COMPLEMENT 3A RECEPTOR BY THE TRICHOTHECENES

DEOXYNIVALENOL AND SATRATOXIN Gl

' This work has been submitted for publication in a similar form with authors Chung,
Y.J., Yang, G.H., Islam, 2., and Pestka, J .J . in Toxicology.

67

ABSTRACT

The trichothecenes are a group of mycotoxins that target leukocytes and have a wide
range of immunomodulatory effects. Differential display analysis was applied to assess the
effects of the trichothecenes deoxynivalenol (vomitoxin, VT) and satratoxin G (SG), on
mRNA in the RAW 264.7 macrophage cell line. Cells were incubated with VT (1 pg/ml)
or SG (5 ng/ml) for 2 h and total RNA then subjected to RT-PCR with a set of oligo(dT)
primers. Resultant cDNA was amplified using an oligo (dT) downstream primer and an
arbitrary decanucleotide upstream primer to make 3sS-labeled PCR products. After
separation" of the products in denaturing polyacrylamide gels, 23 differentially expressed
cDNA fragments were isolated and sequenced. Two of these were identified as known genes,
namely, macrophage inflammatory protein-2 (MIP-2), a potent neutrophil chemoattractant
involved in tissue injury and inflammation, and complement 3a receptor (C3aR), a
proinflammatory mediator. Both MIP-2 and C3aR mRNAs were up-regulated by VT while
only MIP-2 mRNA was induced by SG. Using commercially available antibodies, MIP-2
protein was also found to be induced by both VT and SG in RAW 264.7 cell cultures. When
mice were treated with VT (12.5 mg/kg), splenic MIP-2 mRNA and serum MIP-2 levels
were increased. MIP-2 mRN A and serum MIP-Z levels were synergistically increased when
mice were co-treated with VT and LPS. Up-regulation of MIP-2 and C3aR are consistent
with previous reports of trichothecene-induced inflammatory gene up-regulation and suggest

that the specific genes affected may depend on trichothecene structures.

68

INTRODUCTION

The trichothecene mycotoxins are a group of sesquiterpenoid fungal metabolites that
are commonly found as contaminants of grain-based foods and indoor air. These mycotoxins
include some of the most potent protein synthesis inhibitors known (Ueno, 1983a).
Exposures of farm animals and humans to trichothecenes have been reported worldwide and
have been related to outbreaks of alimentary toxic aleukia, vomiting, gastroenteritis,
leukocytosis, and circulatory Shock (Yagen and Joffe, 1976; Ueno, 1983b; Luo et al., 1990
Arnold et al., 1986; Li et al., 1999). Trichothecenes can markedly alter immune function in
experimental animals (Bondy and Pestka, 2000).

Deoxynivalenol (VT, vomitoxin) is produced by F usarium graminearum, and
frequently found in cereals (Yuwai et al., 1994; Rotter et al., 1996; Ryu et al., 1996).
Previous work in our laboratory has demonstrated that VT modulates immune function in
mice. One of the most prominent effects of dietary VT is dysregulation of immunoglobulin
A (IgA) production (Pestka et al., 1989; Dong et al., 1991), which is highly analogous to
human IgA nephropathy (D'Amico, 1987). Overproduction of IgA by VT, observed in vivo,
is mediated through alteration of cytokine production by macrophages and helper T cells
(Azcona-Olivera et al., 1995a; Li et al., 1997; Yan et al., 1997, Zhou et al., 1997; Wong et
al., 1997; Yan et al., 1998). Expression of various cytokine genes including interleukin (IL)-
6, IL-2, IL-4, IL-5, IL-1 [3, and TNF-0t is also up-regulated by VT. The mechanisms by which
VT induces cytokine gene expression involve increased binding of transcription factors
(Ouyang et al., 1996; Li et al., 2000; Wong et al., 2002) and increased mRNA stability (Li

et al. , 1997; Wong et al., 2001). At high exposure levels, VT induces apoptosis in lymphoid

69

organs (Pestka et al., 1994; Zhou et al., 2000) and in macrophage and leukemic cell lines
(Yang et al. , 2000a). Down-regulation of GRP78/BiP (a 78-kDa glucose-regulated protein)
and cochaperone P58 (IPK) by VT may also be involved in VT-induced apoptosis (Yang et
al,2000b)

Satratoxins, a group of macrocyclic trichothecenes, are mainly produced by
Stachybotrys atra, which grows on cellulose materials in water-damaged buildings (Bata et
al., 1985; Johanning et al., 1996). Satratoxins are 10 to 100 times more toxic than are other
groups of trichothecenes in mouse LD50 studies, HeLa cell cytotoxicity tests, and rabbit
reticulocyte protein synthesis inhibition assays (U eno, 1983a). These toxins have been
etiologically associated with stachybotryotoxicosis in horse and sheep in Central Europe
(Hintikka, 1978; Harrach et al., 1981; 1983; Harrach et al.; Bata et al., 1985). Clinical signs
of this disease include leukopenia, thrombocytopenia, hemorrhage, arrhythmic heartbeat, and
death (Forgacs, 1972). Outbreaks of Stachybotiys-associated diseases in water-damaged
homes have been reported (J ohanning et al., 1996; Etzel et al., 1998). Recently, the toxigenic
S. atra was reported to be involved in pulmonary hemorrhage and hemosiderosis in infants
living in S. atra-contaminated homes (Elidemir et al., 1999). Leukocytes are remarkably
sensitive to macrocyclic trichothecenes. At low exposure levels, these toxins have been
shown to superinduce IL-2 production in EL-4 thymoma cells (Lee et al., 1999) and TNF-0t
production in RAW 264.7 murine macrophage cells (Chung et al. , 2002). In addition, these
toxins impair blastogenesis of lymphocytes in the presence of mitogens (Pestka and Forsell,
1988; Hughes et al., 1990) as well as induce apoptosis in macrophages (Yang et al. , 2000a).

In order to expand understanding of how trichothecenes affect gene regulation,

70

murine RAW 264.7 macrophage cells were treated with VT or satratoxin G (SG) and
expressed genes were assessed by differential display PCR (DD-PCR) (Liang and Pardee,
1992). This cell line was employed since it has been previously used for studies on VT or
SG-induced cytokine production as well as apoptosis studies (Wong et al., 1998; Yang et al.,
2000a; Chung et al., 2002). The results indicate that both toxins up-regulated expression of
macrophage inflammatory protein-2 (MIP-2), which is a chemokine responsible for
chemotaxis of neutrophils to inflammation site, whereas VT selectively up-regulated
complement 3a receptor (C3aR), which is a receptor involved in activation through
complement. Induction of these two genes might contribute to trichothecene-induced
immunotoxicity.

This study was to test the hypothesis that VT or SG induces MIP-2 and/or C3aR

expression in RAW 264.7 murine macrophage cells

MATERIALS AND METHODS

Reagents and cell culture. All chemicals were reagent grade or better and were
purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise noted.
Satratoxin G was kindly provided by Dr. Bruce Jarvis (U. Of Maryland). The murine
macrophage RAW 264.7 cell line was obtained from the American Type Culture Collection
(Rockville, MD). Cells were maintained at 37°C in a 7% CO2 humidified incubator in
Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% (v/v) fetal bovine
serum (FBS; Gibco BRL, Gaithersburg, MD), 1% (v/v) NCTC 135 (Gibco BRL), 1 mM

sodium pyruvate, 100 U/ml penicillin, and 100 pg/ml streptomycin (Gibco BRL).

71

RNA preparation. Total RNA was extracted from RAW 264.7 cells by the method
of Chomczynski and Sacchi (1987) using TrizolT'M reagent (Gibco BRL) according to the
manufacturer’s instructions. Total RNA content was determined spectrophotometrically at
260 nm.

Differential display analysis. Differential display (Display Systems Biotech, Vista,
CA) was performed on total RNA isolated from RAW 264.7 cell cultures treated with or
without VT (1 pg/ml) or SG (5 ng/ml) for 2 h according to the manufacturer's instructions
with some modifications. Briefly, 7 down stream primers were used in combination with 12
upstream primers. For first-strand cDNA synthesis, cDNA master mix was prepared by
mixing 24 pl of 5X first strand cDNA synthesis buffer, 39 pl of 500 pM dNTP mix, and 3
pl of RNasin (40 units/ pl). A mixture (1 1 pl) containing total RNA (1 pg), oli go (dT) primer
(75 pmol, 5-T11VV-3, where V represents A, C, or G), and dNTP (2.5 nmol) was heated to
70 °C for 10 min, then put on ice. cDNA master mix (16.5 pl) and reverse transcriptase M-
MLV (1.5 pl, 30 units) were added to the mixture, which was subsequently incubated at 42
°C for 1 h, and for 5 min at 95 °C, then put on ice. H20 (61 pl) was added and the resultant
cDNA solution was aliquoted to 15 pl per vial for following PCR.

For differential display RT-PCR (DDRT-PCR), a master mix for 72 DDRT-PCR
reactions was prepared by mixing 175.5 pl of 10X PCR buffer, 35.1 pl of 25 mM MgC12,
8.85 pl of [a-3SS]-dATP (12.5 mCi/ml; NENTM Life Science Products, Boston, MA), 21.2
pl of 500 pM dNTP mix, 16.5 pl of TAQ FL DNA Polymerase (Display System Biotech; 5
units/pl), and 796.1 pl of H20. To six tubes containing 13 p1 of cDNA (each two tubes for

control, VT treatment, and SG treatment) and 27 pl of 25 pM downstream primer 5-T1 lVV-

72

3 (where V represents A, C, or G), 162 pl of the DDRT-PCR master mix were added. Then,
these premixed components (15 pl) were mixed with 5 pl of 2 pM arbitrary decanucleotide
upstream primer. PCR reactions were performed in a GeneAmp PCR System 2400 (Perkin-
Elmer, Foster City, CA) using the following parameters: 40 cycles of 94 °C for 30 S, 40 °C
for 90 s, and 72 °C for 60 s for amplification followed by a final extension step at 72 °C for
7 min.

For electrophoresis, the DD-PCR sample (10 pl) was mixed with 8 pl of denaturing
loading dye (95% (v/v) fonnamide, 20 mM EDTA (pH 8), 0.05% (w/v) bromphenol blue,
and 0.05% (w/v) Xylene Cyanol FF), and were denatured by incubating at 75 °C for 3 min.
Each sample (4 pl) was applied to a denaturing 5% (w/v) polyacrylamide gel (Long Ranger
gel solution, FMC Bioproducts, Rockland, ME) in 1.2X TBE buffer, and the gel was run at
50 Watt in 0.6x TBE buffer. The gel was dried and subjected to autoradiography using
Kodak BioMax MR film (Eastman Kodak, Rochester, NY).

Cloning and sequence analysis of differential display bands. cDNA bands of
interest in dried gel were superimposed on autoradiography film and the corresponding gel
slice was incubated in 400 pl of H20 at 100 °C for 15 min. Eluted cDNA was further
amplified by PCR in 50 pl reaction mixture containing 1X PCR buffer, 1.5 mM MgC12, 250
pM dNTP, 10 pmol of each downstream and upstream primer used in DDRT-PCR, 2.5 U
T aq polymerase, and 2 pl of eluted DNA. PCR parameters were the same as described
above. Amplified DNA fragments were purified with Qiaex II Gel Extraction Kit (Qiagen,
Valencia, CA), and were cloned into the plasmid vector pCRII using a TA Cloning Kit

(Invitrogen, San Diego, CA). The cloned vectors were purified with a Wizard® Plus

73

Miniprep Kit (Promega, Madison, WI) and sequenced using T aq-cycle sequencing and dye
terminator chemistry at the Michigan State University Sequencing Facility. Similarity of
DNA sequences was compared with the GenBank and the EMBL databases using BLAST
(http://www.ncbi.nlm.nihgov/BLASTL National Center for Biotechnology Information,
NCBI).

RT-PCR for mRNA expression for in vitro study. For first-strand cDNA
synthesis, 10 pl of total cellular RNA (0.50 pg) was heated to 70 °C for 5 min and chilled
on ice. cDNA master mix containing 5 pg of oligo(dT)”,18 primer (Gibco BRL), 1 mM
dNTPs, 1X first-strand cDNA synthesis buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3mM
MgClz, Gibco BRL), 10 mM DTT, 20 U of RNase inhibitor (Boehringer-Mannheim,
Indianapolis, IN), and 100 U of M-MLV reverse-transcriptase (Gibco BRL) was added to
make a final volume of 30 pl/reaction. This reaction mixture was incubated at 37 °C for 60
min, then heated at 70 °C for 15 min and chilled on ice. The RT product was diluted 10-fold
with distilled H20 and stored at -20 °C.

Specific primer sets used for cDNAs of MIP-2 and C3aR were synthesized from
Macromolecule Synthesis Facility at MSU (forward primer for MIP-2 5'-
GAACAAAGGCAAGGCTAACTGA-3', reverse primer for MIP-2 5'-
AACATAACAACATCTGGGCAAT-3', forward primer for C3aR 5'-
CCTATGATTTCCAGGGGGAT-3', reverse primer for C3aR 5'-
CCAAGAGGGCATACAGGAAA-3', forward primer for B-actin 5'-
CACACCCGCCACCAGTTC-3', and reverse primer for B-actin 5'-

ACGCACGATTTCCCTCTCA-3'). PCR was performed using a GeneAmp PCR system

74

9600 (Perkin-Elmer) in a final volume of 25 pl. A master mixture was made to contain 5
pmol of each primer, 125 pM dNTPs, 10 mM Tris-HCI (pH 9.0), 50 mM KCl, 2.5 mM
MgC12, and 0.5 U of T aq DNA polymerase (Gibco BRL) per each reaction tube. cDNA
template (5 pl) was added to 20 pl of master mixture. PCR amplification for MIP-2, C3aR,
and B-actin was performed using 23 to 31 cycles of denaturation (30 s at 94 °C), annealing
(45 s at 59 °C), and extension (60 s at 72 °C) with a final extension for 10 min at 72°C. The
PCR products were electrophoretically separated on 2 % (w/v) agarose gel containing 0.2
pg/ml ethidium bromide. Fluorescence intensity of each band was captured with a Molecular
Imager® F X (Bio-Rad, Hercules, CA). Data were presented for multiple cycles to verify that
endproduct plateau was not attained. I
MIP—2 protein quantitation. Production of MIP-2 in RAW 264.7 supematants and
mouse sera was quantified by ELISA using modifications of the procedure of Wong et al.
(1998). Briefly, microtiter strip wells (Irnmunolon TV Removawell; Dynatech Laboratories
Inc., Chantilly, VA) were coated with 50 pl/well of lpg/ml purified antibody to MIP-2
(rabbit anti-recombinant murine; PeproTech Inc., Rocky Hill, NJ) in 0.1 M sodium
bicarbonate buffer (pH 8.2) overnight at 4 °C. Plates were washed three times with 0.01M
phosphate-buffered (pH 7.2) saline containing 0.2% (v/v) Tween 20 (PBST) to remove
unbound capture antibodies. Wells were then incubated with 300 pl of PBST containing 3%
(w/v) bovine serum albumin (Amresco, Solon, OH) (BSA-PBST) for 30 min at 37°C to
block nonspecific protein binding and washed four times with PBST. Fifty p1 of standard
recombinant murine MlP-2 (PeproTech) or samples diluted in 10% (v/v) FBS DMEM were

added to appropriate wells and plates were incubated for 1 h at 37°C. After washing four

75

times with PBST, 50 p1 of biotinylated rabbit anti-mouse MIP-2 antibody (PeproTech),
diluted in BSA-PBST (1 pg/ml), was added to each well and plates were incubated for 1 h
at room temperature. After washing 6 times with PBST, 50 pl of streptavidin-horseradish
peroxidase conjugate (1.5 pg/ml in BSA-PBST; Sigma) was added and incubated for 1 h at
room temperature. After washing 8 times with PBST and 2 times with deionized H20, bound
peroxidase conjugate was detected by adding 100 pl/well substrate solution consisting of 25
ml of 0.1 M citric-phosphate buffer (pH 5.5), 400 p1 of tetrarnethylbenzidine (TMB) (F luka
Chemical Corp., Ronkonkoma, NY; 6 mg/ml in dimethylsulfoxide, Sigma), 100 pl of 1%
H202. To stop the reaction, 100 pl/well of 2N H2804 was added and absorbance was
measured at 450 nm on a Vmax kinetic microplate reader (Molecular Devices, Menlo Park,
CA). MlP-2 was quantified from standard curve using the Softmax curve-fitting program
(Molecular Devices).

MIP-2 expression in mouse. All animal handling was conducted in accordance with
recommendations established by the National Institutes of Health. Experiments were
designed to minimize the number of animals required to adequately test the proposed
hypothesis, and were approved by the Michigan State University Laboratory Animal
Research Committee. Male B6C3F1 (C57B1/6JXC3H/HeJ) mice (7 weeks) obtained from
Charles River (Portage, M1) were used for experiments. Mice were acclimated for at least 1
wk, housed 3 per cage under a 12 h light/dark cycle, and provided standard rodent chow and
water ad libitum.

Food and water were withdrawn from cages l h before toxin administration. In a

typical experiment, mice were given vehicle (VH) (i.p.) + VH (p.o.) [VH], VH (i.p.) + VT

76

(p.o.) [VT], LPS (i.p.) + VH (p.o.) [LPS], LPS (i.p.) + VT (p.o.) [LPS + VT]. LPS was
dissolved in tissue culture-grade, endotoxin-free water (Sigma), aliquoted and stored at -80
°C. VT was also dissolved in tissue culture grade, endotoxin-free water and stored at 4 °C.
Escherichia coli LPS was injected i.p. (0.1 mg/kg bw: 250 pl/mouse). VT was gavaged p.o.
(12.5 mg/kg bw: 250 pl/mouse) 5 min after LPS injection.

For serum collection, blood was collected and allowed to clot overnight at 4 °C.
Serum was collected and stored at -80 °C for MIP-2 ELISA.

Total RNA was extracted from Spleen with TrizolTM reagent (Gibco BRL) according
to the manufacturer’s instructions. RNA (100 ng) from each sample was reverse transcribed
to cDNA and MIP-2 cDNA was amplified as described previously.

Statistics. A one-way analysis of variance (ANOVA) using Bonferoni’s test for
parametric data was performed with Sigma Statistical Analysis System (J andel Scientific,

San Rafael, CA). A p value of less than 0.05 was considered statistically significant.

RESULTS
mRNA differential display analysis of VT- and SG-treated RAW 264.7 cells.
Genes differentially expressed after VT or SG treatment of RAW 264.7 cells were identified
using DD-PCR. RNAs derived from VT (1 pg/ml)- or SG (5 ng/ml)- treated cells for 2 h
were compared to those of untreated cells. These concentrations were used because these
partially (SO-75%) inhibit protein synthesis and have been found effective at inducing
cytokine gene expression. Using seven degenerate anchored oligo (dT) primers, two pools

of cDNA from each treatment were generated. Then, PCR was performed with cDNA pools

77

using the same oligo (dT) downstream primers in combination with 12 short arbitrary
upstream primers. A total of 140 bands were identified as potentially responsive genes
following toxin treatments. Of 140 bands, twenty-three bands with high band intensities
were chosen, reamplified, subcloned into a T/A vector, and sequenced. After comparisons
of cDNA sequences to nucleic acid sequences in EMBL databases at NCBI, two cDNA
fragments (Figure 4.1) were identified as known genes with >99% homology. The others
showed no strong homology (i.e., >85%) to any existing nucleic acid sequences. The two
known genes were macrophage inflammatory protein-2 (MIP-2), a chemokine that attracts
neutrophils to inflammation sites, and complement 3a receptor (C3aR), which is
transmembrane receptor activated during complement activation.

VT and SG induction of MIP-2 and C3aR mRNA expression in RAW 264.7
cells. RT-PCR was used to confirm MIP-2 and C3aR gene expression. Total RNA was
isolated from control and VT or SG-treated RAW 264.7 cells and subjected to RT-PCR. For
dose-response experiments, cells were treated for 3 h with a range of concentrations of VT
(0 to 1000 ng/ml) or SG (0 to 25 ng/ml). Lipopolysaccharide (LPS, 500 ng/ml) induction
was used as a positive control for MIP-2 (Kopydlowski et al., 1999) and C3aR (Drouin et
al., 2001) mRNA expression. MIP-2 and C3aR cDNAs were dose-dependently increased
by VT over a range of 100 to 1000 ng/ml (Figure 4.2). MIP-2 cDNAs were dose-dependently
increased by SG over a range of 10 to 25 ng/ml (Figure. 4.3) whereas C3aR cDNAs were not
induced over a range of 2.5 to 25 ng/ml of SG (Figure 4.3).

For time course experiments, cells were treated with VT (250 ng/ml) or SG (10

rig/ml) and mRNAs were analyzed over a Six hour period. MIP-2 cDNAs were time-

78

A Control VT SG B

MIP-z-p C3aR->

   

Figure 4.1. Sequencing gel section from mRNA DD-PCR. RAW 264.7 cells were cultured
in the presence of VT (1 pg/ml) or SG (5 ng/ml) for 2 h. After cDNA synthesis from total
RNA, 3sS-labeled PCR products were synthesized using a combination of upstream and
downstream primers. Resultant PCR products were subjected to electrophoretic separation.
(A) Primers used: #3 downstream primer (T11AG) and #6 upstream primer (B) Primers
used: #8 downstream primer (T1 1GC) and #5 upstream primer. Arrows indicate mRNA
transcripts that are highly expressed in RAW 264.7 cells.

79

25 cycles 27 cycles 29 cycles

 

 

27 cycles 29 cycles 31 cycles

 

—....R

9 9 Q 0 o o 0
3% Q '9 '1‘? '90 3% 9 $9,139.90 3% 9:9 rf? .90 (VT. ng/ml)
23 cycles 25 cycles 27 cycles

 

t- B—actin

 

Q Q Q
3% Q '90 £30.99 3% Q GQq‘? .90 (VT.ng/ml)

Figure 4.2. Dose-dependent induction of MIP-2 and C3aR mRNAs expression by VT in
RAW 264.7 cells. RAW 264.7 cells (2.5 x 105 cells/ml) were treated with various
concentrations of VT for 3 h. Total RNA was subjected to RT-PCR for variable cycle
periods as shown using murine specific MIP-2 C3aR and B-actin primers, respectively. PCR
products were separated on 2% (w/v) agarose gel and visualized using ethidium bromide (0.2
pg/ml). LPS (500 rig/ml) was used as a positive control for M1P-2 and C3aR. Data are a
representative of two independent experiments.

80

25 cycles 27 cycles 29 cycles

 

- . - - ---- m hMlP-Z
<2e ° 03’ ’9 '33 Qc°° 45° ’9 13’3” ° (C? '9 0‘? (SG,ng/ml)

27 cycles 29 cycles 31 cycles

 

0:9

 

QCoo 9.3.19390

'9 ti” 39 ° qf? '9 ri? (SG,ng/ml)

23 cycles 25 cycles 27 cycles

 

' ' ' t- B-actin

 

(SG, ng/ml)

Figure 4.3. Dose-dependent induction of MIP-2 mRNAs expression by SG in RAW 264.7
cells. RAW 264.7 cells (2.5 x 105 cells/ml) were treated with various concentrations of SG
for 3 b. Total RNA was subjected to RT-PCR for variable cycle periods as Shown using
murine specific MIP-2 C3aR and D-actin primers. Detection of agarose gel separation of
cDNA bands of PCR products were explained in Figure 2 legend. LPS (500 ng/ml) was used
as a positive control for MIP-2 and C3aR mRNA. Data are a representative of two
independent experiments.

81

dependently increased by VT and over a 6 h period while C3aR cDNAs were-increased by
VT at 2 h and remained constant until 6 h (Figure 4.4). MIP-2 cDNAs were also time-
dependently increased by SG over a 6 h period, whereas C3 aR cDNAs were not affected by
SG (Figure 4.5).

Further studies for MIP-2 protein expression by VT or SG were performed using
commercially available antibodies whereas examination of C3aR protein expression was not
possible due to unavailability of specific antibodies.

VT and SG induce MIP-2 protein production in RAW 264.7 cells. ELISA was
used to quantify induction of MIP-2 protein expression by VT and SG in RAW 264.7 cells.
After 12 h, VT significantly induced production of MIP-Z between 100 and 1000 ng/ml with
peak levels observed at 250 ng/ml of the toxin (Figure 4.6A). SG at concentrations between
2.5 and 10 ng/ml also induced production of MIP-2 with peak effects observed at 5 ng/ml
(Figure 4.6B). Maximum levels of MIP-2 (32 ng/ml) induced by VT were 8 times higher
than maximum levels of MIP-2 (3.8 ng/ml) induced by SG. This result suggests that VT
appeared to be a more potent inducer of MIP-2 than SG when presented at optimal
concentrations for stimulation.

For time course experiments, cells were treated with VT (250 ng/ml) or SG (5 ng/ml)
for 6, 12, 24 h, then MIP-2 protein in cell culture supernatant was quantified by ELISA. MIP-
2 production by VT (Figure 4.7A) and SG (Figure 4.73) was significantly increased up to
24 h. Maximum levels of MIP-2 produced by VT at 24 h incubation were 12 times higher
than those levels induced by SG at the same incubation time, which also supports the

observation that VT was a more potent inducer of MH’-2 than SG.

82

27 cycles 29 cycles 31 cycles

 

 

LPSO 2 4 6LPSO 2 4 6LPSO 2 4 6(hr,VT)

27 cycles 29 cycles 31 cycles

 

{IC3aR
LPSO 2 4 6LPSO 2 4 6LPSO 2 4 6(hr,VT)

 

23 cycles 25 cycles 27 cycles

 

 

_. -_- --- w- an I~ .— -- qt— nu.- alum—uh” hB-actin

LPSO 2 4 6LPSO 2 4 6LPSO 2 4 6(hr,VT)

Figure 4.4. Time-dependent induction of MH’-2 and C3aR mRNA expression by VT in
RAW 264.7 cells. RAW 264.7 cells (2.5 x 105 cells/ml) were treated with VT (250 ng/ml)
over a 6 h period. Total RNA was subjected to RT-PCR using murine specific MIP-2, C3 aR,
and B-actin primers, respectively. LPS (500 ng/ml) at 2 h was used as a positive control for
MIP-2. Detection of cDNA bands and agarose gel separation of PCR products were
explained in Figure 2 legend. Data are a representative of two independent experiments.

83

27 cycles 29 cycles 31 cycles

 

hMlP—Z
LPSO 2 4 6LPSO 2 4 6LPSO 2 4 6(hr,SG)

 

27 cycles 29 cycles 31 cycles

 

 

LPSO 2 4 6LPSO 2 4 6LPSO 2 4 6 (hr,SG)

23 cycles 25 cycles 27 cycles

 

.. hB-actin
LPSO 2 4 6LPSO 2 4 6LPSO 2 4 6(hr,SG)

 

Figure 4.5. Time-dependent induction of MIP-2 mRNA expression by SG in RAW 264.7
cells. RAW 264.7 cells (2.5 x 105 cells/ml) were treated with SG (10 ng/ml) over a 6 h
period. Total RNA was subjected to RT-PCR using murine Specific MIP-2, C3aR and B-actin
primers, respectively. LPS (500 ng/ml) at 2 h was used as a positive control for MIP-2.
Detection of cDNA bands and agarose gel separation of PCR products were explained in
Figure 2 legend. Data are a representative of two independent experiments.

84

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

40 6
35 < *
5 4
30 ~11
'5 25 . E 4 ' T
E 20 g 31
‘7‘ * 0'1 *
E 15 - E T
2 2 2 *
10 ~ 1: T
* 1
5‘ rfi
o . . . . . o . . . .
O 100 250 500 1000 0.0 2.5 5.0 10.0
VT (rig/ml) Satratoxin G (ng/ml)

Figure 4.6. Effect of VT or SG on MIP-2 protein production by RAW 264.7 cells for dose-
dependent study. Cells (2.5 x 105 cells/ml) were cultured with various concentrations of (A)
VT or (B) SG for 12 h. MIP-2 production in supernatant was assayed by ELISA. Data are
mean :1: SEM of triplicate cultures. Values marked with an asterisk (*) differ significantly
from control values (p < 0.05). Data are a representative of two separate experiments.

85

 

 

 

 

 

 

 

 

 

 

80 7 ,
70‘ +Control
6‘ —o—SG
60 54 '
Am A
5 E“
J0 23 .
N 1
013° 3
220 52«
10« 1‘
0* 0.
0 5 10 15 20 25 30 0 5 10 15 20 25 II)
Hows Hous

Figure 4.7. Effect of VT or SG on MIP-Z protein production by RAW 264.7 cells for time-
course study. Cells (2.5 x 105 cells/ml) were cultured for various time periods with (A) VT
(250 ng/ml) or (B) SG (5 ng/ml). MIP-Z production in supernatant was assayed by ELISA.
Data are mean i SEM of triplicate cultures. Values marked with an asterisk (*) differ
significantly from vehicle (p < 0.05). Data are a representative of two separate experiments.

86

MIP-2 expression by VT, LPS and VT+LPS in vivo. Mice were treated with VH,
LPS, VT or VT + LPS and splenic expression for MIP-2 mRNA was assayed 3 h later. As
observed by Kopydlowski et. al. (1999), MIP-Z mRNA expression was induced by the
treatment of LPS and this served as a positive control. VT also independently induced MIP-2
mRNA at this time point (Figure 4.8A). When mice were co-treated with LPS and VT,
more MIP-2 mRNA was induced than single treatment in spleen.

After 3 h toxin treatment, sera were collected and subjected to MIP-2 protein assay
by ELISA (Figure 4.8B). VT significantly induced MIP-2 protein (20 ng/ml) at 3 h and LPS
also significantly induced MIP-Z protein (14 ng/ml). When mice were treated with both LPS
and VT, MIP-2 concentrations in sera was significantly higher than expected additive

responses.

DISCUSSION

VT, SG and other trichothecenes are protein synthesis inhibitors that target actively
dividing cells such as leukocytes (Ueno, 1983a). Trichothecene mycotoxins can dose-
dependently cause cytokine up-regulation and apoptosis in lymphoid tissues in mouse
(Bondy and Pestka, 2000). Here, we employed differential display to identify additional
genes that might be involved in immune dysregulation by these toxins in a murine
macrophage model. The capacity of trichothecenes to induce MIP-2 and C3aR mRN A may
contribute to immunotoxicity.

Macrophage inflammatory proteins (MIP) are structurally related Chemokines that

induce migration of specific subsets of leukocytes (Driscoll, 1994). MIPS are approximately

87

A Vehicle LPS VT LPS+VT

 

-- MIP—z. 25 cycles

up

 

a. ‘- ..... 'I' d- -- hMlP-Z, 27 cycles

 

 

 

 

 

 

 

 

 

 

 

 

 

100
B
**
A 80 " 1
E
8’
v 60
‘7‘
E
2
E 40 -
E
(D 'k
w 20 - * L
O I 1 I I
LPS (0.1 mg/kg) - + — +
VT(12.5 mg/kg) — — + +

Figure 4.8. Effect of VT on MIP-Z expression in vivo. Mice were treated with vehicle, LPS
+ vehicle, vehicle + VT or LPS + VT for 3h. (A) Spleen was collected afier treatment. Total
RNA was subjected to RT-PCR (n = 3) using murine MIP-2 primers for 25 and 27 cycles of
PCR. (B) Blood was collected at 3 h treatment and MIP-2 concentrations (mean :l: SEM, n
= 3) in serum were measured by ELISA. Values marked with an asterisk (*) differ
significantly (p < 0.05) from vehicle values. Values marked with an double asterisk (**)
differ significantly (p < 0.05) from additive expected responses of LPS + VT. Data are a
representative of two separate experiments.

88

6-8 kd, heparin binding proteins and three MIPS (MIP-la, MIP-IB and MIP-2) have been
identified according to their similar structure and function (Sherry et al. , 1988; Wolpe et al. ,
1988; Wolpe et al., 1989). MIPS are induced by various stimuli such as LPS,
proinflammatory cytokines, and oxidative stress in a wide range of cells including alveolar
macrophages, mast cells, peritoneal macrophages, epithelial cells, and fibroblasts (Burd et
al., 1989; Driscoll et al., 1993; Kopydlowski et al., 1999; Shi et al., 1999). MIP-la and
MIP-lB can activate both neutrophils and mononuclear cells and they possess
proinflammatory properties (Driscoll, 1994). MIP-2 is a potent neutrophil chemoattractant
and epithelial cell mitogen and it is involved in acute pulmonary inflammation (Huang et al.,
1992; Driscoll et al., 1995).

Several in vivo studies demonstrate that MIP-2 is involved in tissue injury and
inflammation. Expression of MIP-2 mRNA and accumulation of neutrophils in the lung
were associated with development of lung edema during hepatic ischemia/repercussion in
mice. Passive immunization against MIP-2 with neutralizing antibody reduced lung edema
indicating that MIP-2 is involved in tissue injury (Yoshidome et al. , 1999). LPS instillation
into rat lung up-regulates MIP—2 mRNA in a time dependent manner. Furthermore, MIP-2
is responsible for neutrophil accumulation in lungs (Schmal et al., 1996). When the effect
of elevated MIP-2 was also studied in a cecal ligation and puncture (CLP) model of septic
peritonitis in CD-1 mice, the chemokine was significantly increased in peritoneal fluid,
serum, liver and lung afier CLP and this increase correlated with severity of sepsis. Passive
immunization prior to CLP with antibody to MIP-2 decreased both mortality and neutrophil

percentage in peritoneal fluid (Walley et al. , 1997). Overall, these studies demonstrate that

89

increased MIP-2 expression and neutrophil accumulation can mediate tissue damage and
inflammation. The previous observation by our laboratory that ingestion of VT by mice
significantly increases neutrophil levels in blood (Forsell et al., 1986) may relate to MIP-2
induction.

In this experiment, satratoxin G, a major macrocyclic trichothecene produced by S.
atra, induced MIP-2 expression of both mRNA and protein levels in RAW 264.7
macrophage model. Since neutrophils induced by MIP-2 are involved in acute pulmonary
inflammation and hemorrhage-induced acute lung injury (Huang et al., 1992; Driscoll et al.,
1995; Abraham et al., 2000), the capacity of SG to induce MIP-2 may be important in
relation to Stachybotrys-associated diseases. Pulmonary hemorrhage and hemosiderosis in
infants have been associated with toxigenic S. atra contaminated homes (Etzel et al. , 1998).
Exposure of S. atra to humans resulted in various disorders in respiratory, immune, and
central nervous systems (Johanning et al., 1996). Although the specific etiologic
involvement of macrocyclic trichothecenes hasnot yet been verified, the capacity to induce
MIP-2 as well as other cytokines (Lee et al., 1999; Chung et al., 2002) provides a feasible
mechanism for Stachybottys-associated lung injury.

Induction of MIP-2 expression by VT and SG might be mediated through activation
of mitogen-activated protein kinases (MAPKS). SG at 2.5 to 10 ng/ml activates all three
major MAPKS; the extracellular signal-regulated kinase (ERKS or p42/p44), the c-Jun
amino-terminal kinase (JNKS, also known as stress-activated protein kinases, SAPK), and
p38 MAPK in RAW 264.7 murine macrophage cells (Yang et al., 2000a). VT at 250 ng/ml

also induces phosphorylation of the three MAPKS in RAW 264.7 cells (Moon and Pestka,

90

2002). MIP-2 induction by soluble intercellular adhesion molecule-1 in mouse astrocytes is
mediated through activation of ERKs (Otto et al., 2002). Production of MIP-2 by
staurosporine in rat peritoneal neutrophils is mediated through both p38 and ERK pathways
(Xiao et al., 1999). Also, p38 kinase plays an important role in LPS-induced murine
pulmonary inflammation in which MIP-2 draws neutrophils into airways (Nick et a1. , 2000).
In that study, inhibition of p38 kinase by M39, a novel p38 kinase inhibitor, blocked MIP-2
and TNF-a production in vitro and, furthermore, systemic inhibition of p38 kinase
significantly decreased in neutrophil accumulation and the release of TNF-a in the airspaces.
Thus, firrther study is needed to relate MAPK activation to trichothecene-induced MIP-2
expression and potential downstream pathologic effects.

MIP-2 is considered to be a fimctional analog of human IL-8 (Sherry et al., 1992).
Interestingly, VT readily induces IL-8 production in a clonal human macrophage U-937 cells
(Sugita-Konishi and Pestka, 2001). Neutrophil recruitment to sites of inflammation due to
IL-8 is involved in adult respiratory stress syndrome, idiopathic pulmonary fibrosis,
rheumatoid arthritis, and hepatic ischemia-repercussion injury (Lynch et al., 1992; Donnelly
et al., 1994; Koch et al., 1994; Colletti et al., 1995). Increased IL-8 expression may be related
to progression of immunoglobulin A nephropathy (IgAN). For example, a significant
increase of lL-8 in sera of IgAN patients was detected compared to healthy controls. IgA
autoantibodies to IL—8 are frequently detected in IgAN patients (Lai et al. , 1996). In addition,
Sekikawa et al., (1998) observed the expression of IL- 8 both in its transcript and the protein
levels in renal biopsy specimens obtained from patients with IgAN and lupus nephritis. The

expression of IL-8 mRNA was significantly correlated to the number of neutrophils in the

91

glomerulus. In our laboratory, a VT-induced mouse model of IgAN has been used to study
progress of this disease as well as cellular and molecular mechanisms of trichothecene-
induced immunotoxicity. As a functional analog of human IL-8, MIP-2 may play an
important role in progression of VT-induced IgAN in the mouse.

C3aR is a transmembrane receptor that is functionally coupled to G proteins (Roglic
et al., 1996), and is distributed in both myeloid cells (monocytes/macrophage, neutrophils
and eosinophils) and nonmyeloid cells (mast cells, glial cells, astrocytes, and smooth muscle
cells) throughout various tissues including lung, heart, kidney, liver, testis, and brain (Legler
et al., 1996; Martin et al., 1997; Gasque et al., 1998; Davoust et al., 1999; Sayah et al., 1999).
C3aR is activated upon binding C3a ligand, which is one of the anaphylatoxins produced by
proteolytic cleavage during complement activation (Hugli, 1990). C33 ligand, on binding
the C3aR, mediates numerous biological responses including lysosomal enzyme secretion
from neutrophils, histamine release from mast cells, smooth muscle contraction, and
modulation of the humoral and cellular immune responses (Glovsky et al., 1979; Showell et
al., 1982; Stimler et al., 1983; Morgan, 1986).

C3a can modulate immune response by stimulating or suppressing cytokine
production. When the effects of C3a on IL-6 gene expression and protein production were
studied in the presence of LPS in human PBMC (Fischer et al., 1999), both IL—6 mRNA and
its protein were increased with a co-treatment of C3a and LPS whereas IL-6 was not
enhanced with C33 alone. In a study with human astrocytomas, stimulation by C3a enhanced
IL-6 mRNA level and protein level while IL-lB, TNF-a and TGF-B mRNA expression

remained unchanged (Sayah et al. , 1999). IL-6 mRN A expression was blocked by treatment

92

of pertussis toxin or C3aR antibody, which indicates that the IL-6 response was related to
C3a. Regulation of B cell functions by C3a was studied by measuring IgG, TNF-oz, and IL-6
production in Staphylococcus aureus Cowan strain I (SAC)/IL—2-activated B cells (Fischer
and Hugli, 1997). Incubation of SAC/IL-Z-activated B cells with C3a suppressed polyclonal
immune response, IL-6 and TNF-or production. Monsinjon et al. (2001) studied the effect
of C3a on IL-8 production in the epithelial cell line ECV 304. Binding of C3a to the
epithelial cells resulted in H.-8 production in time- and dose dependent manners.
Pretreatment of ECV 304 cells with pertussis toxin inhibited C3a-induced IL-8 mRNA and
its protein. Taken together, C3a has immunomodulatory effects upon binding C3aR in
various cell types.

C3aR expression may potentiate or attenuate pathologic responses. For example,
expression of C3aR plays a role in lung inflammation. When mice are treated with LPS or
ovalbumin (OVA), both C3aR protein and mRNA Significantly increase in bronchial
epithelial and smooth muscle cells fi'om mouse lung (Drouin et al., 2001). These
observations suggest the possible involvement of C3aR in the pathology of diseases such as
sepsis and asthma. On the other hand, in a study with targeted-disruption of C3aR in mice,
C3aR played an anti-inflammatory role in endotoxemia (Kildsgaard et al., 2000). In that
study, C3aR"‘ mice were i.v. challenged with increasing doses of LPS and displayed
increased mortality to endotoxin-induced shock compared to their wild-type mice. Plasma
IL-IB levels were significantly elevated in the C3aR‘" mice compared with wild-type
littennates after LPS challenge. This latter result indicates that C3aR attenuates the

pathological effects of septic shock. Thus, the role of C3aR may function in both pro- and

93

anti-inflammatory manners in models of shock.

Although the observation that VT and SG, two protein synthesis inhibitors (PSls),
increased MIP-2 protein levels is paradoxical, several studies support the capability of
translational arrest by PSI to induce early response genes including cytokines. For example,
VT enhances expression of cytokine mRN A in vivo (Azcona-Olivera et al., 1995 a; Zhou et
al., 1997) and in vitro (Dong et al., 1994; Wong et al., 1998). Cycloheximide, a prototype
protein synthesis inhibitor, also induces cytokine gene expression (Dong et al., 1994;
Azcona-Olivera et al., 1995b). In those studies with VT and/or cycloheximide, elevated
cytokine mRNA levels by PSI contribute to subsequent increase in cytokine protein levels.
Increased cytokine gene expression by PSIs can be partly explained by both transcriptional
and post-transcriptional mechanisms. Transcriptional up-regulation might result from
inhibition of labile negative regulator synthesis (Fong et al., 1995; Ye and Young, 1997;
Yang and Pestka, 2002). At the post-transcriptional level, increased cytokine mRNA
stabilities by VT have been reported (Li et al. , 1997; Wong et al., 2001). This phenomenon
can be explained by inhibition of synthesis of labile RN ases that facilitate RNA degradation
(Shaw and Kamen, 1986) and/or labile proteins that bind 3'-untranslated region to regulate
mRNA degradation (Baker et al., 2000; Chambers and Kacinski, 1994; Madireddi et al.,
2000). Similarly, VT and SG-induced MIP-2 production in this study may be related to
transcriptional and post-transcriptional controls by the PSIS.

In summary, we have isolated VT- and SG-responsive genes, MIP-2 and C3aR, from
a murine macrophage cell line using DD-PCR. These findings provide further insight into

potential mechanisms of trichothecene-induced tissue injury and immunotoxicity. Additional

94

studies of the mechanisms MIP-2 and C3aR induction by trichothecenes are necessary to
understand their roles in immunopathological effects of the toxins. In addition, DD-PCR is
subject to a number of difficulties, most notably the use of many multiple primer
combinations as well as the generation of many clones with no known relationship to
existing genes. Further study of trichothecene-altered gene regulation could be greatly
enhanced by application of microarray technology which has evolved significantly since the

onset of this DD-PCR study.
ACKNOWLEDGMENTS

This work was supported by Public Health Service Grants ES—0335 8 (JJP) and ES-

09521 (J JP) and by the Michigan Agricultural Experiment Station.

95

CHAPTER V

ROLE OF MITOGEN-ACTIVATED PROTEIN KINASES IN VOMITOXIN-

INDUCED TNF-a MESSENGER RNA EXPRESSION IN MURINE

MACROPHAGE RAW 264.7 CELLS

96

ABSTRACT
Upregulation of proinflammatory cytokine gene expression by vomitoxin (VT,
deoxynivalenol) is transcriptionally and post-transcriptionally controlled. To better

understand the molecular basis for this observation, the effects of VT-induced mitogen-
activated protein kinases (MAPKS) on TNF-on gene expression were studied in murine
macrophage RAW 264.7 cells. VT was found to induce phosphorylation of p38 kinase,
extracellular signal-regulated kinases (ERKS), and c-Jun amino terminal kinases (INKS) in
a dose-dependent manner. Induction of TNF-on mRN A expression was significantly reduced
in lipopolysaccharide (LPS) plus VT treated cells at 3 hr in the presence of the p38 kinase
inhibitor, SB 203580 (2 pM), or the MEK-ERK inhibitor, PD 98059 (10 uM), whereas the
induction of TNF-on mRNA was not affected in the presence of INK inhibitor, SP 600125
(1 1.1M). To investigate the role of MAPKS on VT-enhanced TNF-on promoter activity, a
luciferase reporter gene driven by murine TNF-on promoter was used. LPS+VT-induced TNF
reporter gene expression was significantly reduced at 12 hr in the presence of 0.2 to 2 uM
of SB 203580, 100 uM of PD 98059, and 10 uM of SP 600125. To study the effects of
MAPKS on VT-induced TNF-0L mRNA stability, cells were exposed to VT and LPS in
asynchronous fashion. In the asynchronous model, cells were pretreated with LPS (1 ug/ml)
for 4 hr, medium removed, and medium containing a transcription inhibitor, 5,6-dichloro-
beta-D-ribofuranosyl-benzimidazole (DRB), MAPK inhibitors, and/or VT (250 ng/ml) were
added. VT-induced TNF-or mRN A stabilization was abolished in the presence of SB 2035 80

(2 uM) while the stabilization by VT was not affected in the presence of PD 98059 (10 pM)

97

or SP 600125 (1 pM). To determine whether activation of MAPKS was involved in the
regulation of LPS+VT-induced TNF—or production, cells were incubated with LPS (100
ng/ml), VT (250 ng/ml) or LPS (100 ng/ml)+VT(250 ng/ml) for 18 hr in the presence of

inhibitors. ELISA of supernatant revealed that LPS+VT-induced TNF-or production was
significantly reduced with 0.2 to 20 uM of SB 203580, 1 to 100 uM of PD 98059, or 0.1 to
10 uM of SP 600125. These results demonstrate that activation of MAPKS by VT are critical

in VT-induced TNF-a expression in macrophage cells.

INTRODUCTION

Trichothecenes are a group of structurally related sesquiterpenoid metabolites
produced by various strains of fungi including F usarium and Stachybotzjys (Grove, 1988;
Grove, 1993; Grove, 1996). These mycotoxins bind to eukaryotic ribosomes and
consequently inhibit protein synthesis (Ueno, 1983, 1984). More than 180 trichothecenes
have been identified, and of these, deoxynivalenol (vomitoxin, VT) is one of the most
common contaminants in cereal grains (Rotter et al., 1996). VT is resistant to degradation
during processing (Jackson and Bullerman, 1999) and, therefore, is often encountered in
human and animal food. Notably, VT can alter immune function experimentally (Bondy and
Pestka, 2000).

Depending on dose and frequency of exposure, VT can be both immunosuppressive
and immunostimulatory. Irnmunosuppression by high doses of VT is manifested by rapid
onset of leukocyte apoptosis (Islam et al. , 2002; Pestka et al. , 1994; Yang et al. , 2000; Zhou

et al., 2000; Zhou et al., 1999). On the other hand, low doses of VT exposure enhance

98

expression of various cytokines in vitro and in vivo (Yan et al., 1997; Yan et al., 1998; Zhou
et al., 1997; Azcona-Olivera et al., 1995a; Li et al., 1997; Wong et al., 1998). Exposure to
VT increases IL-2, IL-4, IL-5 and IL-6 mRNA and protein levels in T cells (Ouyang et al.,
1996; Warner et al., 1994). Superinduction of proinflarnmatory cytokines such as lL-6,
TNF-or, and IL- 1 [3 has also been observed in VT-treated macrophages (Miller and Atkinson,
1986; Wong et al. , 1998). Acute exposure of mice to VT increases IL-6, TNF-0., IL-l B, and
interferon (IFN)-y mRNA expression (Azcona-Olivera et al., 1995b; Zhou et al., 1997,
1998).

Upregulation of proinflammatory cytokines such as TNF-a and IL-6 plays an
important role in VT-induced pathologic consequences. TNF-or is a key cytokine in
triggering various physiological and pathological processes. For example, TNF-or mediates
biological responses such as cell death, cytokine and chemokine induction, antiviral activity,
and sepsis (Beutler, 1999; Sedgwick et al. , 2000; Smith et al. , 1990). IL-6 produced by VT-
stimulated macrophages in Peyer's patch drives IgA production from B cells (Yan et al.,
1998) and this overproduction of IL-6 is strongly suspected to contribute to dysregulation of
IgA production (Dong etal., 1991; Pestka et al., 1989). Furthermore, IL-6-deficient mice
are refractory to IgA dysregulation by dietary VT (Pestka and Zhou, 2000). This
dysregulation of IgA production is highly analogous to human IgA nephropathy (D'Amico,
1987)

Although the exact mechanisms for superinduction of proinflammatory cytokine
genes by VT are not fully understood, studies with RAW 264.7 murine macrophage cells to

VT have indicated the following effects: increased binding activities of transcription factors

99

such as AP- 1 , NF-KB, NF—IL6 (Wong et al. , 2002); increased IL—6, TNF-or, and IL-1 [3 mRN A
expression (Wong et al. , 1998); stabilization of IL-6 and TNF-or mRNA (Wong et al. , 2001);
and increased soluble IL-6 and TNF-or in culture supematants (Wong et al. , 1998). Recently,
VT was found to induce activation of mitogen-activated protein kinases (MAPKS) (Moon
and Pestka, 2002; Yang et al. , 2000) which may contribute to both activation of transcription
factors and mRNA stability.

MAPKS transduce extracellular Signals to intracellular responses in leukocytes and
other cells. Three distinct, but partially “cross-talking” MAPKS have been characterized in
the mammalian system (Tibbles and Woodgett, 1999). These include the extracellular
signal-regulated kinase (ERKS or p42/p44), the c-Jun amino-terminal kinase (JNKS, also
known as stress-activated protein kinases, SAPK), and p38 MAPK. The MAPK signaling
pathways consist of a series of kinases which sequentially activate and consequently
phosphorylate downstream kinases to transduce extracellular stimuli into intracellular
responses. Typical MAPK cascades involve the activation of a MAP kinase kinase kinase
(MAPKKK), which phosphorylates and activates a MAP kinase kinase (MAPKK) which in
turn phosphorylates MAPK(S). Then, activated MAPKS phosphorylate downstream cellular
substrates such as transcription factors and other kinases (Kyriakis and Avruch, 2001). The
major three MAPKS are known to be involved in TNF-or expression following treatment of
macrophages with bacterial lipopolysaccharides (LPS) in transcriptional and post-
transcriptional manners (Brook et al. , 2000; Mahtani et al., 2001; Swantek et al. , 1997; Zhu
et al., 2000).

The purpose of this study was to test the hypothesis that l NF-a expression by VT is

100

mediated by differential activation of p38 kinase, ERKS and/or INKS. Specifically, the
effects of VT on MAPK activation, as well as the relationship of MAPKs to VT-induced
mRNA expression, TNF—or promoter activity, TNF-or mRNA stability, and TNF-or protein

production were assessed in RAW 264.7 murine macrophage cells.

METHODS AND MATERIALS

Reagents and plasmids. All chemicals were reagent grade or better and were
purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise noted. The p38
MAPK inhibitor SB203580, MEKl inhibitor PD98059, and INK inhibitor SP600125 were
purchased from Calbiochem (San Diego, CA). A transcriptional inhibitor, 5,6-dichloro-beta-
D-ribofuranosyl-benzimidazole (DRB) was purchased from F luka Co. (Madison, WI).
Murine TNF-oz promoter luciferase reporter construct (pCDNA-tnf-luc) was kindly provided
by Dr. W. Zhu (The Scripps Research Institute, La Iolla, CA). The murine TNF promoter
construct received was originally derived from a XEMBL3 genomic clone containing most
of the mouse (strain C57B1/6) TNF locus (Shakhov et al., 1990). A genomic fragment
(2,223-nt fragment containing the distal coding sequence of lyrnphotoxin [exon IV] and 28
nt upstream of the initiation site within the TNF S'—untranslated region) was cloned to the
luciferase coding sequence in pcDNA3 vector (Beutler and Brown, 1991; Zhu et al., 2000).
Beta-galactosidase reporter construct (pCMV-gal) was kindly provided by Dr. T.
Zacharewski (Michigan State University, East Lansing, MI). All plasmids were purified with
Endo-Free Plasmid Prep Kit (Qiagen, Valencia, CA).

Cell culture. The murine macrophage RAW 264.7 cell line was obtained from the

101

American Type Culture Collection (Rockville, MD). Cells were maintained at 37 °C in a 6%
CO2 humidified incubator in Dulbecco’s Modified Eagles Medium (DMEM) supplemented
with 10% (v/v) heat inactivated fetal bovine serum (F BS; Atlanta Biologicals Inc., Norcross,
GA), 100 U/ml penicillin, and 100 ug/ml streptomycin (Gibco BRL).

For MAPK studies, cells (2.5 x 105 cells/ml) were incubated in 60 mm tissue culture
plates (Fisher Scientific Co., Corning, NV) in a volume of 6 ml for 24 hr. Supernatant was
replaced with medium containing LPS (Salmonella typhimuriumJ 00 ng/ml), VT (250 ng/ml)
or LPS (100 ng/ml) + VT (250 ng/ml). Cells were incubated for intervals, and then subjected
to lysis for MAPK activation assay. For dose-dependency experiments, media containing
LPS (100 ng/ml), VT (100 to 1000 ng/ml), and combination of LPS and VT were added and
cells analyzed as described above.

For mRN A induction studies, cells (2.5 x 105 cells/ml) were incubated in 6 well tissue
culture plates (Fisher Scientific Co., Corning, NV) in a volume of 1.5 ml for 24 hr.
Supernatant was replaced with media containing LPS (100 ng/ml), VT (100 - 500 ng/ml) or
a combination of LPS and VT. Cells were incubated for 2 and 7 hr and total RNA was
extracted.

For mRNA stability studies, cells were pretreated as described above. Supernatant
was replaced with media containing LPS (1000 ng/ml) and incubated for 4 hr. Then, media
containing DRB (50 uM), VT (250 ng/ml), and/or MAPK inhibitor were added. Cells were
incubated for various time intervals and then subjected to total RNA isolation.

For TNF-or production studies, cells (2.5 X105 cells/ml) were cultured for 24 hr in

flat-bottomed 24-well tissue culture plates (Fisher Scientific Co.) with each well containing

102

500 pl of cell suspension. Then, supematants were replaced with medium containing MAPK
inhibitors and VT (250 ngml) and/or LPS (100 ng/ml). After 18 hr incubation, supematants
were subjected to enzyme-linked immunosorbent assay (ELISA).

MAPK phosphorylation by Western blot analysis. Cells were washed with ice-
cold phosphate buffer, lysed in lysis buffer (1% [w/v] SDS, 1.0 mM sodium ortho-vanadate,
and 10 mM Tris, pH7.4), and sonicated for 10 seconds. After determination of protein
concentration using Bio-Rad Dc protein assay kit (Bio-Rad, Hercules, CA), equal amounts
of protein were resolved by SDS-PAGE (10%[w/v] gel). Proteins were then transferred to
polyvinylidene difluoride (PVDF) membrane (Du Pont, Boston, MA). Membranes were
blocked either 1 hr at room temperature or overnight at 4 °C in 5% (w/v) bovine serum
albumin (BSA) in TBST (20 mM Tris-HCl, pH 8, 137 mM NaCl containing 0.1% (v/v)
Tween 20). After two washes in TBST, membranes were probed with specific phospho-p3 8,
phospho-ERK, or phospho-INK antibodies (New England Biolabs, Beverly, MA) diluted in
5% BSA in TBST overnight at 4 °C. After 4 washes, membranes were incubated with
horseradish peroxidase-conjugated secondary antibody for 1 hr, and blots were developed
using an ECL chemiluminescent detection kit (Amersharn Pharrnacia Biotech, Piscataway,
NJ).

RNA extraction. Total RNA was extracted from RAW 264.7 cells by the method
of Chomczynski and Sacchi (1987) (Chomczynski and Sacchi, 1987) using TrizolTM reagent
(Gibco BRL) according to the manufacturer’s instructions.

Ribonuclease protection assay. Total RNA (5 or 10 ug) was used for cytokine

expression analysis by multiprobe RPA (Rionuant mCK3b template set; Pharmingen).

103

Assays were performed according to the manufacturer's instruction. Briefly, radioactive
riboprobes were synthesized at 37 °C for 1 hr in vitro from 50 ng of linearized plasmids with
T7 RNA polymerase in the presence of unlabeled ATP, CTP, GTP, UTP and [a-32P]UTP
(PerkinElmer Life Sciences, Boston, MA) using an In Vitro Transcription Kit (Pharmingen).
The probes were digested with DNase I (2000 units/ml) at 37 °C for 30 min, extracted with
phenol: chloroform: isoamyl alcohol (50:50: 1) and chloroformzisoamyl alcohol (50:1),
precipitated with ammonium acetate and ethanol, and washed with ethanol. The purified
antisense RNA probes were dissolved in hybridization buffer. A quantity of 5 or 10 pg of
total RNA from samples was incubated at 56 °C for 12-16 hr in a hybridization buffer with
more than 6 x 10" cpm/reaction. After hybridization, RN ase digestion with Phanningen RPA
RNase A (0.25 U/ul) and T1 (10 U/ul) was carried out for 45 min at 30 °C. The undigested
(protected) RNA was precipitated with ethanol, separated on 6% (w/v) polyacrylamide gel
electrophoresis with 8 M urea at 50 watts constant power for 1 hr. The gel was dried and
subjected to band analysis using Molecular Irnager F X (Bio-Rad). Bands were quantified
using Quality One Software (Bio-Rad). The cytokine template set (mCK3b) included ten
cytokines, TNF-B, lymphotoxin (LT)-B, TNF-or, IL—6, interferon (IFN)-y, IFN-B,
transforming growth factor (TGF)-Bl, TGF-BZ, TGF-B3, macrophage inflammatory factor
(MIF), and two housekeeping genes, L32 and GAPDH, with the following lengths of RNA
transcripts: 389, 351, 316, 284, 257, 232, 208, 191, 171, 154, 141, and 125 bases,
respectively.

Real Time PCR. Total RNA was analyzed to detect TNF-or mRNA level using an

ABI PrismTM 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA).

104

The levels of 18S rRNA were also measured as an indigenous control. Specific primers and
probes for TNF-on and 188 were obtained from PE Appllied Biosystems. Concentrations
used for primers and probes were 200 and 100 nM, respectively, in each reaction. Sixteen
pl of master mix comprising 20 x TNF-0L primers and probe, 20 x 183 primers and probe,
predeveloped assay reagents for PCR and RT (PE Applied Biosystems) was added to 9 pl
of samples containing 100 ng total RNA. Reverse transcriptase and polymerase reactions
were performed in the same tubes. Relative quantities of TNF—0L mRN A and 18S rRN A were
calculated using the comparative threshold cycle number for each sample fitted to standard
curves for TNF-on mRNA and 18S rRNA, respectively, generated according to
manufacturer’s recommendations. Expression levels for TNF-or mRN A in each sample were
normalized to 18$ rRNA.

Cell transfection. RAW 264.7 cells were grown to 80 % confluence and then
transfected with plasmids by the electroporation method of Stacey et al., (1993). Briefly,
cells were harvested and resuspended at a concentration of 3.0 x 107 cells/ml in DMEM with
10% (v/v) heat-inactivated FBS supplemented with 100 U/ml penicillin, and 100 pg/ml
streptomycin (Gibco BRL). Two hundred microliter medium containing 5 x 106 cells was
transferred to 0.4 cm electroporation cuvettes (Bio-Rad) with 44 pl of TE (10mM Tris, 1 mM
EDTA, pH 7.4) buffer containing plamid DNA (3 pg of pCDNA-tnf-luc, 3 pg of pCMV - gal,
and 5 pg of control vector [pCDNA]). Cuvettes were held at room temperature for 5 min and
electroporated at 960 pF, 300 V using a Bio-Rad Gene Pulser with capacitance extender.

These conditions generated a time constant of 46-48 ms.

105

Luciferase and B-galactosidase assay. Transfected cells were diluted in 12 ml
culture medium, plated in 12 well culture plates (1 ml/well), and incubated 24 hr.
Supernatant was removed and fresh media containing MAPK inhibitors and LPS (100 ng/ml)
and/or VT (250 ng/ml) were replaced and incubated 12 hr. Cells were then washed with ice-
cold PBS once and lysed with 200 pl of lysis buffer (Promega, Madison, WI). After a brief
centrifugation, supernatant was subjected to luciferase and B-galactosidase assay. Luciferase
activity was measured with a luminometer (Turner Designs Co. Model 20e, Sunnyvale, CA)
after brief mixing of supernatant with luciferase assay substrate solution (Promega). For B-
galactosidase assay, equal volmnes of supernatant and 2x assay buffer (Promega) were mixed
in a 96 well culture plate and incubated at 37 °C for 30-60 min. After stopping reaction by
adding 1M Tris buffer, absorbance was read at 405 nm on a microplate reader (Molecular
Devices, Menlo Park, CA). Luciferase activity was normalized against B-galactosidase
activity by dividing activity of luciferase by activity of B-galactosidase.

TNF-a production. Supematants were collected at 18 hr and assayed for TNF-a
by ELISA using Opt BIATM mouse TNF-on set (Pharmingen, San Diego, CA) according to
manufacturer's instruction.

Statistics. A one-way analysis of variance (ANOVA) using Bonferroni’s test for
parametric data or a student's t-test was performed with Sigma Statistical Analysis System
(Jandel Scientific, San Rafael, CA). A p value of less than 0.05 was considered statistically

significant.

106

RESULTS

VT induces TNF-or, IL-6, and IL-IB mRNA in the presence of LPS in RAW 264.7
macrophage cells (Wong et al. , 1998). To assess the effect of VT+LPS on activation of p38
kinase, ERK1/2, and INKl/2, RAW 264.7 cells were incubated with LPS (100 ng/ml), VT
(250 ng/ml), or LPS+VT over a 8 hr period and whole cell lysates were subjected to Western
blot analysis (Figure 5.1). Since activation of MAPKS requires specific phosphorylation at
threonine and tyrosine residues, antibodies specific for phosphorylated p38, ERK1/2, and
INK1/2 were used to detect their activation. As shown in Fig 5.1, LPS induced
phosphorylation of p38 kinase and JNKl/Z with a maximal activation at 30 min and
diminished their activation at 2 hr. Activation of ERK1/2 was maximal at 30 min and
prolonged with a lesser activation until 8 hr. These results were consistent with observations
from Chen and Wang (1999). VT alone induced phosphorylation of ERK1/2 and INK1/2
with a maximal activation of at 30 min and diminished their activation at 2 hr. Activation
of p38 kinase was maximal at 30 min and prolonged with a lesser activation until 8 hr.
Similar results were observed at a study of Moon and Pestka (2001) (Moon and Pestka,
2002). Cotreatrnent of LPS and VT induced activation of all three MAPKS with a peak
activation at 30 min and prolonged their activation until 8 hr for p38 kinase and ERK1/2 and
2 hr for INK1/2.

For dose-dependency experiments, RAW 264.7 cells were incubated with LPS (100
ng/ml), VT (100 -1000 ng/ml), or LPS (100 ng/ml)+VT(100 - 1000ng/ml) for 2 hr and whole
cell lysates were subjected to Western blot analysis (Figure 5.2). VT induced dose-

dependent activation of all three MAPKS whereas cotreatment of LPS and VT also induced

107

LPS VT LPS + VT

 

p-p38 —’ 7"- ---- “--.~_-..

1’43“ 3 m: = .. 3:2:
—> on. - -- .—
-JNK ..
p —' .2: — — :2 ”-
Time(hr) 0 0.5 2 4 8 O 0.5 2 4 8 0 0.5 2 4 8

Figure 5.1. Time-dependent activation of p38, ERK1/2, and INK1/2 by LPS, VT and
LPS+VT in RAW 264.7 macrophage cells. Cells were treated with 100 ng/ml of LPS, 250
ng/ml of VT or cotreatment of LPS and VT for a period of times. Whole cell lysates were
prepared and subjected to Western blotting using antibodies specific for phosphorylated form
of p3 8, ERK1/2, or INK1/2 as described in Materials and Methods. Data are a representative
of two independent experiments.

108

LPS — + — +

 

'" _ O to O O o tn 0 O
._ N In .— — N n --
p-p33 —> «Imp—-m-M
p-ERK

 

p-INK

Figure 5.2. Dose-dependent activation of p38, ERK1/2, and INK1/2 by VT and LPS+VT
in RAW264.7 macrophage cells. Cells were treated with 100 ng/ml of LPS as control, VT
(100-1000 ng/ml) or LPS (100 ng/ml)+VT (100-1000 ng/ml) for 2 hr. Whole cell lysates
were subjected to Western blotting using antibodies specific for phosphorylated form of p3 8,
ERK1/2, or JNK1/2 as described in Materials and Methods. Data are a representation of two
independent experiments.

109

phosphorylation of three MAPKS with Slightly increased activities compared to VT alone
treatment.

To investigate and confirm genes affected by VT and cotreatment with LPS, RAW
264.7 cells were incubated with LPS, VT, and LPS+VT for 2 and 7 hr and total RNA was
subjected to ribonuclease protection assay using multiprobes as described in material and
methods (Figure 5.3A). LPS (100 ng/ml) alone induced LT-B, TNF-on, IL-6, IFN-y, TGF-B 1,
and TGF-B3 mRNA at both 2 and 7 hr. Induction of these genes was consistent with other
studies for LT-B (Browning et al., 1997), TNF-or, IL-6 (Wong et al., 1998), IFN-y (Wang et
al., 2000), TGF-Bl , and TGF-B3 (Gosiewska et al., 1999). VT alone also induced TNF-a,
IL-6, IFN-y, TGF-Bl, and TGF-B3 mRNA at 2 hr but much less at 7 hr. These results are
consistent with previous studies for TNF-or, (Azcona-Olivera et al., 19953; Wong et al.,
1998), IL-6, IFN-y (Azcona-Olivera et al., 1995a; Zhou et al., 1997), TGF-B (Azcona-
Olivera et al. , 1995a). Cotreatrnent of LPS and VT enhanced the induction of TNF-or, IL-6,
IF N-y, TGF-B l , and TGF-B3 mRN A at both 2 and 7 hr. TNF-or mRN A was most profoundly
induced by both LPS and VT at 2 and 7 hr in RAW 264.7 cells. After normalization with
GAPDH mRN A, relative intensities of TNF-or mRNA were described in Figure 5.3B. VT
dose-dependently induced TNF-on mRNA at 2 hr and lessened induction at 7 hr. When LPS
was cotreated with VT, TNF-or mRNA levels at 7hr remained as high as the levels at 2 hr.

To assess the effect of MAPK inhibitors on LPS+VT induced TNF-or mRNA
expression, RAW 264.7 cells were pretreated with SB 203580 (2 pM, p38 kinase inhibitor),

PD 98059 (10 pM, MEKl-ERK inhibitor), or SP 600125 (1 pM, INK inhibitor) for 30 min.

110

 

A $20
08‘ ¢°
0 2 hr 7 hr
TNF-B — .-
LT-B —-
TNF-a —- ” M ,, g .. LT-B
lL-6 ---I- U—w—“d' «Iv—u-“ c-TNF-a
IFN 7 —‘ , ":6
fl
lFN-B —u. ._ lFN-y
TGF-pz — -
TGF-B3 __.__ .- TGF-B1
MlF - ~ 0- TGF-BZ
.- TGF-B3
L32 — ..

“'""""""’"’ “9‘3-.. u.-- c-MIF
GAPDH—a ~~

p.3"'.'.a.‘~..'-m
-.---'Q.'-:"-..‘ «GAPDH

LPS(100ng/ml)—+-——+++—+_-_+++
VT(ng/mll — — 100 250 500100250 500- — 100250 500100 250 500

Figure 5.3.A. Cytokine mRNA expression in LPS-, VT-, and LSP+VT treated RAW264.7
cells. Cells were treated with LPS (100 ng/ml), VT (100-500 ng/ml), or LPS+VT for 2 and
7 hr. Total RNA was subjected to RNase protection assay using multiprobes as described
in Material and Methods. The results are a representative of two separate experiments.

111

 

 

 

 

 

 

 

 

 

 

250

<

E _2hr

5200‘ 1:17hr

?

LL

E150.

“5

Q

2100..

22

.5

m

.2

E 50‘

0

°‘ Ill 1

04 HID 'l . .~
\ o o o o o o
«0 o o o o 6: ,9
(s. N 63 '\ q,
6) 39$ 4"" 8‘ 94" Of" 9,4"
3 3 3

Figure 5.3.B. The bands of TNF-0t and GAPDH mRNA were quantified using
Phospholrnager and are shown as a relative intensity of control sample at 2 hr (set at 100
with LPS treatment at 2 hr).

112

Then, cells were incubated with LPS (100 ng/ml), VT (250 ng/ml), or LPS+VT for 3 hr.
Total RN As were isolated and subjected to real time PCR. In the presence of SB 203580 and
PD 98059, LPS+VT-induced TNF-or mRN A expression was significantly suppressed while
LPS-induced TNF-0t mRNA level was not affected (Figure 5.4). VT-induced TNF-or mRNA
expression was significantly suppressed in the presence of PD 98059.

The effects of MAPKS on TNF-or gene expression were further studied by
investigating TNF-0t promoter activity. TNF-(1 promoters have binding sites for AP-l and
other transcription factors which are known downstream targets of MAPKS (Garcia et al.,
1998; Hazzalin et al., 1998; Minden and Karin, 1997). VT induces AP—l, NF-KB, and NF-
IL6 binding activities in vitro (Li et al., 2000; Wong et al. , 2002). These increased binding
activities can potentially be regulated by MAPK activation (Chakraborti and Chakraborti,
1998; Hambleton et al., 1996; Koj, 1996). To investigate the role of MAPKS on VT-
enhanced TNF-or promoter activity, a luciferase reporter gene driven by murine TNF
promoter was used. LPS, VT, and LPS+VT all Significantly induced TNF-or promoter
luciferase activity in RAW 264.7 cells (Figure 5.5A, 5.5B, and 5.5C). Cotreatrnent of LPS
and VT Si gnificantly increased TNF-0t promoter activity compared to mean additive
response. As shown in Figure 5.5A, the p38 kinase inhibitor, SB203580 (0.2, 2, and 20 pM),
significantly inhibited LPS-, VT- and LPS+VT- induced TNF reporter gene expression in a
dose-dependent manner. Interestingly, MEK-ERK inhibitor, PD98059, significantly
increased LPS-induced TNF reporter gene expression (Figure 5 .SB). VT-, LPS+VT-induced

TNF reporter gene expression were significantly decreased with 100 pM of PD98059. The

113

 

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Figure 5.4. Effect of MAPK inhibitors on LPS-, VT-, and LPS+VT- induced TNF-or mRN A
expression in RAW 264.7 macrophage cells. Cells were pretreated with SB 203580 (2 pM),
PD 98059 (10 pM), or SP 600125 (1 pM) for 30 min, and then incubated with LPS (100
ng/ml), VT (250 ng/ml), or LPS+VT for 3 hr. Total RNAS were isolated and subjected to
Real Time PCR. Data are representative of two separate experiments. An asterisk indicates
a Significant difference from control with p value < 0.05.

114

 

 

 

 

 

 

 

 

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Figure 5.5. Effect of MAPK inhibitors on LPS-, VT-, and LPS+VT- induced TNF reporter
gene expression in RAW 264.7 macrophage cells. A, cells were transfected with murine
TNF reporter. After 24 hr, cells were incubated with LPS (100 ng/ml), VT (250 ng/ml), or
LPS (100 ng/ml)+VT (250 ng/ml) in the presence of p38 inhibitor, SB203580 (0.2, 2, and
20 mM) for 12 hr. Luciferase activities were measured. Cotransfected B-galactosidase was
used to normalize the transfection efficiency. Data are representative of two separate
experiments. B, the same conditions as in A except using PD98059 (1, 10, and 100 pM).
C, the same conditions as in A except SP600125 (0.1, 1, and 10 pM). An asterisk, T, or 1
indicates a significant difference from control group (no inhibitor), from control (no
treatment), from an expected mean additive effect with p value <0.05, respectively.

115

INK inhibitor, SP600125, significantly reduced LPS-induced TNF reporter gene expression
at 1 and 10 pM while it reduced LPS+VT-induced TNF reporter gene expression at 10 pM
(Figure 5.5C). VT-induced TNF reporter gene expression was not changed by the JNK
inhibitor.

VT can increase the stability of TNF-or mRNA (Wong et al., 2001). To determine
whether activation of MAPKS contributes to this increased stability, RAW 264.7 cells were
pretreated with LPS (1 pg/ml) for 4 hr. After washing with culture medium, cells were
treated with VT (250 ng/ml), p38 inhibitor (SB203580, 2 pM) in the presence of DRB, a
transcription inhibitor, over several time intervals. DRB treatment suppressed TNF-0t
mRNA expression at 30, 90, and 180 min, resulting in TNF-a mRNA half-life (tm) of 45
min (Figure 5.6A). GAPDH mRNA expression was not changed with DRB treatment
because of their long half life. VT at 250 ng/ml in the presence of DRB increased TNF-0L
mRNA stability, resulting in TNF-0t mRNA tm of more than 180 min. This result was
consistent with the finding of Wong et al., (2001). When p38 inhibitor, SB203580 (2 pM),
was coincubated with DRB and without VT treatment, TNFo mRNA levels were rapidly
reduced, resulting in TNF-0t mRNA t,,2 of 11 min. In other words, p38 inhibitor reduced
TNF-0L mRNA t,,2 from 45 min to 1 1 min. This result indicates that activation of p38 kinase
is required to stabilize TNF-0L mRNA. This observation is consistent with findings from
other studies indicating that p38 kinase is involved in stabilization of LPS-induced TNF-or
mRNA (Brook et al., 2000; Wang etal., 1999). However, increased TNF-0L mRNA t,,2 (>

180 min) by VT was reduced to 22 min in the presence of p38 inhibitor. When comparing

116

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Figure 5.6. Effect of SB203580 on VT-induced TNF-a mRNA expression in LPS-treated
RAW 264.7 cells. Cells were pretreated with 1 pg/ml of LPS for 4 hr and medium was then
removed. Fresh medium containing VT (250 ng/ml), DRB (50 pM), and/or SB203580
(2pM) was added to cultures and incubated for a period of times. Total RNA was subjected
to RNase protection assay using multiprobes including TNF-or. A, blot from RNase
protection assay. B, relative TNF-or mRNA levels after normalization with GAPDH gene
expression determined by densitometry of blot from RNase protection assay. C, differential
induction at each time points by subtracting percent remaining TNF-0t mRNA of DRB-
treated cells from percent remaining TNF-(X. mRNA of VT-treated cells. Data are
representative of two separate experiments.

117

differential percent remaining TNF-or mRNA in the absence of p38 inhibitor to those of
TNF-or mRNA in the presence of p38 inhibitor over time, the difference due to VT were
abrogated over a period of time in the presence of p38 inhibitor (Figure 5.6C), indicating that
VT-induced TNF-0L mRNA stability was impaired by p38 inhibitor.

The MEKl inhibitor, PD98059 (1 OpM), was used to investigate whether the ERK] /2
pathway is involved in mRN A stabilization. DRB treatment resulted in TNF-0t mRN A half-
life (tm) of 45 min (Figure 5.7A). VT increased TNF-or mRN A half life to greater than 180
min. When MEKI inhibitor was coincubated with DRB and without VT treatment, TNF-0L
mRNA stability were slightly reduced, resulting in TNF-0t mRNA tl,2 of 38 min. This
observation indicates that activation of ERK1/2 might be involved in stabilizing TNF-or
mRNA. Similar findings were reported that MEKl inhibitor destabilizes TNF-or mRNA
(Dumitru et al., 2000; Rutault et al. , 2001). Increased TNF-0L mRNA t1,2 (> 180min) by VT
was reduced to 60 min in the presence of MEKI inhibitor. When comparing differential
percent remaining TNF-0L mRNA in the absence of MEKl inhibitor to those of TNF-or
mRNA in the presence of MEKl inhibitor over time, the difference due to VT were not
changed over a period of time (Figure 5.7C), indicating that VT-induced TNF-or mRNA
stability was not likely affected by the presence of MEKl inhibitor.

The INK inhibitor, SP600125 (lpM), was used to investigate whether INK1/2
pathway is involved in mRN A stabilization. DRB treatment resulted in TNF-0t mRNA half-
life (tm) of 60 min (Figure 5.8A). VT increased TNF-0t mRNA stability, resulting in t,,2 of

more than 180 min. When INK inhibitor was coincubated with DRB and without VT

118

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Figure 5.7. Effect of PD98059 on VT-induced TNF-or mRNA expression in LPS-treated
RAW264.7 cells. All experimental conditions were the same as described in figure 5.6
legend except the inhibitor. PD98059, 10 pM was used instead. A, blot from RNase
protection assay. B, relative TNF-or mRNA levels after normalization with GAPDH gene
expression determined by densitometry of blot fiom RNase protection assay. C, differential
induction at each time points by subtracting percent remaining TNF-or mRNA of DRB-
treated cells from percent remaining TNF—0c mRNA of VT-treated cells. Data are
representative of two separate experiments.

119

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Figure 5.8. Effect of SP600125 on VT-induced TNF-a mRNA expression in LPS-treated
RAW264.7 cells. All experimental conditions were the same as described in figure 5.6
legend except the inhibitor. SP600125, 1 uM was used instead. A, blot from RNase
protection assay. B, relative TNF-a mRNA levels after normalization with GAPDH gene
expression determined by densitometry of blot from RNase protection assay. C, differential
induction at each time points by subtracting percent remaining TNF-oz mRN A of DRB-
treated cells from percent remaining TNF-0c mRNA of VT-treated cells. Data are
representative of two separate experiments.

120

treatment, TNF-0L mRNA stability was not affected. This observation indicates that
activation of JNK1/2 might not be required for stabilization of TNF-0L mRNA. When
comparing differential percent remaining TNF-a mRNA in the absence of JNK inhibitor to
those of TNF-0L mRNA in the presence of JNK inhibitor over time, the difference due to VT
were not much changed over a period of time (Figure 5.8C), indicating that VT-induced
TNF-a mRNA stability was not affected by the presence of JNK inhibitor.

To determine whether activation of p38 kinase, ERK1/2, or JNKl/Z was involved in
the regulation of VT-induced and LPS+VT-induced TNF-0L secretion, the p38 inhibitor
(83203580), the MEKI inhibitor (PD98059), or the JNK inhibitor (SP600125) was used,
respectively. RAW 264.7 cells were incubated with LPS (100 ng/ml), VT (250 ng/ml) or
LPS (100 ng/ml)+VT(250 ng/ml) for 18 hr in the presence of inhibitors. Cell culture
supematants were subjected to TNF-a assay by ELISA. As shown in Figure 5.9A and 5.98,
LPS-, VT-, LPS+VT- induced TNF-0L productions were significantly inhibited dose-
dependently at 18 hr in the presence of SB203580 and PD98059 in RAW 264.7 cells. In the
presence of INK inhibitor, SP600125, LPS- and LPS+VT-induced TNF-a production were
significantly and dose-dependently reduced at 1 8 hr whereas VT-induced TNF-0L production

was not affected.

DISCUSSION
VT superinduces various inflammatory mediators such as proinflammatory cytokines,

chemokines, and prostaglandins which have been suggested as major contributors to

121

 

 

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Figure 5.9. Effect of MAPK inhibitors on LPS-, VT-, and LPS+VT- induced TNF-a
production in RAW 264.7 macrophage cells. A, cells were incubated with LPS (100 ng/ml),
VT (250 ng/ml) or LPS (100 ng/ml)+VT(250 ng/ml) for 18 hr in the presence of 0.2, 2, and
20 uM of SB203580. TNF-0L levels in the supernatant were measured by ELISA. Data are
the means of triplicate culture supematants i SEM. The results are representative of two
separate experiments. B, the same conditions as in A except using PD98059 (l , 10, and 100
M). C, the same conditions as in A except SP600125 (0.1, 1, and 10 M). An asterisk
indicates a significant difference from control with p value < 0.05.

122

immunopathogenic events induced by the toxin (Bondy and Pestka, 2000; Chung et al.,
2002; Moon and Pestka, 2002). Induction of the inflammatory mediators are closely related
to activation of MAPKS (Dong et al. , 2002). Activation of MAPKS by VT has been observed
previously in RAW 264.7 cells (Moon and Pestka, 2002; Yang et al., 2000). In this study,
activation of MAPKS by VT was prolonged in the presence of LPS and contributed to
superinduction of proinflammatory cytokine, TNF-a, in both transcriptional and post-
transcriptional manners. These data suggest that MAPKS play a major role in TNF-0L
superinduction by VT in RAW 264.7 cells.

Cycloheximide (CHX), a prototype protein synthesis inhibitor, also superinduces the
inflammatory mediators. For example, cytokines such as interleukin (IL)-2, IL-5, and IL-6
mRNA and proteins are superinduced by CHX in phorbol 12-myristate 13-acetate (PMA)
stimulated EL—4 cells (Azcona-Olivera et al., 1995b; Dong et al., 1994). IL-8 is a potent
neutrophil chemoattractant and its mRN A and protein levels are superinduced by TNF-a and
LPS in the presence of CHX in lung epithelial cells and bone marrow-derived mononuclear
cells, respectively (Dibb et al. , 1992; Roger et al. , 1998). Those results are further explained
by increased transcript and increased mRN A stability followed by enhanced binding activity
of transcription factors such as AP-l and NF-KB (Roger et al., 1998). Cyclooxygenase-Z
(COX-2) is an important enzyme required for synthesis of prostaglandin which play an
important role in inflammation (Vane et al. , 1998). CHX mediates superinduction of COX-2
expression by increased transcript but not by mRNA stabilization in IL-lB-stimulated
pulmonary type 11 A549 cells (Newton et al., 1997). In that study, increased COX-2

transcript is suggested to be due to NF-KB binding to COX-2 promoter KB sites and

123

activation of JNK. Interestingly, VT superinduces the inflammatory mediators in a similar
manner; increased transcripts (Azcona-Olivera et al. , 1995b; Dong et al. , 1994; Wong et al. ,
1998), extended mRNA half life (Li et al., 1997; Wong et al., 2001), enhanced binding
activity of transcription factors (Li et al., 2000; Wong, 2000), and activation of MAPKS
(Moon and Pestka, 2002; Yang et al. , 2000). Thus, protein synthesis inhibitors seem to share
common pathways, increased binding of transcription factors and increased mRN A transcript
stabilization that ultimately contribute to superinduction of inflammatory mediators.

VT-induced MAPK activation may relate to a“ribotoxic stress response”. Iordanov
et al., (1997) proposed that translational inhibitors such as anisomycin, ricin, and a-sarcin
activate JNKI by binding to or altering the structure of 28S ribosomal RNA, resulting in a
“ribotoxic stress response”. Trichothecene-induced ribotoxic stress response is known to
activate JNK and p38 kinase and consequently induces apoptosis (Shifrin and Anderson,
1999). In addition, activation of ERK1/2, p38 kinase, and JNKl/2 by VT and satratoxins is
related to induction of COX-2 expression and apoptosis (Moon and Pestka, 2002; Yang et
al., 2000). Shiga toxin also triggers the ribotoxic stress response resulting from activation
of JNK1/2 and p38 kinase and consequently produce TNF-a in the human monocyte cell line
THP-l (Foster and Tesh, 2002). Taken together, the ribotoxic stress response by
translational inhibitors initiates activation of MAPKS resulting in apoptosis or inflammatory
responses. These observations are consistent with our data that activation of MAPK by VT
contributes to superinduction of TNF-a through the ribotoxic stress response.

One of major functions of MAPK is to activate transcription factors (Chang and

Karin, 2001; Davis, 2000). For example, p38 kinase phosphorylates activating transcription

124

factor-2 (ATP-2) (Zhu and Lobie, 2000) and Elk-1(Zhu and Lobie, 2000). JNK can also
phosphorylate many transcription factors including Elk-1 (Cavigelli et al., 1995), ATP-2
(Botteron and Dobbelaere, 1998), and c-Jun (Hambleton et al., 1996). ERK can induce
activation of Elk-1(Babu et al., 2000) and C/EBP-B (Zhu et al., 2002). ATF-2 binds to the
CAMP response element (CRE) (Yamada et al., 1997). Elk-1 is a c-fos regulatory
transcription factor which recognizes the AP-l binding site (Papavassiliou, 1994). c-Jun is
one of Jun family transcription factors which bind to the AP-l site (Hambleton et al. , 1996).
Activation of C/EBP-B leads to enhanced transactivation potential of the factor (Ramj i and
Foka, 2002). In addition, activation of nuclear factor KB (NF -KB) through phosphorylation
of IicBa is related to activation of all three MAPKS (Lee et al. , 1997; Schwenger et al. , 1998;
Zhao and Lee, 1999). The aforementioned AP-l, CRE, and NF-KB binding sites are known
to be important in cytokine gene expression in leukocytes. Consistent with these
possibilities, our laboratory recently reported that VT increased binding activity of AP-l
through activation of c-Jun and c-Fos, of NF-IL6 through activation of C/EBP-B, of NF-KB
through activation of p50 and c-Rel subunits (Wong et al. , 2002). Therefore, increased TNF-
a promoter reporter activity by VT and/or LPS in this study might be related to increased
binding activity of the transcription factors such as AP-l , NF-IL6, and NF-KB resulting from
activation of MAPKS since TNF-or promoter is known to possess binding sites for these
factors (Pauli, 1994).

Activation of MAPKS by VT might also contribute to stabilization of TNF-a mRN A.

The sequences in the 3'-untranslated region (3'-UTR) of TNF-or mRNA is responsible for

125

mRNA stability and translational repression (Chen and Shyu, 1995; Han et al., 1991). This
region contains multiple repeats of an AUUUA motif (AU-rich element, ARE), which are
also found in early response genes such as cytokines and protooncogenes (Chambers and
Kacinski, 1994; Schiavi et al., 1992; Wang et al., 1997). The ARES are known to be
recognized by several RNA binding proteins (Bohjanen et al., 1992; Zhang et al., 1993).
Recently, proteins, which bind to ARE of TNF-a mRNA, have been identified; as TIAR
(Gueydan et al. , 1999), HuR (Sakai et al., 1999), and tristetraprolin (TTP) (Lai et a1. , 1999).
TIAR is cytoplasmic protein and binds to TNF ARE independently of LPS stimulation of
macrophages. This protein might be involved in TNF translational repression (Gueydan et
al., 1999). HuR, a member of ELAV—like protein family, is important in translocation and
protection of ARE-containing RNA (Fan and Steitz, 1998). It is a TNF ARE binding protein
which stabilized TNF-a mRNA (Dean et al., 2001). In that study, HuR-mediated
stabilization was found to be independent on activation of p38 kinase. TTP is a zinc finger
RNA binding protein that is responsible for destabilization of TNF-or mRNA (Lai et al.,
1999). This protein is inducible by LPS in macrophages and phosphorylated by MAPK-
activated protein kinase-2 (MAPKAPK2 or MK-2) through activated p38 kinase (Mahtani
et al., 2001). The phosphorylated form of TTP is suggested to have less binding activity to
ARE RNA probe than dephosphorylated TTP (Carballo et al. , 2001). In our study, treatment
with the p38 inhibitor, SB 203580, greatly decreased TNF-or mRNA stability. This suggests
that inhibition of p38 kinase may block phosphorylation of TTP, causing avid binding of
dephosphorylated TTP to TNF ARE, resulting in rapid degradation of TNF-a mRNA.

MAPK inhibitors, SB 203580, PD 98059, or SP 600125 have been used to block

126

activation of p38 kinase, MEKl/Z (upstream of ERK1/2), or JNK1/2, respectively. Efficacy
of these inhibitors was tested by checking phosphorylated state of their substrate, ATP-2,
RSK”, or c-Jun for p38 kinase, ERK1/2 or JNK1/2, respectively, by Western blot analysis
on total cell lysate. At highest concentrations of inhibitors used (20 uM of SB 203580, 100
uM of PD 98059, and 10 uM of SP 600125), phosphorylation of these substrates following
LPS treatment was reduced by 40 to 50 %. Since the substrates of p38 kinase, ERK1/2, and
JNK1/2 can be also phosphorylated by alternate routes such as JNK for ATP-2 activation
(Tindberg et al., 2000), p38 kinase for RSK9O activation (Merienne et al., 2000), p38 kinase
for c-Jun activation (Y amagishi et al., 2001), it would not be possible to completely block
their phosphorylation. In order to accurately measure activities of MAPKS,
immunoprecipitation from whole cell extracts using MAPK-specific antibodies could be
applied.

Taken together, the results presented here indicate that VT may superinduce TNF-0L
expression by increasing its transcripts and mRN A stability through activation of MAPK.
Trichothecene-induced activation of MAPK may play a critical role in superinduction of
proinflammatory cytokine, TNF-0L, and succeeding pathologic events. Further research on
RNA binding protein, TTP, for stabilization is desirable to better understand VT-induced

cytokine superinduction.

127

CHAPTER VI

SUMMARY AND CONCLUSIONS

128

In this dissertation, firstly, high antibody titers to macrocyclic trichothecene,
satratoxin G (SG), were readily achieved upon immunization of rabbits with SG bis-
hemisuccinate conjugated to BSA. The antibodies could detect free SG using a CD-ELISA
with a range of detection from 0.1 to 100 ng/ml. The observed antibody cross-reactivity may
facilitate simultaneous detection of other satratoxins and to a lesser extent, other macrocyclic
trichothecenes. Methanol content up to 20% in samples did not largely affect the
immunoassay and this stability expands applicability of the antibodies. Secondly, the
capacity of representative macrocyclic trichothecenes to alter TNF-0t and IL-6 production and
viability was assessed in a murine macrophage model. Macrocyclic trichothecenes can
superinduce the proinflammatory cytokine, TNF-0t, at low cytotoxic concentrations, whereas
these compounds are cytotoxic and reduce cytokine production at higher concentrations.
These immunomodulatory effects were observed at relatively low (ng/ml) concentrations,
suggesting that macrocyclic mycotoxins may pose a hazard to humans exposed to
Stachybotrys. Thirdly, in order to expand understanding of how trichothecenes affect gene
regulation, the VT- and SG-responsive genes, MIP-2 and C3 aR, were isolated from a murine
macrophage cell line using DD-PCR. These findings provide further insight into potential
mechanisms of trichothecene-induced tissue injury and immunotoxicity. Additional studies
of the mechanisms MIP-2 and C3aR induction by trichothecenes are necessary to understand
their roles in immunopathological effects of the toxins. In addition, DD-PCR is subject to
a number of limitations, most notably the use of many multiple primer combinations as well
as the generation of many clones with no known relationship to existing genes. Further study

of trichothecene-altered gene regulation could be greatly enhanced by application of

129

microarray technology which has evolved significantly since the onset of this DD-PCR study.
Lastly, the effects of VT on MAPK activation, as well as the relationship of MAPKS to VT-
induced mRNA expression, TNF-or promoter activity, TNF-0c mRNA stability, and TNF-0t
protein production were assessed in RAW 264.7 murine macrophage cells. The results
indicate that VT may superinduce TNF-a expression by increasing its transcripts and mRN A
stability through activation of MAPK. Trichothecene-induced activation of MAPK may play
a critical role in superinduction of proinflammatory cytokine, TNF-0t, and succeeding
pathologic events. Further research on RNA binding protein, TTP, for stabilization is
necessary to better understand VT-induced cytokine superinduction.

Overall, the studies in this dissertation will contribute to understanding how
trichothecenes can modulate immune function. In the long run, results of these studies would

be used to reduce a health risk due to exposure to trichothecenes.

130

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131

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