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THESlS IMO This is to certify that the dissertation entitled Mechanisms for vomitoxin-induced cytokine superinduction in macrophages presented by Shu-shyan Wong has been accepted towards fulfillment of the requirements for Ph.D. . Food Science and Human degnwin Nutrition and Institute for Environmental Toxicology menu/Zr; Major professor Date July 5th, 2000 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan. State Univzarsity PLACE IN REfURN Box to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 8/01 cJClRC/DateOuepSS—pJS MECHANISMS FOR VOMITOXIN-INDUCED CYTOKINE SUPERINDUCTION IN MACROPHAGES By Shu-shyan Wong 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 2000 ABSTRACT MECHANISMS FOR VOMITOXIN-INDUCED CYTOKINE SUPERINDUCTION IN MACROPHAGES By Shu-shyan Wong Potential mechanisms for superinduction of cytokine expression by the trichothecene vomitoxin (VT) were assessed in the cloned macrophage cell line RAW 264.7. First, the effects of VT on secretion of IL— 1 B, IL-6 and TNF-a protein and expression of their mRN A were measured with and without lipopolysacchride (LPS) activation. Secreted TNF-a was significantly increased after 2 days exposure to VT with and without LPS activation. While VT had no effect on IL-6 secretion in the absence of LPS, it significantly increased IL-6 secretion in LPS-exposed cells. Secreted IL-lB was not detected in cells treated with or without activation. Immunostaining of intracellular cytokines in conjunction with flow cytometric analysis suggested that the percentage of the cells producing intracellular TNF-a were significantly increased by VT with and without LPS; whereas, VT increased IL-6 output in LPS-activated cells. Elevated TNF-a mRNA was observed in RAW 264.7 cells incubated with VT. IL-1 [3 and IL-6 mRNAs were increased in cells incubated with LPS. To assess the transcriptional effects of VT, binding activities of the transcription factors which enhance promoter functions of pro-inflammatory cytokine promoters AP-l, NF-KB and NF-IL6 (C/EBP) were studied by electrophorectic mobility gel-shift assay (EMSA). AP- 1 binding was enhanced by VT and specifically targeted the phosporylated c-Jun, JunB, c- Fos, and Fra-2 subunits of AP-l. VT increased p50 and c-Rel of NF-KB/Rel complexes in cells incubated with and without LPS. The binding activities of NF-IL6 (C/EBPa and C/EBPB) were also increased by VT. However when the effects of VT on the activity of the cis-acting elements of the promoter were evaluated by in RAW 264.7 cells with transiently transfected a plasmid containing a TNF-a promoter linked to a CAT reporter gene, VT was found to induce CAT mRN A expression slightly. To assess potential post-transcriptional effects of VT, stability of cytokine mRNAs were assessed by Northern blot analysis in RAW cells treated with the transcriptional inhibitor DRB. TNF-a and IL-6 mRNA half- lives were prolonged from 2- to lO-fold. Taken together, these results suggested that (l) VT is able to superinduce cytokine protein and mRNA expression in macrophages; (g) VT can increase of the binding activities of transcription factors (i) increases in TNF-oc transcription promoter activity were detectable but small; and (5) the ability of VT to stabilize TNF-a and IL-6 mRNA was marked. Therefore, elevated cytokine protein and mRNA expression may primarily attributable to post-transcriptional modulation by VT. Dedicated to my deceased yet unborn child -iv- ACKNOWLEDGMENTS First of all, I would like to thank my dissertation advisor Dr. James J. Pestka, for his guidance, support and assistance throughout my Ph.D. study. I also thank him for giving me the privilege to work in this laboratory. I also wish to thank my other committee members, Drs. John E. Linz, Joseph J. Schroeder, and Kathryn H. Brooks for their valuable advice and comments. Special thanks are expressed to Dr. Richard C. Schwartz for technical guidance in Northern hybridization, to Dr. Hui-Ren Zhou for technical assistance in RT-PCR, Southern analysis and EMSA and cooperation in research, to Dr. Maria L.Marin-Martinez for technical guidance in cell culture and cooperation in research, and to Dr. Hsien-Ming Hu for technical assistance in transfection assay. Thanks also given to all other people in the Food Microbiology laboratory and the Department of Food Science and Human Nutrition. I also thank Jim and Karen for their encouragement and spiritual guidance for all these years. I also value the friendships of Marvin and Shirley, Sarah, Vicky, Xiao-yen, Ching-Cheng, Wei-Kuo and Yi-Hsuan, F ang-Chen and Da-lih, Dora and C.Y., Hye-Wan, Marina and Toshihiro. My special appreciation goes to my husband Bob, for his support, encouragement and patience for all these years. I also like to express thanks to my parents for their support and understanding. Finally, I give thanks to Jesus Christ, my Savior and Lord. -v- TABLE OF CONTENTS Page INTRODUCTION ........................................................................................................... 1 CHAPTER 1. LITERATURE REVIEW ...................................................................... 6 A. TRICHOTHECENES .................................................................................. 7 A.l Introduction ....................................................................................... 7 A2 Vomitoxin ......................................................................................... 9 A3 Cycloheximide ................................................................................... 10 B. MACROPHAGES ......................................................................................... 1 1 Bl Introduction ....................................................................................... 1 1 B2 Macrophage activation ..................................................................... 1 1 B3 Macrophage and cytokines .............................................................. 12 B.4 Regulation of macrophage gene expression .................................... 13 B .4. 1 . Transcriptional control ........................................................ 13 3.4.2. Post-transcriptional control ............................................... 14 C. LIPOPOLYSACCHARIDES .......................................................................... 15 Cl Structure of lipopolysaccharides ....................................................... 15 C .2 Proteins that bind lipopolysaccharides .............................................. 17 C21. Lipopolysaccharide binding proteins (LBP) .................... 17 C22 Cluster differentiation antigen 14 (CD14) ........................ 18 C.2.3. Bactericidal/permeability increasing protein (BPI) ........... 18 C3 Signal transduction by lipopolysaccharides ...................................... 19 C.3.1. CD14 dependent pathway .................................................. 19 C32. CD14 independent pathway .............................................. 20 C4 LPS and cytokine induction ............................................................... 20 D. REGULATION OF IL-6 .................................................................................. 21 D. 1 Introduction ........................................................................................ 21 D2 IL-6 and its regulation ........................................................................ 22 D21. Activator protein-1(AP-1) .................................................. 22 D22 Nuclear factor KB/RCI (NF-KB) ......................................... 24 D23. CCAAT/enhancer binding proteins (C/EBP) .................... 28 -vi- D.2.4. Cyclic AMP response element binding protein (CREB)... 30 D25. Glucocorticoids (GC) as negative regulator ....................... 31 D3. Post-transcriptional control .................................................................. 31 D4. Summary ............................................................................................... 33 E. TNF-a and its regulation .................................................................................. 33 El Introduction .......................................................................................... 34 E2 Receptors and signal transduction pathways ..................................... 35 E.3 Regulation of TNF-or and gene expression ....................................... 38 F. IL-1 [3 and its regulation ...................................................................................... 39 F. 1 . Introduction ......................................................................................... 39 F .2. Receptors and signal transduction ..................................................... 41 F .3. IL- 1 B and its regulation ....................................................................... 42 G. Rationale for this research ................................................................................ 44 CHAPTER II. MODULATION OF IL-lB, IL-6 AND TNF-a SECRETION AND mRNA EXPRESSION BY THE TRICHOTHECENE VOMITOXIN IN THE RAW 264.7 MURINE MACROPHAGE CELL LINE ...................................... 47 ABSTRACT .......................................................................................................... 48 INTRODUCTION .................................................................................................. 49 MATERIALS AND METHODS .......................................................................... 51 Experiment Desrgns ....... 51 Cytokine Assay ......................................................................................... 51 Intracellular cyokine and flow cytometry staining of TNF-or and IL-6 ..... 52 Measurement of cytokine mRN A abundance ............................................. 53 Statistical analysis ....................................................................................... 57 RESULTS ............................................................................................................... 57 DISCUSSION ........................................................................................................ 62 CHAPTER III. EFFECTS OF VOMITOXIN ON ACTIVATION OF TRANSCRIPTIONAL BINDING ACTIVITIES OF CYTOKINES -vii- IN MACROPHAGE RAW 264.7 CELLS ..................................................................... 70 ABSTRACT .......................................................................................................... 71 INTRODUCTION ................................................................................................ 72 MATERLALS AND METHODS ............................................................................ 75 RAW 264.7 cell cultures ............................................................................ 76 Nuclear Extract Preparation ....................................................................... 76 Electrophoretic mobility gel shift assay (EMSA) ....................................... 77 RESULTS .............................................................................................................. 79 DISCUSSION ........................................................................................................ 95 CHAPTER IV. EFFECTS OF VOMITOXIN ON TRAN SCRIPTIONAL ACTIVITY OF CYTOKINE PROMOTER IN MURINE MACROPHAGE RAW 264.7 CELLS .......................................................................................................... 103 ABSTRACT ............................................................................................................ 104 INTRODUCTION .................................................................................................. 104 MATERIALS AND METHODS ........................................................................... 106 RAW 264.7 cell culture ............................................................................. 106 Cell transfection ........................................................................................ 106 CAT ELISA .............................................................................................. 108 TNF-a ELISA assay .................................................................................. 108 RNA extraction and Reverse transcription (RT) ..................................... 1 10 Construction of competitor DNA ............................................................ 1 10 Oligonucleotide synthesis ......................................................................... 1 12 Semi-competitive RT-PCR ........................................................................ l 12 Statistical analysis ...................................................................................... 1 13 RESULTS ............................................................................................................... 1 13 DISCUSSION ........................................................................................................ 124 CHAPTER V. STABILIZING EFFECTS OF VOMITOXIN ON CYTOKINE mRNA HALF -LIFE IN MURINE MACROPHAGE RAW 264.7 CELLS ............ 131 -viii- ABSTRACT .......................................................................................................... 132 INTRODUCTION ................................................................................................. 133 METHODS ............................................................................................................. 135 RAW 264.7 cell culture .............................................................................. 135 Ctyokine assays .......................................................................................... 136 cDNA probe preparation ........................................................................... 137 Northern blotting and mRNA stability analysis ...................................... 137 Statistical analysis .................................................................................... 138 RESULTS ............................................................................................................... 138 DISCUSSION ......................................................................................................... 145 CHAPTER VI. SUMMARY AND FUTURE STUDIES ........................................... 157 LIST OF REFERENCES ................................................................................................ 160 -ix- LISTS OF TABLES Table Page Table 2.1. Primer Sequences for Cytokine cDNA Amplification and Probe Sequences for Southern Blot Detection of Amplification cDNA Product ............ 55 Table 2.2. Effect of VT on pro-inflammatory Cytokine Secretion by RAW 264.7 cells ................................................................................................... 58 Table 3.1. Oligonucleotides for Transcription factors in EMSA ..................................... 78 LISTS OF FIGURES Figge Page Figure 1.1. General chemical structure of bacterial lipopolysaccharides ................ 16 Figure 1.2. Dimer combinations of F os and Jun proteins that capable of forming Stable AP-l transcription factor complexes ................................................ 23 Figure 1.3. Members of the NF-KB family proteins .................................................. 26 Figure 1.4. Activation for the mechanism of the NF-KB activation .......................... 27 Figure 1.5. Model of the C/EBP structure .................................................................. 29 Figure 1.6. Regulation of gene expression by CREB ................................................. 32 Figure 1.7. Schematic representation of the TNF receptors, TNF and associated signaling molecules ...................................................................................... 36 Figure 1.8. Interaction of VT with LPS activation pathway ...................................... 46 Figure 2.1. Flow cytometry analysis of cytoplasmic IL-6 in RAW 264.7 cells after incubation with VT for 24 hr in the presence of LPS .................................. 60 Figure 2.2. Flow cytometry analysis of cytoplasmic TNF-0t in RAW 264.7 cells afier incubation with VT for 24 hr in the absence or presence of LPS ............... 61 Figure 2.3. Relative abundance of IL-1 B mRNA in RAW 264.7 cells after in vitro exposure to VT with and without LPS ............................................................ 63 Figure 2.4. Relative abundance of IL-6 mRNA in RAW 264.7 cells afier in vitro exposure to VT with and without LPS ............................................................ 64 Figure 2.5. Relative abundance of TNF-0t mRNA in RAW 264.7 cells afier in vitro exposure to VT with and without LPS ............................................................ 65 Figure 3.1. Dose and temporal effects of VT on AP-l binding activity in a synchronous exposure model .......................................................................... 80 Figure 3.2. Dose and temporal effects of VT on AP-l binding activity in a delayed synchronous exposure model ............................................................................ 82 Figure 3.3. Specificity of AP-l DNA binding activity in RAW 264.7 cells in -xi- synchronous model and delayed synchronous model ....................................... 83 Figure 3.4. Supershifi EMSA of effects of VT on the AP-l subunit binding in RAW 264.7 cells ........................................................................................................... 84 Figure 3.5. Dose and temporal effects of VT on NF-KB binding activity in a synchronous exposure model ............................................................................ 87 Figure 3.6. Dose and temporal effects of VT on NF-KB binding activity in a delayed synchronous exposure model ............................................................................ 88 Figure 3.7. Specificity of NF-KB DNA binding activity in RAW 264.7 cells in synchronous model and delayed synchronous model ......................................... 89 Figure 3.8. Supershifi EMSA of effects of VT on the NF-KB subunit binding in RAW 264.7 cells ............................................................................................................. 90 Figure 3.9. Dose and temporal effects of VT on NF-IL6 binding activity in a synchronous exposure model ............................................................................. 92 Figure 3.10. Dose and temporal effects of VT on NF-IL6 binding activity in a delayed synchronous exposure model ............................................................................. 93 Figure 3.11. Specificity of NF-IL6 DNA binding activity in RAW 264.7 cells in synchronous model and delayed synchronous model .......................................... 94 Figure 3.12. Supershifi EMSA of effects of VT on the NF-IL6 subunit binding in RAW 264.7 cells .............................................................................................................. 96 Figure 3.13. Regulatory elements of IL-1 0 (A), IL-6 (B) and TNF-0r (C) ........................ 97 Figure 4.1. Construction of the CAT competitor by using internal primers with the splicing by overlap extension (SOE) method modified from Horton et al. (1989) ........................................................................................................... 111 Figure 4.2. Cells transfected with CaPO4 for 16 hr and then incubated for 12 hr before staining for the B-galactosidase activity of pCMV plasmid ............. 1 14 Figure 4.3. Cells transfected with DEAE for 3 hr and then incubated for another 24 hr before staining for the B-galactosidase ......................................................... 1 15 Figure 4.4. Cells transfected with liposome for 3 hr and then incubated for 12 hr before staining for the B-galactosidase ................................................................ 1 16 -xii- Figure 4.5. Effect of VT on CAT and TNF-0t protein expression in the presence of LPS for 2 hr in synchronous model ................................................................. 1 1 8 Figure 4.6. Effect of VT on CAT and TNF-0t protein expression in the presence of LPS for 6 hr in synchronous model ................................................................ 1 19 Figure 4.7. Effect of VT on CAT and TNF-0t protein expression in the presence of LPS for 2 hr in delayed synchronous model ................................................... 120 Figure 4.8. Effect of VT on CAT and TNF-0t protein expression in the presence of LPS for 4 hr in delayed synchronous model ..................................................... 121 Figure 4.9. Effect of VT on CAT and TNF-0t protein expression in the presence of LPS for 8 hr in delayed synchronous model ................................................... 122 Figure 4.10. Effect of VT on CAT and TNF-0t protein expression in the presence of LPS for 24 hr in delayed synchronous model ................................................. 123 Figure 4.11. Effect of VT on the TNF-0t transactivation in the synchronous model ....... 126 Figure 4.12. Effect of VT on TNF-O. transactivation in the delayed synchronous model ...................................................................................................................... 127 Figure 5.1. Effect of VT on TNF-0t production in LPS-treated RAW 264.7 cells ........ 140 Figure 5.2. Effect of VT on IL-6 production in LPS-treated RAW 264.7 cells ............... 141 Figure 5.3. Effects of asynchronous VT-exposure TNF-0t and IL-6 mRNA expression in LPS-treated RAW 264.7 cells .......................................................... 143 Figure 5.3. B,C. mRNA stability for TNF-0t and IL-6 in asynchronous model ................ 144 Figure 5.4. Effects of delayed synchronous VT-exposure TNF-0t mRNA expression in LPS-treated RAW 264.7 cells .......................................................... 147 Figure 5.5. Effects of delayed synchronous VT-exposure IL-6 mRNA expression in LPS-treated RAW 264.7 cells ......................................................... 149 Figure 5.6. The AU-rich elements on the 3'-untranslated region of cytokine gene A, B, C ...................................................................................................................... 152 Figure 5.6. D. The AU-rich elements on the 3'-untranslated region of cytokine gene (Continued) ............................................................................................................ 155 -xiii- INTRODUCTION Trichothecenes are secondary metabolites mainly produced by species of F usarium found in wheat and corn supplies throughout the world (V esonder and Hesseltine, 1980; Ueno, 1985; Rotter et al. , 1996). Trichothecenes have a tetracyclic epoxy-ring at their C12- C13 position and an unsaturated bond at the C9-C10 position (Bamburg, 1983; Betina, 1989). More than 100 different varieties of trichothecenes have been identified (Scott, 1990). Trichothecenes are classified into A, B, and D (macrocyclic) groups which include vomitoxin (VT, deoxynivalenol), T-2 toxin and nivalenol (NIV). Trichothecenes are very stable and do not degrade at high temperature during milling and processing (Scott, 1991). Therefore, they are common contaminants in crops and in food and feed supplies. The trichothecene VT has been shown to cause a wide range of toxicological and immunological effects (Cundliffe et al., 1974; Ueno, 1983; Bamburg, 1983; Forsell et al., 1987; Fitzpatrick et al., 1988; Thompson and Wannemacher, 1990; Bergrnann et al. , 1992). VT can either be immunosuppressive or immunostimulatory depending on the dose and timing of exposure (Pestka and Bondy, 1994). VT has been shown to cause depression in the mitogen-induced proliferative responses of murine lymphocytes (Tryphonas et al., 1986; Robbana-Barnat et al., 1988). In contrast, VT also increases resistance to the mastitis pathogen Mycobacterium and to Staphylococcus in mice (Atroshi et al., 1994). Increased serum IgA, IgM and IgG levels with mastitis mice gavaged with VT for a week was observed (Atroshi et al., 1994). Dong and Pestka (1993) also showed that dietary exposure of VT in mice elevated serum IgA, circulating IgA immune complexes and mesangial IgA deposition in the kidney. Therefore it was suspected that these effects of VT might -1- contribute to the alteration of the function of immune cells including macrophage, T cells, or B cells. Superinduction of cytokines by VT has been observed by several researchers. For example, Miller and Atkinson (1986) demonstrated the superinduction of inflammatory cytokine IL-1 in peritoneal macrophages treated with VT. Recently, increased mRNAs for interleukin-2, -4, -5 and/or -6 and their secreted proteins were observed upon exposure to VT and cycloheximide (CHX) in viva (Warner et al. , 1994; Azcona-Olivera et al. , 1995a; Ouyang et al., 1996). Transient increases of mRNAs for pro-inflammatory cytokines associated with macrophages, IL-1 [3, IL-6 and TNF-a, were also shown upon exposure to VT in vivo (Azcona-Olivera et al., 1995b; Zhou et al., 1997). Macrophages are pivotal regulators of inflammation and immunity (Solbach et al., 1991) and play an important role in regulating T cell function, proliferation, regulation of antibody and production of mediators for celiular immunity. The aforementioned macrophage cytokines may be related to the pathogenic manifestations associated with trichothecene exposure. For example, IL-1 [3, IL-6 and TNF-0t are cytokines observed to mediate septicemia and fatal circulatory shock-like syndrome by LPS (V ogel and Hogan, 1990). IL-1 0, IL-6 and TNF-0t also have been associated with anorexia and weight loss, or cachexia (Abbas et al. , 1994; Schobitz et al., 1994). Thus, feed refusal and reduced weight gain observed in the trichothecene-exposed animals (Rotter et al. , 1996) might be mediated by superinduction of these pro-inflammatory cytokines. Also, IL-6 has been shown to be involved in the development of IgA responses (VanCott er al. , 1996) and is able to drive IgA production when added to the Peyer’s patch B cell cultures (Y an at al., 1997). Taken together, these results suggest that superinduction of those cytokines in macrophage by VT might contribute to hyperelevation of the IgA production. However, the superinduction of cytokines by VT in macrophage cells has not yet been studied. Therefore, it was important to assess the effects of VT on the cytokine protein and mRNA expression in the macrophage cells. The RAW 264.7 murine cell line was employed for this purpose in my research. Gene expression is regulated at the transcriptional, and post-transcriptional level. Transcriptional control has been suggested to be a main regulatory mechanism of macrophage activation (Yu et al. , 1990; Paulnock, 1994). Previous study (Ouyang et al. , 1996) revealed that the activity of transcription factor NF-KB increased in IL-2 gene expression in EL-4 cells treated with VT. NF-KB also plays an important role in induction of various gene expression in macrophages, such as M-CSF, G-CSF, TNF-0t, IL-1 [3, and IL-6 (Akahane et al. , 1994; Baeuerle and Henkel, 1994). NF-IL6 and AP-l have also been cited as important transcription factors in myeloid specific genes (Clark and Gordon, 1998). Thus, it is possible that VT can superinduce IL-1 [3, TNF-0t and IL-6 in macrophage cells by altering the activity of these transcriptional factors. Therefore, it is useful to study the effects of VT on the binding activity of those transcription factors on the promoters in macrophage RAW 264.7 cells. cis-acting elements also play important roles in the transcriptional control of the cytokine mRNA expression. Transcriptional activity of the TNF-0t gene was enhanced by CHX in human monocytes (Osipovich et al., 1993) and the macrophage cell line, J774 (Akahane et al., 1994). Transcription of the IL-2 gene also increased 2-3 fold after exposure to CHX in previously activated EL-4 cells (Zubiaga et al. , 1991 ). The extent of the transcription in mammalian cells can be studied by using a plasmid containing the desired promoter linked to a reporter gene; promoter activity is measured based on expression of the reporter protein. Therefore, it was desirable to employ a promoter construct to fully understand the effects of VT on the promoter activity of these cyokines. In addition to the transcriptional control, post-transcriptional control also plays a key role in the regulation of the cytokine superinduction. Protein synthesis inhibitors, such as CHX, have been shown to increase the half-life of TNF-0t mRNA in macrophages (Celada et al., 1989; Taffet et al. , 1989) in the presence of the transcriptional inhibitor actinomycin D (Act-D). Fort et al. (1987) also demonstrated that cytokine mRNA degradation seems to resume quickly after the removal of protein synthesis inhibitors such as CHX. VT may also superinduce expression of cytokines in macrophages by stabilizing their mRNAs. Thus, it was important to evaluate the stabilization effects of VT on the cytokine mRNA expression in the trichothecene-mediated cytokine superinduction. The four aforementioned areas were studied in this dissertation, which is comprised of seven parts. Chapter I reviews literature on trichothecenes, macrophages, lipopolysaccharides, and the regulation of the IL-6, TNF-a and IL-lB gene expression. Chapter H describes the effects of VT on the cytokine protein and mRNA expression in murine macrophage RAW 264.7 cells. Chapter III describes the effects of VT on the binding activity of transcription factors AP-l , NF-IL6 and NF-KB in RAW 264.7 cells.. Chapter IV describes the effects of VT on the transcriptional activity of TNF-0t promoter in RAW 264.7 cells. Chapter V describes the stabilizing effects of VT on cytokine mRNA half-life in RAW 264.7 cells. Chapter VI summarizes these inter-related studies and makes suggestions for future research. In addition, the appendix contains three reprints of published work to which I have contributed either as a primary author or as a collaborator. CHAPTER 1 LITERATURE REVIEW A. Trichothecenes AJ. Introduction Trichothecenes are toxic secondary metabolites produced by genera of fungi such as F usarium, Myrothecium, Stachybotrys, T richoderma and T richothecium that can be found in food grains or environmentally (Schoental, 1983; Ueno, 1985; Vesonder and Hesseltine, 1980; Rotter et al. , 1996). They have a sesquiterpenoid structure which is characterized by the presence of an unsaturated bond at the C9-C10 position and a tetracyclic 12,13-epoxy ring (U eno et al. , 1973; Schoental, 1983; Betina, 1989, Rotter et al. , 1996). Trichothecenes are often produced during temperature cycling under high humidity (Gu et al., 1991; Sherwood and Peberdy, 1972). Trichothecenes have been classified into A, B, and D (macrocyclic) groups according to their fimctional groups. Trichothecenes have been to induce acute shock-like toxicity as well as chronic effects. They can cause gastrointestinal disturbances such as vomiting, diarrhea, and inflammation, feed refusal, abortion, dermal irritation and hematological sequelae (anemia and leukopenia) (Coulombe, 1993). Ingestion of thrichothecenes has been shown to cause outbreaks of alimentary toxic aleukia (ATA) disease in the former Soviet Union, India, China and Japan (Joffe, 1978; Bhat et al., 1989; Ueno, 1987; Luo et al., 1990). Dietary exposure to trichothecenes has been associated with high esophageal cancer incidence in South Africa and China (Marasus et al. , 197 7; Luo et al. , 1990). The underlying mechanism for trichothecene toxicity relates to their ability to be potent protein synthesis inhibitors in eukaryotic cells (U eno et al. , 1968; Rosenstein and Lafarge-Frayssinet, 1983; Suneja et al. , 1983). They inhibit all steps of protein synthesis: initiation, elongation and termination by binding to the 60 S ribosomal subunit (Ueno, 1983). They also inhibit DNA synthesis in -7- vitro. T-2 (30t-hydroxy-413,l 5-diacetoxy-80t-(3-methylbutryloxy)-12,13-epoxytrichothec-9- ene) toxin, one of the most potent trichothecenes, is also a protein synthesis inhibitor. It is found as a natural contaminant in foods and feeds (U eno, 1986) as is VT. It can be produced by different species of F usarium, Myrothecium, T richothecium, T richoderma and Stachybotrys (Schoental, 1985). T-2 was first identified in moldy corn that causes a fatal hemorrhagic disorder in lactating cows (I-Isu et al., 1972). As a protein synthesis inhibitor, it inhibits protein synthesis in mammalian cells (Ueno, 1983; Cannon et al., 1976) by binding to ribosomes (McLaughlin et a1. , 1977). T-2 toxin reduces weight gain, and causes emesis, inflammation, internal hemorrhage, and damage in the bone marrow and thymus (Saito and Ohtsubo, 1974; Hayes and Schiefer, 1980). A.2. Vomitoxin (Deoxynivalenol) Vomitoxin (VT) was first isolated from barley and characterized by Yoshizawa and Morooka (1973) in Japan. It was given the name because of its observed emetic effect on swine.It is also known as ‘Rd toxin’ in barley and corn infected with F usarium sp. (V esonder et al., 1973; Vesonder and Ciegler, 1979) from the mid-western United States and Canada. VT is mainly produced by strains of F usarium graminearum and F. culmarum, which are pathogens in wheat and corn. When F usarium grows on wheat, it produces scabbed wheat, which is soft and shriveled, often with pink discoloration on the kernels and flowering head (V esonder and Hesseltine, 1980). Environmental conditions such as low temperature and high humidity encourage the growth of mold on crops and VT production, thereby increasing the chances of VT contamination. VT is a very stable compound and does not degrade at high temperature during milling and processing and readily enters into finished food products -3- (Scott, 1991). Therefore, VT is a common contaminant of com and wheat worldwide and widely found in food and feed. VT has been shown to cause a wide range of toxicological and immunological effects. Low and acute doses of VT are known to cause anorexia and emesis in animals. A significant increase of serotonin was observed in the cerebrospinal fluid following oral administration of VT (Fitzpatrick et al. , 198 8). VT is also reported to cause necrosis of the intestinal tract, bone marrow and lymphoid tissues, and kidney and heart lesions at very high doses (2100mg/kg) (Forsell et al., 1987). VT can either be immunosuppresive or immunostimulatory depending on the dose and timing of exposure (Pestka and Bondy, 1994). In vivo exposure to VT depresses mitogen-induced proliferative responses of murine lymphocytes (Robbana-Barnat et al. , 1988; Tryphonas et al. , 1986). It has also been shown that both single and multiple gavage of mice with VT increase resistance to the mastitis pathogens Mycobacterium and to Staphylococcus (Atroshi et al., 1994). Elevated serum IgA, IgM and IgG levels have been found in mastitis mice gavaged with VT for a week (Atroshi et al., 1994). Similarly, Forsell et al. (1987) detected a dramatic increase in serum IgA, but found decreases in serum IgM and IgG in mice fed with VT. Dong and Pestka (1993) also demonstrated that dietary exposure of mice to VT can elevate serum IgA, circulating IgA immune complexes and mesangial IgA deposition in the kidney. These features are highly analogous to the human glomerulonephritis, IgA nephropathy (D’Amico, 1987). VT is able to stimulate in vitro IgA production. Prior exposure to VT in vitro enhances CD4+ cell mediated help for IgA production by B cells through elevated cytokine production (Warner et al. , 1994). VT can also increase the percentage of total T cells, CD4+ -9- cells and the CD4+/CD8+ cell ratio in Peyer’s patches (PP) (Pestka et al., 1990a, b) and spleen. Increased mRN As of interleukin-2, -4, -5 and/or IL-6 (T-helper cytokines) and their secreted proteins have been observed upon exposure to VT or cycloheximide (CHX) in viva (Warner et al. , 1994; Azcona-Olivera et al. , 19953; Ouyang et al. , 1996). Thus, VT-induced elevation of IgA production might be related to the superinduction of various T helper cytokines. Superinduction of inflammatory cytokines such as IL-1 has been observed in peritoneal macrophages treated with VT (Miller and Atkinson, 1986). Oral exposure of mice to VT causes transient increases in mRNAs for cytokines IL-1 B, IL-6 and TNF-0t within 2 hr (Azcona-Olivera et al., 1995b; Zhou et al., 1997). A.3. Cycloheximide Cycloheximide (CHX), a glutarimide antibiotic (Pestka, 1971) is the most well- studied protein synthesis inhibitor. It binds to 80$ ribosomes in eukaryotic cells (Cannon et al., 1994). CHX inhibits protein synthesis in yeast and mammalian cells (Kerridge, 1958; Young et al., 1963; Ennis and Lubin, 1964; Gorski and Axman, 1964). It has been shown to inhibit translocation of peptidyl-tRNA into the the ribosomes. CHX is thought to be involved in the inhibition of the initiation, elongation and termination steps of protein synthesis. CHX superinduces cytokines such as TNF-0t and IFN-y (Efrat and Kaempfer, 1984; Cockfield et al., 1993; Faggioli et al., 1997). CHX also superinduces IL-2, IL-6, IFN-y and TNF-0t mRNA both in viva and in vitro (Efrat and Kaempfer, 1984; Zubiaga et al., 1991; Cockfield et al. , 1993; Osipovich et al. , 1993; Akahane et al. , 1994; Biragyn and Nedospasov, 1995; F aggioli et al., 1997). Increased cytokine mRNA half-life has also been observed in macrophages treated with CHX (Celada et al., 1989; Taffet et al., 1989; and -10- Zuckerman et al., 1991). CHX also induces the activity of the transcription factor NF-KB (Menegazzi et al. , 1996). The induction may be related to cytokine superinduction observed in CHX-treated cells or animals. B. Macrophages 13.1. Introduction Macrophages are multi-functional cells involved in the regulation of both innate and acquired immunity (Van Rooij en et al., 1996). Mononuclear phagocytes originate from pluripotent stem cells in bone marrow (Auger and Ross, 1992; van F urth et al., 1986a). A monoblast, the most immature cell, can divide into two pro-monocytes. The pro-monocytes then divide into two monocytes each. Monocytes migrate fiom the bone marrow and into the blood within 24 hr after they are formed. The peripheral monocytes then migrate into tissues in which they differentiate and become macrophages (van F urth et al. , 1986b). The constant influx of the monocytes into the tissues requires a constant efflux from the tissue and/or cell death in the tissues. Macrophages are important regulators in inflammation. They are able to perform phagocytosis, intracellular killing, chemotaxis, antigen processing and presentation, as well as secretion of cytokines and prostaglandins (Solbach et al. , 1991 ; Angus and Ross, 1992). 8.2. Macrophage activation Upon stimulation, macrophages can be activated and involved in inflammation. There are two different kinds of extracellular stimulation (Schneider and Dy, 1985). The components that present in the immune system such as cytokines, irnmunoglobulins and -11- complement are the major and most effective stimuli (Schneider and Dy, 1985; Paulnock, 1994). Non-immunological components, such as endotoxin, pyron polymer and polyanions, are additional sources of stimulation. The binding of these components by receptor molecules on macrophages lead to activation. Several different receptors on the macrophage have been identified. These include CD 14, CD33, CD18, Mac-1, and a variety of cytokine receptors (Angus and Ross, 1992). Activation of macrophages involves at least two different steps (Schneider and Dy, 1985; Ohmori and Hamilton, 1994). During the priming step, resident macrophages are stimulated with a cytokine such as interferon-y (IFN-y). Further encounter with endotoxin or other signals will trigger their differentiation into fully activated, cytotoxic macrophages. Activation of macrophages can increase their secretory protein products (>100 varieties of protein) (Adams and Hamilton, 1984; Nathan, 1987) including monokines and prostaglandins that alter morphology (causing an increase in size, spreading and membrane ruffling), modify receptor expression, and increase oxidative metabolism (Schneider and Dy, 1985; Auger and Ross, 1992). These reactions will in turn enhance chemotaxis, phagocytosis and cytotoxicity in the host inflammatory system. 8.3. Macrophage and cytokines Macrophages can release many different cytokines including interleukin (IL)-1, tumor necrosis factor (TNF)-0i, IL-6, IL-8 and IFN-y (Ohmori and Hamilton, 1994). IL-1 is a low molecular weight protein that can be produced by many cells, but macrophages are major producers (Cavaillon and Haeffner-Cavaillon, 1990). IL-l can increase proliferation and activation of T and B lymphocytes, macrophages and other cells. It also increases postaglandin production by macrophages and enhances their cytotoxicity. IL-l also promotes B cell maturation and division and induces acute phase proteins in -12- hepatocytes (Dayer and Burger, 1994). This cytokine is also known as “ endogeneous pyrogen” because it can cause fever (Dinarello, 1994). TNF-0t, also known as “cachectin”, is primarily produced by activated macrophages. It is named for its ability to kill tumor cells (Laskin and Pendino, 1995). TNF-0t has been found to be involved in septic shock and inflammation (Dayer and Burger, 1994). It regulates acute phase protein gene expression and the P450 activity during cellular proliferation and apoptosis. TNF-0t also stimulates release of other cytokines such as IL-1 and IL-6. It can cause severe weight loss and also death. Like IL- 1 , it can also induce fever. Another important cytokine produced by macrophages is IL-6. Both IL-1 and TNF- 0t upregulate IL-6 production while IL-4 is known to inhibit IL-6 production. IL-6 enhances proliferation of both immature and mature T cells and an important differentiation factor for B cells and can induce immunoglobulin (Ig) secretion. IL-8 is a chemokine that attracts neutrophils and T-cells (Wuyts et al., 1998). It inhibits IFN-y production by natural killer (NK) cells. Three different kinds of TGF-B (type 1,2,3) are produced by macrophages (Derynck and Choy, 1998). It is immunosuppresive by inhibiting IL-2 production. TGF-B is found to be involved in wound healing and also plays a role in fibrosis. B. 4. Regulation of macrophage gene expression Gene expression is controlled at different levels which include transcriptional, and Post-transcriptional mechanisms (Manthey and Vogel, 1994). B. 4. 1. Transcriptional control Relative to transcriptional control of macrophage cytokine gene expression, several -13- cis elements have been found in the cytokine genes. For TNF-ct gene, the region 5' of the transcription start site and the 3' untranslated region (UT R) are the important regulatory regions (Collart et al., 1990; Drouet et al., 1991). Different motifs such as a KB site, cytokine-1 (CK-l) site, and activator protein-l (AP-l) site have been found (Ohmori and Hamilton, 1994). There are at least three KB motifs in the human TNF-0t gene (Ohmori and Hamilton, 1994). In the IL-lB gene, several motifs have also been found in the promoter region. These include KB, cyclic AMP(cAMP), AP-l and the nuclear factor (NF) -IL6 sites. Deletion of the NF-IL6 site reduces IL-1 [3 gene expression (Shirakawa et al., 1993). In the IL-6 promoter, NF-KB, NF-IL6 and the cAMP sites appear to be important. Several trans acting proteins are also involved in cytokine transcription regulation. These are NF-KB/Rel proteins, NF-IL6 (or C/EBP) proteins and AP-l proteins (Ohmori and Hamilton, 1994; Valledor et al. , 1998). When macrophages are activated, these proteins are induced and bind to the related motifs on the cytokine promoters. B. 4. 2. Post-transcriptional control Post-transcriptional control has been suggested to be important in the regulation of cytokine genes in macrophages (Paulnock, 1994; Ohmori and Hamilton, 1994). Notably, AU-rich sequences in the 3'-untranslated region (UT R) of many cytokines and oncogenes contribute to the mRNA stability of those genes (Caput et al., 1986; Shaw and Kamen, 1986). Examples of where such sequences are found include TNF-0t, IL-1 [3, IL-6 and c-fos genes. These sequences can confer instability in the resultant mRNAs and decrease the formation of mature transcripts in the cytoplasm. Different proteins that recognize the AU- rich sequences have been identified: AU-A, AU-B, AU-C and hnRNP (heterogeneous -14- nuclear ribo-nucleoprotein) or known as AUFl (Bohjanen et al., 1991; Chen and Shyu, 1995; Ross, 1995). AU-A, a 34-kd protein, is abundant in nuclear extracts and regulates mRNA metabolism. AU-A was found to induce stabilization of c-myc mRNA in vitro by binding to the AU-rich response elements (ARE) (Ross, 1996). The 30-kd AU-B is another cytoplasmic factor that binds ARE in the 3' UTR of lymphokine mRNAs and promotes degradation of their mRN As (Bohj anen et al. , 1991). The induction of this protein requires protein synthesis. Similarly, the protein AUFl enhances mRNA degradation by binding to the ARE site in vitro assay (DeMaria and Brewer, 1996; Kiledjian et al., 1997). C. Lipopolysaccharide C.1. Structure of lipopolysaccharide Lipopolysaccharide (LPS), a component found on the outer membrane of Gram negative bacteria like the Enterobacteriaceae (Escherichia coli and Salmonella typhimurium) (Mayeux, 1997), consists of a polysaccharide region and a lipid A region (Fig. 1.1). The polysaccharide region is comprised of “O-Antigen”, a polymer of oligosaccharides and a “core” region of oligosaccharides (Schletter et al., 1995; Henderson et al., 1996). The “O- Antigen” is a variable region which confers serologic specificity to the Gram negative microbes. The core oligosaccharide consists of an inner structural region and an outer region (hexose region). The core region has less diversity than the “O-Antigen” (Schletter et al., 1995). The lipid A region of LPS is the least variable portion and is responsible for the biological properties induced by endotoxin (Vogel and Hogan, 1990; Watson et al., 1994). The lipid A component consists of a phosphorylated glucosamine disaccharide that is substituted with ester- and amide-linked fatty acids, including hydroxylated fatty acids. -15- . O ’0 91:9 \\‘ COCOIIOOOOVz’é/lxtb¢3\\‘ n a t l—-“—--—J H ' Polysaccharid?‘ ——' Lipid ——' O: Monosaccharide O: Phosphate ww : Fatty Acid Figure 1.1. General chemical structure of bacterial lipopolysaccharides. (Adapted from Ulevitch and Tobias, 1995) -16- It has been shown that any changes in the hydrophilic backbone or in the hydrophobic region will lead to a partial or total loss of the LPS activity (Rietschel et al., 1994). C.2. Proteins that bind lipopolysaccharides C.2.1. Lipopolysaccharide binding proteins (LBP) LBP, (lipopolysaccaride-binding protein), is a 60-kd glycoprotein synthesized in the liver and released into the circulation (Schumann, 1992; Ulevitch and Tobias, 1995; Henderson et al. , 1996; F enton and Golenbock, 1998). It is constitutively expressed in liver, but can be fiirther induced by LPS and other stimuli. It serves as a transport protein mainly presenting LPS monomers from bacteria to CD14 (F enton and Golenbock, 1998). It recognizes LPS by interaction with the lipid A moiety of LPS. The lipid A moiety has a high affinity binding to the LBP protein with a kd of 10'9 (Su et al., 1995). The amino terminal of the LBP is the binding site for LPS and the carboxyl terminal is the site for CD14 interactions. It also serves as an opsonin for bearing particles such as the Gram negative bacteria. Normally, 5-10 rig/ml of LBP is expressed in human serum. During the acute phase response, the concentration of LBP is induced up to 200 rig/ml (Tobias et al., 1992). Anti-LBP antibodies can suppress low LPS dose-induced endotoxemia shock in mice (Gallay et al. , 1994). When LPS is bound to LBP, 1000-fold less LPS (0.1 ng/ml) is required to stimulate monocyte secretion of TNF-or (Su et al. , 1995). IL-6 can induce expression of LBP in the serum and TNF-0t, IL-1 and dexamethasone can synergize with IL-6 in this induction. LBP plays a different role in directing or transporting LPS to bind to the high density lipoprotein (HDL) particles. This binding will lead to a marked reduction in the clearance rate of LPS but decreases the toxicity of LPS. Therefore, binding to HDL is a detoxification process of LPS (Ulevitch et al., 1990). -17- C.2. 2. Cluster differentiation antigen 14 (CD14) Cluster differentiation antigen 14 (CD14), a myeloid differentiation antigen, is mainly expressed on the cell surface of monocytes, basophils and granulocytes (Wright et al., 1990; Chaby and Girard, 1993; Kielian and Blecha, 1995). The CD14 gene is found on the long arm of chromosome 5 which carries genes also encoding for IL-3, GM-CSF, CSF- 1, CSF-lR and PDGF-R (Goyert et al., 1988). There are two types of CD14, the membrane bound CD14 (mCD14) and the soluble CD14 (sCDl4) found in the serum (Bazil et al., 1986). mCDl4 is a 55-kd glycoprotein which is anchored to the membrane by a glycophosphatdylinositol (GPI) glycan moeity (Kielian and Blecha, 1995). sCD14 lacks the GPI which penetrates into the membrane. Binding of the LPS to mCD14 induces the production of IL- 1 , IL-6 and TNF-0t by monocytic cells. The binding of LPS to CD14 can induce the secretion of free radicals such as 02', H202, OH and singlet oxygen (Watson et al. , 1994), monokines such as IL-6, IL-8, and TNF-0t, and arachidonic acid metabolites. An increase of the concentration of 02' enhances phagocytosis of macrophages which in turn increases the ability to kill pathogenic microorganisms. Stable transfection of CD14 has been shown to increase the cell’s sensitivity to LPS by 10,000-fold (Lee et al., 1992). Anti- CD14 antibodies cannot block the high LPS dose-induced stimulation and binding to the protein (Beaty et al., 1994). C.2. 3. Bactericidal/permeability increasing protein (BPI) BPI binds specifically to the lipid A moiety of LPS. The BPI gene is located on the chromosome 20 as is the LBP gene (Su etal., 1995). BPI is a 55-kd membrane associated protein found in neutorphil granules and is released upon activation by LPS and cytokines (Marra et al., 1992). Two domains have been found in this protein: N—terminal and C- -18- terminal domains (Gray et al. , 1989). The N-terminal domain binds LPS and the C-terminal domain is a transmembrane region that anchors the holoprotein in the granule membranes. It has 44% identity with LBP protein and it possess LPS neutralizing ability (F enton and Golenbock, 1998). BPI concentration is induced from 5 to 20 ug/ml upon activation by LPS (Horwitz et al., 1995). C3. Signal transduction by lipopolysaccharides (13.1 CDI4 dependent path way LBP catalyzes the binding of monomeric LPS to mCD14. After binding to CD14, the LPS-LBP-CD14 complex activates two different protein kinase system: protein tyrosine kinase (PTK) aVIayeux, 1997) and mitogen activated protein kinase (MAP kinase) (Weinstein et al., 1992). Activation of PTK leads directly to the activation of the IKBa kinase and phosphrylation of the IKBa. This phosphorylation results in the dissociation of the IKBa from the NF-KB. NF-KB then translocates fi'om the cytoplasm to the nucleus and binds to the KB sites on the related genes, which in turn induces transcription of the targeted genes (Mukaida et al., 1996). PTK is required for induction of IL-1, IL-6 and TNF-0t expression. For the MAP kinase pathway, dual phosphorylation of threonine and tyrosine residues of MAP kinases is required. This is mediated by the MAP kinase kinases (MEKs). When cells are stimulated with LPS, the LPS-LBP-CD14 complex causes activation of MEKs and consequently phosporylates the MAP kinases including p38, ERK1 and ERKZ (the 42 and 44-kd isoforrns of MAP kinases), and c-JUN-N-terminal kinase (JNK)-1 (Schletter et al., 1995). In cell types which lack membrane-bound CD14 (mCD14), the sCD14 in the serum -19- replaces the mCD14 for the binding of the LPS-LBP complex (Mayeux , 1997). It is postulated that a putative receptor that recognizes the complexes existed on the CD14 negative cells (Schletter et al., 1995). Cell types which are deficient in CD14 can also respond to the LPS stimulation but require higher concentrations of LPS (>100 rig/ml LPS) (Mayeux, 1997). (3.3.2. C014 independent pathway In the CD14 independent pathway, a transient increase of intracellular Ca2+ concentration is found in many cell types including macrophages. LPS cause a rapid increase of [Ca2+] which is generated from the activation of myoinositol 1,4,5-triphosphates (InsP3) (Mayeux, 1997). The released [Ca 2+] activates nitric oxide synthase (NOS) resulting in the synthesis of nitric oxide (NO) and cGMP. This is a critical step in the pathway which ultimately leads to cell injury because the generated NO causes formation of oxidizers peroxynitrite anions (ONOO’) and hydroxyl radical (HO') (Mayeux, 1997). This kind of pathway requires a high concentration of LPS or lipid A of the LPS. C. 4. LPS and cytokine induction LPS is also known as “endotoxin”, because of its role in pathophysiological phenomenon associated with Gram negative infections and the induction of responses that are toxic in nature. Different cytokines can be induced by LPS and they are able to mediate various aspects of endotoxicities such as fever, which is an indicator of inflammation. Several cytokines that are involved in LPS-induced fever are IL-l, TNF-or and IFN-a (Watson et al., 1994). They are also known as “endogenous pyrogcns” and are mainly produced by macrophages (V ogel and Hogan, 1990). LPS is also involved in septicemia. -20- This disease is one of the leading causes of death in the United States (Stone, 1994; Dal Nogare, 1991). Sepsis is the clinical term for the presence of bacteria or their toxins in the blood or tissues. D. Regulation of interleukin-6 (IL-6) D. I. Introduction Interleukin 6 (IL-6) is a multifunctional cytokine which has a molecular weight of 21 to 28-kd (Hirano, 1994). This glycoprotein produced both by lymphoid and non- lymphoid cells, can regulate immune responses, acute phase responses and hematopoiesis. It is also known as B cell stimulatory factor 2 (Hirano et al., 1985). There is 65% homology between human and mouse IL-6 DNA. IL-6 can be produced by a variety of cells such as macrophages, T and B cells, and endothelial cells. Diverse stimuli can induce production of IL-6 and these include LPS, IL-l , TNF-0t, IFN-y, viruses (human immunodeficiency virus), and others (Hirano, 1994). However, agents such as IL-4, IL-13, and glucocorticoids inhibit IL-6 production. In a negative regulatory role, IL-6 can inhibit the production of both IL-1 and TNF-0t There are two IL-6 receptor components: the 80-kd or chain which binds IL-6 and the 130-kd [3 chain (gpl30) for which transduces the signal (Y amasaki et al., 1988; Taga et al. , 1989). Binding of the IL-6 to its receptor leads to the dimerization of gpl30, and activates Janus tyrosine kinases (J AKl , J AK2, J AK3, Tyk-2) associated with the receptor. These kinases phosphorylate STATS (signal transducers and activators of transcription). Upon phosphorylation, STAT s translocate to the nucleus as homodimers or heterodimers. They then bind to promoters and activate transcription of genes associated with acute phase -21- responses, differentiation and growth arrest. STATS have also been shown to activate junB transcription. IL-6 plays an important role in the immune response. IL-6 is the key factor for antibody production in B cells. Increases of 3-10 fold in IgG, IgM and IgA antibody production have been seen in mononuclear cells treated with recombinant human IL-6 (Cox and Gauldie, 1997). IL-6 and IL-1 synergistically enhance the growth and differentiation of the murine B cells (Vink et al., 1988). Also, IL-6 increases IgA production in murine Peyer’s Patch B cells (Beagley etal., 1989) and involved in T cell function. IL-6 stimulates proliferation of thymocytes and T cells (Cox and Gauldie, 1997) and enhances delayed-type hypersensitivity in mice (Jayaraman et al., 1990). IL-6 is suggested to be involved in glomerulonenephritis as it acts as an autocrine factor for the mesangial cells (Horii et al., 1989; Ruef et al., 1990). It also acts as a hemopioetic factor and is involved in the acute phase response (Cox and Gauldie, 1997). D. 2. IL-6 and its regulation Key cis elements in the IL-6 promoter include a c-fos serum responsive element (c- fos SRE) site and binding sites for activator protein-1 (AP-l), NF-KB, C/EBP (NF -IL6), CANIP responsive element binding protein (CREB) (Tanabe et al., 1988; Ray et al., 1988; Sehgal, 1992). Glucocorticoids (GCs) have also been identified as the important transcription factors in regulation of IL-6. D.2.1. Activator protein-1 (AP-1) AP-l is a family of transcription factors consist of F os (c-Fos, F ra-l , F ra-2, and Fos B) and Jun (c-Jun, Jun B, Jun D) proteins. The Fos/Jun heterodimers or homodimers are known to bind to a DNA consensus sequence (5'-TGAC/GTCA-3') (TPA response element -22- JunD + . + + + ._______~ Furs - - - - Figure 1.2. Dimer combinations of Fos and Jun proteins capable of forming stable AP-l transcription factor complexes. (+) means Jun and/or Fos proteins able to form stable homo or hetero—dimers. (-) means Fos proteins are not able to form stable proteins by associating with each other (Adapted from Liebermann et al. , 1998). -23- (TRE)) (Adcock, 1997) (Fig. 1.2). AP-l is related to the members of other transcription factor family such as activating transcription factor (ATF) and cyclic adenosine monophosphate (CAMP) response element binding protein (CREB). AP-l sites can be found in the genes encoding pro-inflammatory cytokines, cell growth, hematopoietic differentiation (Liebermann et al., 1998), T and B cell activations (Foletta et al., 1998), and apoptosis (Liebermann et al. , 1998). The expression of AP-l is regulated by both transcriptional and post-transcriptional modifications via phosphorylation and dephosphorylation of the proteins (Pennypacker, 1998). The genes encoding Fos/Jun proteins are immediate early genes which do not require de novo protein synthesis (Angel and Karin, 1991). Extracellular stimuli such as UV light, inflammatory cytokines and stress induce AP-l protein production (Liebermann et al., 1998). Recently, it has been shown that LPS can induce activation of JNK thus resulting in the AP-l in macrophage RAW 264.7 cells (I-Iambleton et al., 1996). Cytokine expression in Th1 or Th2 cells can also be regulated by AP-l (F oletta et al. , 1998). The regulated cytokines include IL-2, IF N-y, TNF-[3, IL-4, lL-5, IL-6 and IL-10. AP-l sites have also been found in the IL-6 promoter region at -61 bp to -55 bp and -280 bp to -274 bp (Ray et al., 1990). It is reported that these elements responsed to several agents such as serum, forsokolin and phorbol esters (Ray et al., 1989). However, they are not significant in the induction of IL-6 but might be important in down regulation of IL-6 gene expression. D.2.2. Nuclear factor kappa B (NF ~16) NF-KB was first identified as a B cell nuclear factor that bound to the immunoglobulin K light chain enhancer (Sen and Baltimore, 1986). It is now known that NF-KB binding sites can be found in promoters of many genes such as those encoding IL-2, -24- IL-6, GM-CSF, ICAM-1, and class I MHC (Baldwin, 1996). NF-KB is activated by a variety of stimuli, including LPS, exotoxin, TNF, IL-l, viruses, parasites, T and B-cell mitogens, stress, and phorbol esters (Baeuerle and Henkel, 1994; Baldwin, 1996). NF-KB is a heterodimer protein belonging to the Rel homology family. The Rel family includes proteins such as NF-KBl(p50/p105), NF-KBZ(p52/p100), p65 (Rel A), c-Rel, Rel B and dorsal (Baeuerle and Henkel, 1994; Adcock, 1997) (Fig. 1.3). The Rel homology domain (RHD) can be found at the N-terminus in all of these proteins. This domain is capable of DNA binding, dimerization and interaction with IKB (Baldwin, 1996; Adcock, 1997). The p105 and p100 proteins contain copies of the ankyrin repeat at their c-terminus. These repeats function as protein-protein interaction motifs which are required for the interaction with DNA binding subunits on the NF-KB. RelA, c-Rel and Rel B have potent transactivation domains which are important in transcriptional activation. p50 and p52 are poor transactivators but are the main DNA binding subunits (Adcock, 1997). Heterodimers of NF-KB can bind to the DNA consensus sequence 5'-GGGRNNYYCC-3' with high affinity (Rzpurine; szyrimdine) (Baeuerle and Henkel, 1994; Baldwin, 1996). NF-KB is regulated as an inactive complexes by binding to an inhibitory molecule (IKB) in the cytoplasm (Fig. 1.4). When cells are activated with agents such as LPS or PMA, the IKB molecule is phosphorylated and degraded, resulted in its dissociation from NF-KB and translocation of NF-KB into the nucleus and binding to the targeted genes (Baldwin, 1996). There are at least five different IKB proteins found in cells: IKBa, IKBB, IKBy, Bcl3, and IKB R(Baldwin, 1996; Adcock, 1997). They contain the ankyrin repeats as in the c-terminal domain of p105 or p100 (Baldwin, 1996). NF-KB plays a key role in immune responses -25- Rel B _:l] C-Rel [—1] P55 (RelA) (I. p105 lie-2513’] . lllllllgl p100 llllllI_l L“- ”H" I Figure 1.3. Members of the NF-KB family of proteins (Adapted from Dumont et al., 1998). -26- (B-Mto IKB 9nCOdan Genes '5' -—> (84110 ‘ ox“ encmng genes E \ 18-1110 01 er 08083 i Degraded Figure 1.4. Activation for the mechanism of NF-KB activation (adapted from Finco and Baldwin, 1995) -27- due to its ability to turn on various inflammatory genes. NF-KB sites are present in IL-2, IL-20t receptor, IL-3, IL-4, IL-6 and c-fos gene. NF-KB can be found in B and T cells, and macrophages (Lenardo and Baltimore, 1989; Baeuerle and Henkel, 1994). An NF-KB site has been found at position -73 to -64 bp in the IL-6 promoter and is required for induction of IL-6 (Shimizu et al., 1990). When cells are stimulated by IL-1, NF-KB binds to its specific site and activates transcription of the IL-6 mRNA. Glucocorticoids (GCs)seems to inhibit IL-6 expression by binding the NF-KB p65 to its GC receptor. D. 2. 3. Nuclear factor (NF )-IL6 or CCAA T/enhancer binding proteins (C/EBP) NF-IL6 (C/EBP) was first isolated fiom a rat liver cDNA library (Landschulz et al. , 1988). Six different kinds of C/EBP proteins have been identified, C/EBPa, C/EBPB, C/EBPS, C/EBPy, C/EBPs and C/EBP§(Yamanka et al., 1998). They all have a DNA binding domain close to their c-terminal regions and a transacting domain in their N—terminal regions (Wedel and Ziegler-Heitbrock, 1995). The leucine zipper and basic region are capable of forming dimers (Fig. 1.5). C/EBPa mostly is expressed in liver and adipose tissue. It transactivates genes such as insulin-responsive glucose transporter (GLUT4) and regulates adipocyte differentiation. C/EBPB is also known as NF-IL6, a nuclear factor that mediates the IL-6 response in human glioblastoma cells. It is expressed in almost every tissue. C/EBP B is responsible for the regulation of genes encoding for the acute phase response and cyokines. It is induced by stimuli such as IL- 1 , LPS, TNF or IL-6. It can serve as both an activator and repressor. C/EBPy is a protein that binds to the immunoglobulin heavy chain enhancer. It is expressed in early B-cells. C/EBP5 is also called NF-IL6B. It is highly expressed in lung and has no intron (Lekstrom-Himes and Xanthopoulos, 1998). -23- NH2 NH2 Figure 1.5. Model of the C/EBP structure (adapted form Wedel and Ziegler-Heitbrock, 1995) -29- It is induced by LPS, IL-1, or IL-6 and is a stronger activator than C/EBPB and mostly regulates genes involved in the immune responses and inflammation. C/EBPe is normally expressed in the organs or cells involved in the immune system such as myeloid and lymphoid lineages (Lekstrom-Himes and Xanthopoulos, 1998). There are four isoforms of C/EBPs mRNA after splicing. These four isoforms encode three different proteins of the C/EBPe with molecular weight of 32.2-kd, 27.8-kd and 14.3-kd. C/EBP binds to the DNA consensus sequence of 5'-T(T/G) NNGNAA(T/G)—3' (Akira et al., 1992; Yamanaka et al. , 1998).C/EBP (NF -IL6) site is located at -158 bp of IL-6 promoter with a 14-bp palindromic sequence 5'-ACATTGCACAATCT-3' (Isshiki et al. , 1990). The C/EBP can be induced upon stimulation with LPS, IL-1 or IL-6. C/EBP can bind to the respective site on the IL- 6 promoter and induces expression of IL-6 as heterodimers or homodimers. C/EBP can also interact with NF-KB for maximal expression of IL-6. D. 2. 4. Cyclic AMP response element binding protein (CREB) CREB is the transcription factor that regulates cyclic AMP (CAM) and calcium- dependent genes. This protein can bind to conserved regions of the 8-nucleotide sequence of 5'-TGACGTCA-3' which is located within 100 bp from the TATA box of the targeted genes (Montminy, 1997; Short et al. , 1986). CREB has been identified as a 43-kd protein which is phosphorylated by protein kinase A (PKA) at its consensus site of RRPSY (Gonzalez and Montrniny, 1989). The phosphorylation at one serine (set-133) leads to the dimerization of the CREB proteins on the CRE sites of the promoter of the targeted genes. Increases in cAMP levels or intracellular calcium levels activate CREB. The activation leads to the activation of the calmodulin-dependent kinase IV (CaMKIV), which in turn phosphorylates CREB and consequently stimulates its binding to the CRE site (Fig. 1.6) -30- (Larnprecht, 1999). There are different forms of CREB that have been identified, a, B and 8. They bind to the CRE site with different affinities (Montminy, 1997). cAMP inducing agents such as PGE2 seems to enhance transcription of IL-6. D.2. 5. Glucocorticoids (GCs) as negative regulators GCs can act as both activators or rcpressors of gene transcription. GCs are steroids that exert effects on immunoregulation by binding to their GCs receptor (GR). This receptor usually binds to the 90-kd heat shock protein (l-Isp90) in the cytoplasm (Mka et al. , 1990). Upon activation with GCs, the GR dissociates from the Hsp90, translocates to the nucleus and binds to the targeted DNA (Berg, 1989). The DNA binding domain has a zinc finger structure which can bind to DNA (Berg, 1989). The GC response element (GRE) has a 15 bp consensus sequence of 5'- GGTACANNNTGTTCT-3' palindrome (Drouin et al. , 1992). GREs are located in the 5' flanking region of GRE responsive genes. Binding of GR on this element leads to the alteration of the transcriptional activity (Schmidt et al. , 1994). GR can inhibit cytokine gene expression by binding on the GREs and thus prevent the binding of the transcription factors to their putative binding sites. The GR can also bind directly to transcription factors and abrogate their binding to the DNA and thus inhibit transcription (Paliogiarri et al. , 1993; Caldenhoven et al., 1995). The repressive effects of the GR have been studied widely in the NF-KB transcription by binding to NF-KB directly or by binding to the GRE site to enhance IKBa transcription (Dumont et al., 1998). GREs can be found in the IL-6 promoter. It has been shown that GCs inhibit IL-6 expression by binding to the AP-l and the NF-KB p65 (Ray et al., 1990; Ray and Prefontaine, 1994). D.3. Post-transcriptional control -31- CAMP Ca1+ E CBP TATA ‘ Figure 1.6. Regulation of gene expression by CREB. Phosphorylation of CREB on Set-133 is the critical step in CREB activation (adapted from Lamprecht, 1999). -32- The AU-rich regions in the 3'-untranslated region play a key role stabilizing IL-6 mRNA expression. It is known that the IL-1- and TNF-a-modulated IL-6 mRNA half-lives are different (Ng et al., 1994). Stabilization of mRNA plays a major role in IL-6 mRNA superinduction (Elias and Lentz, 1990; Roger et al. , 1998a). Ultraviolet (UV) B upregulates IL-6 production by enhancing IL-6 mRNA stability (de Vos et al., 1994). IL-lO has been reported to inhibit IL-6 mRN A expression by increasing degradation of the IL-6 transcripts (Takeshita et al. , 1996). GCs such as Dex can inhibit IL-6 production by decreasing mRNA stability of IL-6 (Amano et al., 1993). Therefore, post-transcriptional control is an important regulation in IL-6 gene expression. D.4. Summary In the regulation of IL—6 gene expression, several factors are involved. First, the positive acting transcription factors NF-KB, NF-IL6 (C/EBP), AP-l, and CREB contribute to IL-6 transcription. It is suggested that cooperative effects of NF-KB and NF-IL6 are the most important factors in transcriptional regulation of IL-6. Also, the effects of negative regulators such as GCs are important in the transcription of IL-6 expression. There are multiple regulatory sites for some of these factors which might indicate a role of these factors in the regulation of IL-6 gene expression. Recently, the regulation of mRNA decay was also recognized as an important mechanism in regulating cytokine gene expression. As with other cytokines, the AU repeats in the 3' non coding region of IL-6 mRNA confer instability and thus may play a part in IL-6 protein and mRNA expression. In the future, more work should focus on both transcription and post-transcriptional control of this gene. E. Tumor necrosis factor-alpha ( T NF -c;l and its regulation -33- E.1. Introduction TNF-0t is a 26-kd protein that synthesized and anchored in the cell membrane. When it is released fiom the cell surface, it is cleaved to a 17-kd protein by enzymes (Dayer and Burger, 1994; Beyaert and Fiers, 1998). The secreted protein folds back onto itself into two B-pleated sheets (Jones et al. , 1989). Secreted TNF-0t was found to cause necrosis in some mouse tumors and was the mediator causing cachexia associated with hyperglyceridema in rabbits chronically infected with T typsanoma brucei (Goodwind and Guy, 1973; Rouzer and Cerami, 1980). Monocytic cells are the main source of TNF-0t in viva. Other cells such as basophils, eosinophils, NK cells, and polyrnorphonuclear leukocytes can also produce TNF (V ilcek and Lee , 1991; Tracey, 1994). The TNF family includes two related proteins, TNF-or (cahcetin) and TNF-B (lyrnphotoxin) which bind to the same cell surface receptors (V ilcek and Lee, 1991). TNF-0L plays a major role in beneficial effects as an immunostimulant and also mediate detrimental effects such as wasting during chronic infections. TNF-0t is the main mediator in the development of septic shock caused by Gram negative bacteria (Tracey et al., 1987). TNF-or can activate neutrophils, mast cells, macrophages and endothelial cells in vitro and is considered to be a pro-inflammatory cytokine. In vitro, it can cause selective cytolysis of tumor cells and induces differentiation of monocytes and secretion of IL-1 and IL-6, and induces B cell proliferation and increased antibody production. In vivo, TNF-0t also enhances lipolysis and muscle wasting. It induces shock and is a major mediator in endotoxin-mediated shock. TNF-0t induces fever and is regarded as an endogenous pyrogen. It also induces acute phase responses and stimulates the production of the -34- procoagulant factor. TNF-0t is involved in the pathogenesis of lupus nephritis. It is angiogenic and promotes proliferation of fibroblasts. It also stimulates production of synovial and macrophages (resting) collagenase and PGE2 which contributes to the inflammation and tissue destruction (Dofferhoffet al. , 1991 ; Pauli, 1994; Dayer and Burger, 1994; Beyaert and F iers, 1998). [5.2. Receptors of T NF and signal transduction path ways There are two types of murine TNF-0t receptors which are involved in signal transduction, mediated by TNF-0t: the 55-kd(TNFR1) and the 75-kd (TNFR2) (Beutler and van Huffel, 1994) (Fig. 1.7). These glycoproteins are of similar length but differ in their carbohydrate content. The extracellular domains of these two receptors are similar in sequence (Beyaert and Fiers, 1998). Both receptors bind TNF-or and TNF-B equally well and are members of the nerve growth factor receptor (N GFR)/TNF R superfamily (Dayer and Burger, 1994). The extracellular domain of each of the two receptors contains four cysteine- rich repeats (Dembic et al., 1990). The intracellular domains (or cytoplasmic signaling components) of both receptors are distinct from each other, therefore a different signaling apparatus may connect with each of the two receptors (Beyaert and Fiers, 1998). It has been suggested that the TNFRl mediates cytotoxic signals (apoptosis) and most of the TNF- or responses (Beyaert and Fiers, 1998), while the TNFR2 is involved in T cell proliferation and inhibition of the early hematopoisis (Beyaert and F iers, 1998). It has been shown that binding of TNF-0t to the TNF receptor leads to the formation of the disulfide-linked receptor aggregates. Each TNF trimer contains three cognate receptor binding sites and the change in receptor conformation that results from engagement of the -35- Figure 17 Schematic representation of the TNF receptors, TNF and associated signaling molecules (adapted from Ledgerwood et al., 1999). -35- TNF trimer is sufficient to initiate signal transduction (Beutler and van Huffel, 1994). The multimerization of the receptors with the ligands leads to the internalization and degradation of the ligands in the lysosomes. The intracellular domain of the TNFRl can be divided into a membrane distal domain and membrane proximal domains (Beyaert and F iers, 1998). The membrane distal domain is known as the “death domain” (Tartaglia et al., 1991; Ledgerwoood, 1999). The death domain is responsible for the cytotoxicity. A 34-kd cytoplasmic protein known as TNF receptor-associated death domain protein (TRADD) has been identified and contained death domain at its c-terminus (l-Isu et al. , 1996). This protein interacts with the death domain of the TNFRI. TRADD is an adaptor that can recruit other signaling proteins such as FADD (Pas-associated death domain protein), TRAF2, and RIP (a serine/threonine protein kinase with a death domain), to TNFRl. TRAF2 binds to the c- terrninal domain of the TNFRl and cause NF-KB activation. It also recruits the NF—KB - inducing kinase (NIK) to the membrane, which in turn activates the IKB kinase, a kinase that phosphorylates the IKB thus releasing active NF-KB. Both FADD and RIP are death domains and mediate TNFRl receptor-induced apoptosis. TNF-0t induces a variety of signal transduction pathways. The activation of protein kinase C (PKC) (Brenner et al., 1989), the release of Ca2+ and increases of inositol 1,4,5- triphosphate have all been demonstrated following stimulation with TNF-0t (Kronkel et al. , 1991). Activation of the phospholipase A2 and the release of arachidonic acid by TNF-0t has also been observed. TNF-0t also activates protein kinase A (PKA) and causes increased levels of cAMP (Zhang et al., 1988). Tyrosine kinases might also be involved in the activation by TNF-0t (Donato et al., 1989). TNF-0t can induce a variety of proteins -37- including the induction of IL-6. Other transcription factors activated by TNF-0t are NF-KB, AP-1(c-fos and c-jun), NF-IL6 , CREB and interferon regulatory factors 1 and 2 (lRF -1 and -2) (V ilcek and Lee, 1991). E. 3. Regulation of T NF -agene expression Different stimuli can induce the production of TNF-0t including LPS, synthetic lipid A, enterotoxin, IL-l , GM-CSF, phorbol esters, oxygen radicals, and TNF itself (Dofferhoff et al. , 1991; Pauli, 1994). LPS is the most studied stimulant that induces TNF-0t production in monocyte/macrophage cells. Many agents can inhibit TNF-0t mRNA expression such as dexamethasone (DEX), cAMP, protein kinase inhibitors (staurosporine or H7), cyclosporin A, and prostaglandin E2 (Pauli, 1994). TNF-0t expression is controlled at different levels: transcriptional, post-transcriptional and translational (Beutler et al. , 1986). TNF-0t is encoded by a single copy gene that is approximately 3 kb pairs long. It consists of four exons and three introns. In hrunan, several regulatory sites had been found in the TNF-0r promoter region. These include the AP-l sites (-108/-101 bp), the three KB sites (-600/-57 6 bp, - 242/ -199 bp, -1 18/-89 bp), the cyclic AMP-responsive element (CRE) (-95/—36 bp), SP-l sites, a TATA box and a Y box (Pauli, 1994). In mice, TNF-0t consists of four KB sites on its promoter. LPS-responsiveness is abrogated with the mutation of all four KB sites. Deletion of the KB site from a promoter construct leads to reduction of the reporter activity. This suggests that these KB sites are important in the regulation of transcription of TNF-0t. It has been shown that TNF-0t gene transcription is enhanced 3- fold upon LPS stimulation (Beutler et al. , 1986). The deletion of the KB sites from the TNF- or promoter will reduce its LPS responsiveness (Goldfeld et al. , 1990, 1991). An AP-l site -38- has been found at -108 to -101 bp of palindrome sequence of TGAGCTA, and is PMA- responsive. Deletion of this site markedly reduces the transcription of TNF—or (Rhoades et al., 1992; Pauli, 1994). An NF-IL6 binding site is also found in the promoter of TNF-0t with a consensus sequence of 5'-GGATTTGGAAAGTT-3' (Akira et al., 1992). The role of dexamethasone as an inhibitor for TNF-0'. expression suggests that GCs might be involved the transcription of this gene (Pauli , 1994). cAMP has been shown to be involved in inhibiting TNF-or mRNA expression via protein kinases. Apart from transcriptional activation, the regulation of the TNF-0t gene is also found at the post-transcriptional level (Pauli, 1994; Vilcek and Lee, 1991). TNF-0t gene consists of the repeating units (TTATTAT) in the 3' untranslated region of its mRN A. The AU rich region has been proved to cause instability and superinducibility upon exposure to protein synthesis inhibitors. GCs such as Dex inhibit TNF-0t production by reducing the amount of the TNF-0r mRNA and by blocking its translation (Beutler et al., 1986). F. IL-lB and its regulation E1. Introduction Interleukin (IL)-1 has been implicated as a major inflammatory mediator in a variety of pathological situations. There are two different forms of IL-1, IL-lor and IL-1 B. They are prototypic multi-fimctional cytokines. IL-1 can mediate different biological activities including pyrogenicity, stimulation of the immune response and release of pro-inflammatory mediators (Cavaillon and Haeffner-Cavaillon, 1990). It is found on the long arm of the band q17 of chromosome 17. The precursors of both IL-1 are 31-kd and lack a signal -39- peptide that enables the protein to insert into the Golgi (Dinarello, 1994). The IL-la precursor is more active and IL-1 B precursor is inactive and needs to be cleaved for its activity (March et al., 1985). Mature IL-lor and IL-1 B are both 17-kd. Though they have different amino acid sequences (<26% similarity), the structure of the two isoforms are closely related at three-dimension level and have similar activities (Dayer and Burger, 1994). Expression of IL-1 can be induced by variety of stimuli such as direct contact with activated T cells, cytokines, thrombin, steroid hormones, bacterial toxin (LPS, lipid A), viruses, mitogens, specific forms of immune complexes, fragments of complements, and crystals (Dayer and Burger, 1994; Tocci and Schmidt, 1997). When stimulated with LPS, there are 5 to 20 times more pro-IL-lor production than the pro-IL-l B (Demczuk et al., 1987; Cavaillon and Haeffner-Cavaillon, 1990). lL-lB can induce a wide variety of biological effects. Its action is similar to those induced by TNF-0t. IL-l B is a potent pyrogen and causes fever, sleepiness, lack of appetite, muscular fatigue (Dayer and Burger, 1994; Tocci and Schmidt, 1997). This suggests that IL-1 B may affect structures in the central nervous system. IL-l B also induces symptoms of shock and is a key mediator in acute phase responses and induces the release of the acute phase proteins (Dinarello, 1984). These include C-reactive protein, serum amyloids, fibrinogen, and various protease inhibitors. Injection of IL-1 B also leads to the release of corticotropin releasing factor (ACTH) and cortisone, which is anti-inflammatory in nature and inhibits cytokine gene expression (Dayer and Burger, 1994). This might be a biological feedback loop. IL-l B also stimulates both B and T cells, causes chemotaxis of neutrophils and monocytes, and release of prostaglandins. It also induces modification of metabolism -40- as it reduces food intake in animals, decreases body weight and exhibits anorectic properties (Dinarello, 1994). IL-l B can induce expression of multiple genes including IL-l B itself , TNF, IL-2, IL-6, IL-8, IL-12, GM-CSF and also cytokine receptor genes such as IL-2, IL-3, and IL-5 receptor. The induction of these genes might be responsible for the biological effects caused by IL-1 B. F.2. Receptors of IL-1 ,6 and signal transduction When monocytes are stimulated, both proIL- 1 or and IL-1 B are produced and released from the nucleus (Cavaillon and Haeffner-Cavaillon, 1990). The proIL-lor is active as a precursor and remains intracellular. The synthesis of the prolL—lor is associated with the microtubules of the cytoskeleton structure. The prolL—lor remains in the cytosol after translation and is then myristoylated. Myristoylated IL-lor is transported to the cell membrane (Kurt-Jones et al., 1985). Membrane IL-lor is biologically active and accounts for only 5% of the total proIL-la (Dinarello, 1997). The myristoylated pro IL-lor can also be cleaved by calpain and released into the extracellular compartment or it can bind to nuclear DNA (Kobayashi et al., 1990). Following synthesis, proIL-lB remains in the cytosol until cleavage by protease. Cleaving is required for its optimal biological activity before transported out of the cells. This specific protease for cleaving IL-lB precursor is known as the IL-lB converting enzyme (ICE). It belongs to the family of the cysteine proteases known as caspases with a molecular weight of 20-kd and it does not cleave the IL- la precursor (Dinarello, 1994). After binding to cells, IL-l B immediately induces several biochemical events. IL- lB binds to the extracellular domain of either type I or type II IL-1 receptor (IL-1R) -41- (Dinarello, 1997). Following binding of the IL-1 B to the receptor, a structural change of the IgG-like domain of the receptor is observed. The binding of IL-1 B to this receptor leads into dimerization of the two cytosolic domains of the two IL-lR. Then activation of the putative GTP binding sites on the cytosolic domains, binding of a G-protein, hydrolysis of the GTP and activation of phospholipase are involved (Dinarello, 1997). Diacylglycerol or phosphatidic acids are generated following the hydrolysis of this phospholipid. Phosphorylation of the JNK and the p38 MAP kinase and downstream phosphorylation of the MAPKAP-kinase-Z and phosphorylation of the heat shock protein 27 (hsp27) occur. Following binding of the IL—1 B to the IL-1 RI, inhibitory KB (I-KB) is phosphorylated and translocation of the NF-KB to the nucleus is observed (DiDonato et al., 1995). E3. IL-1 ,6 and its regulation The IL-lB promoter contains sites for AP-1(c-jun), the octamer binding protein (Octa—l), NF-KB, Pu.l(transcription factor Spi-l), NF-IL6, and a TATA box and CAAT box. AP-l sites have been identified in the upstream region of the IL-lB promoter as TCCGTCA (-610/—604) and ATGTGTCAT (-208/-200) (Cavaillon and Haeffner-Cavaillon, 1990). AP-l helps sustain IL-lB gene transcription in response to PMA . However, no major role has been indicated for AP-l in IL-1 B transcription. The NF-KB site is found on the transcription promoter (~286 to -296) (Cavaillon and Haeffner-Cavaillon, 1990) and further upstream (-2757 bp) (Tocci and Schmidt, 1997) of IL-lB gene with a sequence of 5'-GGGAAATCC-3'. Both sites are required for maximal IL-l B transcription. Agents that inhibit NF-KB activation also block IL-l B transcription. The sequence between -2896 and -2846 bp of IL-1 B promoter is shown to bind the heterodimer of cAMP response element -42- binding protein (CREB) (Dinarello, 1996). Increased cAMP levels induced by agents such as histamine or PGE2 augment IL-l B gene expression and synthesis. However, increased cAMP levels deplete LPS-induced IL-l B gene expression. IL-1 has the octamer binding site in its promoter and a single base difference will affect the binding of Oct (Lakin et al. , 1995). Octamer binding protein can bind to its specific sequence ATGCAAAT or its complement ATTTGCAT (Falkner et al., 1986). The octamer motif is found in various eukaryotic regulatory elements and is responsible for the transcription of variety of genes which include immunoglobulin genes in B cells and cytokine genes in the immune and nervous systems (Schreiber et al., 1989b). Ubiquitous Oct-1 is expressed in a wide variety of mammalian cells and controls the general octamer site-dependent transcription, Oct-2 is only expressed in lymphoid cells and neuronal cells, and regulates B cell-specific expression (He at al. , 1989; Schreiber et al. , 1990 ). Some other octamer proteins also have been found in the embryonic cells and testis. Apart from its role as a transcription factor, Oct-l has also been shown to stimulate DNA replication (Verrij zer et al. , 1990). The octamer motif is located next to the AP-l site on IL-1 B promoter. Transcription of the IL-1 B does not require de novo protein synthesis, suggesting that the activation of pre-existing transcription factors occurs. Cytokines such as IFN-y and GM-CSF synergize with LPS in inducing IL-l gene transcription. Post-transcriptional control of IL-1 B is also implied as the addition of CHX increases the stability of IL-1 B mRNA. IL-l B contains AU—rich regions in its 3'-untranslated region (UTR). IL- 1 0t transcripts are much more stable than those for IL-1 B (Turner et al. , 1988). It has been shown that IF N-y is able to increase IL-l B production by enhancing both IL-1 B -43- transcription and to increase mRNA stability (Arend et al., 1989). Different mRNA half- lifes of IL-1 B were found in monocytes and macrophages. Both IL-4 and GC hormone dexamethasone (Dex) can serve as the negative regulators of IL-1 B by increasing mRNA degradation (F enton, 1992; Lee et al., 1988). The destabilization effect can be blocked by the addition of protein synthesis inhibitors, suggesting that de novo protein synthesis is required for the mRN A degradation (Lee et al. , 1988). It has been indicated that the NF-IL6 sites in IL-lB are sensitive to Dex. In summary, transcription factors such as AP—l , NF-KB, NF—IL6 and Oct are involved in the transcription of IL-1 B gene expression. It has been mentioned that these transcription factors work cooperatively for its maximal gene expression (Adcock, 1997). Post- transcription control also plays another important role in the IL-1 B gene expression as increased mRNA stability can be observed with modulators. However, negative regulators such as IL-4 and GC can increase mRNA degradation of IL-1 B. Therefore, the regulation of IL-1 B is complicated, and future work on the its regulation is needed for better understanding. G. Rationale for this research VT can superinduce cytokine production both in vivo and in vitro systems (Miller and Atkinson, 1986; Azcona-olivera eta1., l995a,b; Ouyang et al., 1996). Therefore it is reasonable to suggest that VT can also superinduce cytokine expression in macrophage cells. Viriyakosol and Kirkland (1995) suggested a model of LPS activation of macrophages (Fig. 1.10) where in a complex of LPS :LBP binds to the CD 1 4 receptor and an unidentified second receptor. The binding of this LPS:LBP:CD14 complex to the hypothetical second receptor -44- then leads to signal transduction. Since CD 14 is incapable conducting the signal transduction itself, other protein kinases such as tyrosine kinase plays a key role in transducing the signal for activation (Weinstein et al. , 1992). When macrophages are activated by LPS, proinflammatory cytokines such as TNF-0t, IL-6, IL-1 B and IL-8 are upregulated (Manthey et al., 1994; Salkowski et al., 1995; Su etal., 1995). It is hypothesized that VT increases the LPS-induced cytokine production by increasing transcription of the cytokines and/or prolonging the mRNA half-life of the cytokines. These interactions of VT with LPS may further enhance the effects of LPS such as endotoxic septicemia, the shock-like syndrome, organ damage and apoptotic cell death. The synergistic effects of LPS and VT probably contribute to the pathogenic manifestations associated with trichothecene exposure. The exact mechanisms of the superinduction of macrophage cytokine gene expression by VT remain unclear. Protein synthesis inhibitors, such as CHX, have been shown to enhance and prolong the normally transient expression of specific gene transcripts via impaired synthesis of high tum-over proteins that down regulate transcription, or via decreased mRN A half-life of those proteins (Sun et al., 1993; Cockfield et al. , 1993; Efrat and Kaempfer, 1984). Previous studies have also revealed increases in activity of transcription factors such as NF- KB in IL-2 gene regulation in the EL-4 cells with VT (Ouyang et al. , 1996). Therefore, it is reasonable to suggest that VT can also superinduce cytokine expression in macrophage utilizing similar mechanisms. The main goal of this dissertation is to better understand the cellular and molecular mechanisms by which VT superinduces cytokines in macrophages and monocytic cells. -45- .3353 cougzoe was :33 H> mo cozofioafi BABE mHZU—O-HVU a setups—won mmZHMOH>U mo ZOHH £9625 Al. ..I. 0:95:32 CHAPTER II MODULATION OF IL-lB, IL-6, TNF-a SECRETION AND MRNA EXPRESSION BY THE TRICHOTHECEN E VOMITOXIN IN THE RAW 264.7 MURINE MACROPHAGE CELL LINE -47- ABSTRACT Oral exposure of mice to vomitoxin (VT) has been previously shown to enhance gene expression of several cytokines associated with macrophage activation. Here, the effects of exposure to VT in vitro on cytokine secretion and mRNA expression were determined in the murine macrophage cell line RAW 264.7. Enzyme-linked immunosorbent assay (ELISA) of supematants revealed that significant increases in secreted tumor necrosis factor alpha (TNF-0t) were observed 2 days after exposure to VT at 100 ng/ml and 250 ng/ml both with and without Lipopolysaccharide (LPS) activation. While VT did not affect IL-6 secretion in the absence of LPS, significantly increased IL-6 production was observed in culture supematants after 1, 2 and 5 days exposure to VT at 250 ng/ml in the presence of LPS. Soluble IL-l B was not detected in control or VT-treated cell cultures with or without LPS activation. Irnmunochemical staining of intracellular cytokines in conjunction with flow cytometric analysis was used to detect the effects of VT on the percentage of positive cells and output per cell. The percentage of cells that produced intracellular TNF-0t were significantly increased at 100 and 250 ng/ml VT with and without LPS whereas increased IL—6 output per cell was observed at 100 and 250 ng/ml VT with LPS. To assess the effects of VT on cytokine mRN A expression, RAW 264.7 cells were analyzed semi-quantitatively using reverse transcription-polymerase chain reaction(RT-PCR) in conjunction with Southern hybridization analysis. Elevated TNF-or mRNA was observed at 100 and 250 ng/ml VT at 6 and 24 hr in the absence of LPS. With the addition of LPS, superinduction of TNF-0t was not observed in the presence of VT. Increased IL-l B and IL-6 mRNAs were observed at 100 and 250 ng/ml VT at 24 hr in the presence of LPS. These results demonstrated that -48- VT could superinduce both cytokine secretion and mRNA levels in macrophage cultures. INTRODUCTION The trichothecene vomitoxin (VT or deoxynivalenol) is produced by several Fusarium species and has been identified as a common contaminant in world wheat and corn supplies (Rotter et al, 1996). VT and other trichothecenes are potent protein synthesis inhibitors that can cause acute shock-like toxicity or chronic effects such as feed refusal in experimental animals (Cundliffe et al, 1974; Bamburg, 1983). Notably, exposure to trichothecenes in viva inhibits DNA and protein synthesis in bone marrow, lymph nodes, spleen and thymus (Rosenstein and Lafarge-Frayssinet, 1983). Trichothecenes can either be immunosuppressive or immunostimulatory depending on the dose and timing of exposure (Pestka and Bondy, 1994). For example, trichothecenes increase susceptibility to bacterial and viral infections, and can impair cell-mediated and humoral immunity (Arnold et al, 1986; Tryphonas et al, 1986). Paradoxically upon dietary exposure to VT, mice exhibit elevated serum. Irnmunoglobulin A (IgA), circulating IgA immune complexes and mesangial IgA deposition in the kidney (Dong and Pestka, 1993). These features are highly analogous to the human glomerulonephritis, IgA nephropathy (D'Amico, 1987). Several cytokines are potentially involved in differentiation of B cells to IgA secretion (McGhee and Kiyono, 1993). Prior exposure to VT in vitro enhances CD4+ cell mediated help for IgA production by B cells through elevated cytokine production (Warner et al, 1994). Ingestion of vomitoxin can also increase the percentage of total T cells, CD4+ cells and CD4+/CD8+ cell ratio in Peyer's patches (PP) (Pestka et al, 1990a,b). Increased mRNAs for and secreted forms of interleukin- (IL-) -2, -4, -5 and/or IL-6 are observed upon -49- exposure to VT or cycloheximide (CHX) (Warner et al, 1994; Azcona-Olivera et al. , 1995a; Ouyang et al, 1996). Thus, VT-induced elevation of IgA production might be related to the superinduction of various T helper cytokines. Macrophages are also pivotal regulators of inflammation and immunity (Solbach et al, 1991). Besides being involved in microbial clearance, they play an important role in regulating T cell function, proliferation, regulation of antibody production and production of mediators for cellular immunity. Many of these activities are mediated through the proinflarnmatory cytokines (Nathan et al, 1987). Since these cytokines may directly or indirectly affect IgA production by modulation of B and T cell function, respectively, macrophages may potentially be to be involved in the VT-induced hyperelevation of IgA. Superinduction of inflammatory cytokines occurs upon addition of other protein synthesis inhibitors such as CHX and has been observed in several macrophage cell lines (Bendtzen, 1983). VT has also been reported to enhance the release of IL-1 in the peritoneal macrophages (Miller and Atkinson, 198 6). Recently, we have determined that oral exposure of mice to VT causes transient elevation of mRNAs for cytokines associated with macrophage (IL-1B, IL-6, and TNF-0t) as well as T helper cell activation (Azcona-Olivera et al. , 1995b; Zhou et al., 1997) within 2 hrs. As yet, the mechanistic basis for VT-stimulated macrophage gene expression in viva remains unknown. The objective of the study was therefore to investigate the toxic effects of VT on cytokine secretion and mRNA using murine RAW 264.7 cells as a cloned macrophage cell model. The results suggested that VT could superinduce both cytokine secretion and mRNA levels in macrophage cell cultures. -50- MATERIALS AND METHODS Experimental design. The RAW 264.7 murine macrophage cell line (TIB 17) was obtained from the American Type Culture Collection (ATCC) (Rockville, MD). Cells were maintained in Dulbecco's modified Eagle's tissue culture medium (DMEM; Sigma Chemical Co., St. Louis, MO) supplemented with penicillin (100 U/ml), streptomycin (100 jig/ml), sodium pyruvate (1 mM), NCTC-135 medium (Gibco BRL, Chagrin Falls, IL) and 10 % (v/v) fetal bovine serum (FBS; Gibco Laboratories, Chagrin Falls, IL) at a temperature of 37 °C in 5 % C02. For toxin exposure, cells (2.5 x 105 per ml) were seeded in 6- or 12-well flat- bottomed tissue culture plates (Corning, NY) in the absence or presence of l jig/ml Salmonella typhimurium lipopolysaccharide (LPS; Sigma) and 0 to 500 ng/ml of VT (Romer Labs, St. Louis, M0) for various time intervals. Cultures were centrifuged at 300 xg for 10 min to separate supernatant from cells. Macrophage cell numbers and viabilities were assessed by trypan blue (Sigma) dye exclusion using a hemacytometer (American Optical, Buffalo, NY) as described by Strober (1991). Cytokine assay. IL-6 and TNF-0t concentrations were measured by ELISA as described by Dong et al (1994) using rat anti-mouse interleukin monoclonal antibodies (Pharmingen, San Diego, CA) and streptavidin horseradish peroxidase conjugate (Sigma). The IL-l B assay was performed by a modified ELISA in which Irnmunolon IV Removawell microtitre strips (Dynatech Laboratories, Chantilly, VA) were coated with 50 141/W611 of monoclonal hamster anti-IL-l B antibody (Genzyme, Cambridge, MA) diluted 1: 1000 in phosphate buffered saline (PB S)and incubated at 4°C by overnight. Plates were washed three -51- times with PBS containing 0.05% (v/v) Tween 20 (Sigma) (PBS-T). To reduce non-specific protein binding, 300 pl PBS containing 1 % (w/v) bovine serum albumin (BSA) was added to each well, incubated for 30 min at 37°C and then washed four times with PBS-T. Reference IL-lB or samples were diluted in DMEM with 10 % (v/v) FBS, and 50 pl was added to appropriate wells. Plates were incubated for 60 min at 37°C and then washed four more times with PBS-T. Rabbit polyclonal anti-IL-l B antibody (Biosource, Camarillo, CA), diluted 1:1000 in PBS containing 1 % (w/v) BSA, was added to each well and the plate incubated at room temperature for 60 min. The plates were then washed six times with PBS- T, and 50 pl of goat anti rabbit IgG antibody conjugated with horse-radish peroxidase (Cappel Laboratories, Durham, NC) diluted 1 : 5000 in PBS with 1% (w/v) BSA was added to each well and incubated for room temperature for 60 min. ELISA plates were wasth eight times with PBS-T. Substrate (100 pl) consisting of 3', 3', 5', 5'- tetrarnethyl benzidine ( 0.1 mg/ml; Fluka Chemical Corp., Ronkonkoma, NY ) in 0.1 M citric-phosphate buffer (pH 5.5) and 0.003 % (w/v) hydrogen peroxide (11202) was added to each well and incubated 20 minutes at room temperature for color development. The reaction was terminated with 100 pl of 6 N sulfuric acid (H2804). Absorbance was measured at 450 nm with Vmax kinetic microplate Reader (Molecular devices, Menlo Park, CA) and interleukins production quantified using the Softmax software (Molecular Devices). Intracellular cytokine and flow cytometry staining of TNF-0t and IL-6. Monoclonal anti-mouse IL-6 (Biotinylated. rat IgG2a, MPS-32c11), biotinylated rat IgG2a (K isotype, R35-95), anti-mouse TNF-0t (PE conjugated; mouse IgGl, MP6-XT22), mouse IgG] (PE conjugated, K isotype, R3-34), and anti-mouse CD32/CD16 (F cy II/III receptors) -52- (rat IgG2b, 2.4G2) were purchased from Pharrningen (San Diego, CA). Cells were cultured as described above and stained for intracellular cytokines by a modification of the method of Sander et al. (1991). Briefly, cells were harvested with cold Dulbecco's phosphate buffered saline (DPBS, Sigma) containing 1 %(v/v) heat inactivated F BS (Gibco) and centrifuged at 300xg for 10 min. The cell pellet was washed with DPBS and blocked with anti- mouse CD32/CD16 F c Block antibodies for 30 min at 4°C for prevention of nonspecific binding. Cells were fixed on ice using cold DPBS containing 4 % (w/v) paraformaldehyde (Sigma) for 40 min. After washing with DPBS, cells were stained for 30 min on ice with fluorochrome conjugated cytokine specific antibodies or biotinylated Monoclonal antibodies (Mab) (Pharrningen) diluted in DPBS containing 0.1 % saponin (Sigma) and 0.1 % sodium azide (Sigma). Isotype controls were incubated at this step too. Cells treated with biotinylated Mab were washed in saponin buffer and subsequently incubated with streptavidin conjugates (Phanningen) for 20 min on ice. Cells were resuspended in DPBS and stored on ice before analyzed. Samples were analyzed with a F ACS Vantage flow cytometer (Becton Dickinson, San Jose, CA) equipped with Argon ion laser, and Consort 32 computer using Lysis 11 Software. The 488 nm Line of the Argon laser was used to excite fluorescein isothiocyanate (F ITC) and phycoerythrin (PE) fluorochromes which were quantified at 530 at 15 and 575 :1: 13 nm respectively. Electronic color compensation was accomplished using single color controls. Data collection was limited to lymphocytes selected sequentially via gates in cell scatter and 1 color fluorescent cytograms. List mode data files of 15,000 - 20,000 cells were gathered. Measurement of Cytokine mRNA abundance. Total cellular RNA was extracted -53- fi'om RAW 264.7 cells by a modification of the single step method (Chomczynski and Sacchi, 1987) using RNA-Stat-60 isolation reagent (Tel test " B" inc., Friendswood, TX). Reverse transcription (RT) for first strand cDNA synthesis was carried out by the method of Kawasaki (1991). For RT, RNA (up to 2 pg) in a maximum of 18.7 pl of Diethyl pyrocarbonate (DEPC) water was mixed with 1.1 pl of 45 mM oligo dT15 mer (Promega Co., Madison, WI) and incubated for 10 min at 70°C. Immediately following incubation, 10.2 pl of RT master mix [comprised of 6 p1 5X RT buffer, 1.5 pl 10 mM of four deoxynucleotides mix (dNTP mix; Boehringer Mannheim, Indianapolis, IN), 0.7 pl ofRNase inhibitor (40 U/ml; BM) and 2 p1 of M-MLV reverse transcriptase (200 U/ p1; Gibco BRL)], was added, mixed and then incubated at 42°C for 1 hr. The reaction was terminated by incubation at 70°C for 10 min and cooling on ice. The RT products were then stored at -80°C. cDNA products were amplified by PCR according as described by Svetic et al (1991) employing the modifications of Azcona-Olivera et al (1995a). Primer and internal Oligonucleotide probes (Table 2.1) were synthesized at Macromolecular Structure Facility (Michigan State University). Bz-microglobulin (BZMG) was used as a " house keeping gene to verify initial quantities of RNA and the integrity of RNA preparation. In brief, 3 pl of RT reaction product was added to a PCR master mix comprised of 6 pl of 25 mM MgCl2 (Perkin Elmer Gene Amp PCR System, Norwalk, CT), 5 pl of 10X PCR buffer (Perkin Elmer), 4 p1 of 10 mM dNTP mix, 2 pl of sense primer (0.2 pg/pl), 2 pl antisense primer (0.2 pg/pl), 0.5 pl of Taq polymerase (5 U/ pl; Boehringer Mannheim) and variable amounts of DEPC- treated water to make up the volume to 50 pl. Following mixing, 75 pl of mineral oil was added and PCR was performed in a 9600 Perkin Elmer thermocycler. The program entailed -54- Table 2.1. Primer Sequences for Cytokine cDNA Amplification and Probe Sequences for Southern Blot Detection of Amplified cDNA Product Sense and antisense primer Probe Cytokine 5’ 3’ 5’ 3’ IL-lB TTGACGGACCCCAAAAGATG“ ATTGTGGCTGTGGAGAA‘ AGAAGGTGCTCATGTCCTCA' IL-6 GTTCTCTGGGAAATCGTGGA‘ TGTGCAATGGCAA’ITCTGAT" TGTACTCCAGGTAGCTATGG’ TNF-0t TCTCATCAGTTCTATGGCCC" CCTGTAGCCCACGTCGTAG‘ GGGAGTAGACAAGGTACAAC" 13ch TGACCGGCTTGTATGCTATC‘ CTCACGCCACCCACCGGAGA“ CAGTGTGAGCCAGGATATAGa References for Oligonucleotides are designated by letter as follows: 'Personal communication from Dr. David Shire, Sanofi Elf Bio Recherches; °Svetic et al.( 1991); cMontgomery and Dalhnan (1991); “Designed in Biochemistry Macromolecular Facility Laboratory with Computer Program OLIGO(National Biosceinces); ‘Wesselingh et al.(1994). -55- an initial denaturation step for 5 min at 95°C, followed by three temperature cycling steps: denaturation for l min at 95°C, primer annealing for 1 min at 52°C, and extension for 3 min at 72°C. This was repeated for an optimized number of cycles for each transcript(15, 18, 20 and 15 cycles for BZMG, IL-l B, IL-6 and TNF-0t , respectively). The final cycle concluded with a 10 min incubation at 72°C. RT-PCR products (10 pl) were electrophoresed on a 2 % agarose gel at 75 V for 6 hr. Following staining with 1 mg/ml of ethidium bromide, the bands were visualized by UV light and photographed using Polaroid Type 55 film (Polaroid Co., Cambridge, MA). Gel denaturation and neutralization as well as DNA transfer on to nylon membranes (Nytran, Schleicher & Schuell Inc., Keene, NH) were carried out by the procedures according to Sambrook et al. (1989). 32P-labelled internal oligonucleotides(specific for each transcript) were prepared using a DNA 3'- end labeling system (Promega Co., Madison, WI). Following cross-linking and prehybridization at 42°C for 2 hr in a buffer containing 6X SSPE, 10X Denhart's, 1% sodium dodecyl sulfate (SDS) and 50 pg/ml of denatured herring sperm DNA, blots were hybridized in a solution (6X SSPE, 10% SDS) containing 32P-labeled probe at 49°C for 18 hr in a hybridization incubator (Robbins Scientific, Sunnyvale, CA). The hybridized membranes were washed once in 6X SSPE containing 0.1 % SDS at 49°C for 20 min and once more in 2X SSPE at room temperature for 10 min with shaking. All oligoprobes had a minimum specific activity of 1 x 109 cpm/ pg and all hybridizations were performed with no less than 107 cpm/ml. Blots were exposed to Kodak X-OMAT-AR film (Eastman Kodak, Rochester, NY) with an intensifying screen at —80°C for variable length periods depending on signal strength. -56— Following autoradiography, resultant gel bands were analyzed using a Scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA) and GS365W Densitometer Program (Hoefer). Each composite band (consisting of three separate samples lanes) was divided to its corresponding BZMG band and the resulting ratio was compared to all other tirnepoints and doses. This facilitated the comparison of relative cytokine mRNA expression across all the experimental groups. Statistical analysis. A one way analysis of variance (ANOVA) using Student- Newman-Keuls tests and Dunnett's test or Kruskal Wallis AN OVA on ranks were applied to parametric data and non-parametric data with application of Sigma-Stat Analysis System (J andel Scientific, San Rafael, CA). A p value of less than 0.05 was considered statistically significant. RESULTS Eflects of VT on cytokine secretion. To determine the potential effects of VT on IL- 1B, IL-6, and TNF-0t release, RAW 264.7 cells were incubated with 0, 50, 100, 250 and 500 ng/ml of VT in the absence or presence (1 pg/ml) of LPS for 1, 2 and 5 days. In the absence of LPS stimulation, IL-l B and IL-6 supernatant levels were negligible at all time and toxin treatments (data not shown). In contrast, TNF—0t levels were significantly increased by VT at 100 and 250 ng/ml without LPS stimulation at days 1 and days 2 (Table 2.2). TNF-0t was not detectable in the supernatant after 5 days. When RAW cells were activated with lpg/ml LPS, significant increases of IL-6 were also detected in the presence of 250 ng/ml of VT at day 1, 2 and 5 incubation time whereas IL-l B was undetectable. TNF-0t was significantly increased by 50 ng/ml VT on day 1, 100 ng/ml VT on day 1 and 2, and 250 VT ng/ml on -57- Table 2. 2. Effect of VT on Proinflarrmatory Cytokine Secretion by RAW 264.7 cells. Day Vomitoxin mm 11-11 (ng/ml) LPS - LPS + LPS + 1 0 0.89:t0.18' 3.44s 0.48' 10.1w 2.39’ 50 0771:009’ 4.30s 0.18” ' 15.32:. 2.56’ 100 1.30i0.12' 4.92: 0.42b 19.983: 4.02 250 2.95: 0.22b 3.06: 0.37a 4754* 4.48b 2 0 1.62:1: 0.03’ 11.48:}: 1.85' <1’ 50 2.46: 0.30’ 15.56:: 4.10’J’ 6.01:1: 0.93’ 100 5.021: 0.23“ 20.14 i 6.83" 11.48 a 9.75a 250 14.00r 0.93c 21.66i 1.73” 42.59a22.13b 5 0 <1 a " <1’ 12.37:: 4.41’ 50 <1 ’ <1’ 13.22:: 0.72’ 100 <1 . <1- rams 3.39a 250 <1 . <1: 2608* 2.97" RAW 264.7 cells (5 x 10’ cells/ml) were cultured with VT and with and without LPS. Values are means :t S.D. (n=3). In the column on the same day, values with different superscripts differ significantly (P < 0.05). IL-6 production of cells treated with 0 pg/ml LPS are all < Ing/ml. -53- day 2. At higher concentrations of VT (500 ng/ml), cell proliferation was markedly inhibited and thus cytokine levels showed a pronounced depression (data not shown). Based on these results, 100 ng/ml and 250 ng/ml of VT were selected for firrther examination of cytokine superinduction in subsequent experiments. Effects of VT on cytoplasmic cytokine levels. To characterize the effects of VT on the intracellular cytokine expression, RAW cells were cultured with O, 100 and 250 ng/ml VT and 1 pg/ml LPS for 24 hr and then intracellular IL-6 and TNF-0t were measured by flow cytometry. Fig. 2.1A shows typical fluorescence profile of IL-6 positive-cells with and without VT treatment. As shown in Fig. 2. 18, the percentages of VT-treated cells containing cytoplasmic IL-6 in RAW cells were not significantly different than controls. However, VT at both 100 and 250 ng/ml significantly increased the level of cytoplasmic IL-6 in cells as indicated by elevated average fluorescence intensities as compared to the control (0 ng/ml) (Fig. 2.1C). Figures 2.2A and 2B contain the fluorescence profiles for TNF-0t positive cells. Here, percentages of cells containing TNF-0t were significantly increased at 100 and 250 ng/ml VT as compared both to control with and without LPS (Fig. 2.2C). In contrast to IL-6, the average level of cytoplasmic TNF-or positive cells was not significantly affected by VT treatment (Fig. 2.2D). Eflects of Wm cytokine gene expression. To relate VT-induced superinduction of cytokine secretion to cytokine mRNA expression, RAW 264.7 cells were treated with combinations of VT and LPS for 2, 6 and 24 hr and cytokine mRNA abundance was measured by using RT-PCR in conjunction with Southern analysis. IL-l B and IL-6 mRNAs -59- 100 CELL COUNT 8 0‘ 1 1 101 102 103 FLUORESCENCE 60 :WOW 12w EWOW -VT1WW ,4..- i =agm ”“1"me g 34w .3- .oo- —-— 0 Eu E 000 §:m4 5 4W1 3 i w an« 0 r 0 Fig.2.l. Flow cytometric analysis of cytoplasmic IL-6 in RAW 264.7 cells after incubation with VT for 24 hr in the presence of LPS. Cells were fixed and stained with specific antibody and subjected to by flow cytometry. Histograms contain data for cells treated with VT are compared with the control non-VT treated cells in the presence (A) of LPS. The data are representative of three experiments. Each histogram represents cell frequency per 15, 000 cells (ordinate) v. irnmunofluorescent (abscissa, log scale). (B) represents percentage of cytoplasmic IL-6+ cells following stimulation with LPS and VT for 24 hr. Data in (C) represents mean fluorescence intensity or average channel number for cytoplasmic IL-6 cells population samples. Results in (B) and (C) are mean :t SEM taken from three replicates cultures of each doses of LPS and VT. Data were analyzed by using the one way ANOVA (P<0.05; Student’s-Newman-Keul’s test). -60- 8 8 8 % OF POSITIVE CELLS (TNF-a) 8 LPS+ . CDV'I’OW CELL COUNT -vnoomm -VT250119I11I -‘ a. 8 8 § 1 L 1 INTENSITY (TNF-a.) 8 10° 1'01 102 o FLUORESCENCE LPS (091ml) Fig.2.2. Flow cytometric analysis of cytoplasmic TNF-0t in RAW 264.7 cells afler incubation with VT for 24 hr in the absence (A) or presence (B) of LPS. The experiments was reported and analyzed as in Fig.2. 1. legend. (A) and (B) indicate histograms of cytoplasmic TNF-0t+ cells following stimulation with VT for 24 hr in the absence or presence of LPS, respectively. (C) and (D) represent percentage cytoplasmic TNF-a ”'cells and mean fluorescence intensity, respectively. -61- were not affected by VT in the absence of LPS (Fig. 2.3A and 2.4A). However, in LPS- stimulated cultures, IL-l B mRN A abundance, as revealed by cDNA hybridization signal after RT-PCR, was 3- and 4-fold higher than controls for cells treated with VT at 100 and 250 ng/ml, respectively after 24 hr (Fig. 2.38). Similarly, relative IL-6 mRNA abundance was increased 2- and 8-fold at 100 and 250 ng/ml VT, respectively in LPS-stimulated cultures at 24 hr (Fig. 2.4B). Consistent with supernatant concentrations, TNF-0r mRNA abundance was increased 2-4 fold at 6 and 24 hr with 100 and 250 ng/ml VT-exposure in the absence of LPS (Fig. 2.5 A, B). With LPS stimulation, TNF-0t mRNA expression increased very early in LPS-treated culture (Fig. 2.5A, C) and diminished rapidly at all three VT doses. DISCUSSION RAW 264.7 cells were selected for study of VT-induced cytokine effects because this clonal macrophage model produces high levels of IL-6 and TNF-0t in culture upon activation with LPS. The results of this study demonstrated that exposure of those cells to VT could enhance mRNA expression of proinflammatory cytokines, IL-1 B, IL-6 and TNF-0r and increase secretion of IL-6 and TNF-0t. These data are consistent with earlier findings that VT exposure elevates expression of macrophage cytokine mRNAs in viva (Azcona-Olivera et al., 1995a,b; Zhou et al., 1997). The three cytokines studied herein exhibited markedly different patterns of mRNA expression and protein secretion following VT exposure. For example, IL-l B gene expression increased within 2 hrs of LPS stimulation but remained elevated in the presence of VT. This is consistent with the findings in viva that IL-lB mRNA abundance increased -62- Vomitoxin 0 100 250 0 100 250 (us/ml) 2hr - c- c‘ 6 hr 24 hr _ A 1.2 1.0 ~ 0 2 81(18- 8 Q ‘7 0.6 - =.-' "6 0.4 ~ x3. :1: 0.2 - —o— VT 0 ng/ml —- — VT 100 ng/ml —A-— VT250 ng/ml B 0.0 1 r T 2 6 24 Hr of Incubation Fig. 2.3 Relative abundance of IL-lB mRNA in RAW 264.7 cells after in vitro exposure to VT with and without LPS. (A) indicates Southern analysis of RT—PCR products form RAW 264.7 cells. Each dose and time point represents three separate lanes prepared from three different replicates; (B) indicates image analysis data for above autoradiographs. Data are percentage of maximal signal based on pooled measurement of three lanes for each dose and time point. -63- LPS- LPS-1- Vomitoxin 0 100 250 0 100 250 (HS/ml) 2hr a“ “C u. 6'" ~ - - 24hr .. w v A 1.4 ~ 0 2 N O. s 3 ’5 O '5 “I O! 0‘2 ‘—-—- VT 100 nglml -£-— VT250 ng/ml B 0.0 . . . 2 s 24 Hr of Incubation Fig. 2.4. Relative abundance of IL-6 mRNA in RAW 264.7 cells after in vitro exposure to VT with and without LPS. Experiment was conducted and analyzed as in Fig.2.3. -64- Vornitoxin 0 100 250 0 100 250 (ng/ml) 2 hr 6 hr 24 hr A LPS - LPS + 1.4 ch2 4 2 $1.0 4 .9 e03 ~ 11- Eos - ‘80 4 ~ g - + VT 0 ng/ml \ “0.2 y —I—> VT 100 nglml B —&— VT 250 ngl C 0.0 I I ' 2 6 24 Hr of Incubation Fig.2.5 Relative abundance of TNF-01 mRNA in RAW 264.7 cells after in vitro exposure to VT with and without LPS. Experiment was conducted and analyzed as in Fig. 2.3. (A) indicates the Southern analysis of RT-PCR product form RAW cells; (B) and (C) indicate image analysis data for above autoradiographs in the absence and presence of LPS, respectively. -65- in spleens and Peyer Patches after gavage with VT for 2 hrs (Azcona-Olivera et al., 1995a; Zhou etal., 1997). The inability to observe IL-lB protein secretion in vitro along with the increases in gene activity might be due to the fact that IL-lB was present as a cell- associated molecule, it bound to receptors or it remained intracellularly located (Bayne et al, 1986; Cavaillon and Haeffner—Cavaillon, 1990). IL-6 mRNA expression in vitro also appeared within 2 hrs of LPS stimulation and the mRN A rapidly disappeared in the absence of VT. With VT, mRNA expression was prolonged after 2 and 6 hrs. Similarly, mRNA expression is increased in viva at 2 hrs and disappeared at 4 hrs after gavage with VT (Azcona-Olivera et al., l995a). Supernatant IL-6 protein was increased at 24 hrs although the mRNA declined at this time point. In contrast, TNF-0t mRNA appeared to be expressed earlier than other cytokines (IL-1 B and IL-6) with and without LPS stimulation and its levels diminished more rapidly. The addition of VT in the absence of LPS may have prolonged TNF-0t mRNA production. In mice, TNF-0r mRNA remained elevated after VT treatment at 2 and 4 hrs (Azcona-Olivera et al., 1995a; Zhou et al., 1997). Altered cytokine expression patterns were further verified by flow cytometric measurement of intracellular changes in IL-6 and TNF-or. The results suggested that the increases of IL-6 in culture following treatment with VT appeared to be due to increased production of IL-6 on a per cell basis and this corresponded to the ELISA results of the culture supematants. In contrast, increases of TNF-or in cells treated with VT appeared to be due to an increase in TNF-0t secreting cell populations. These results suggested that VT could differentially superinduce cytokines both by increasing numbers of cytokine secreting cells or increasing cytokine output per cell. -66- VT’s effects on cytokine production may be generally relevant to pathogenic manifestations associated with trichothecene exposure. For example, because the expressed cytokines (IL-1 B , IL-6 and TNF-01) are identical to those observed to mediate septicemia and fatal circulatory shock induced by LPS (V ogel and Hogan, 1990), these same cytokines may also play a role in shock-like syndrome caused by acute high dose trichothecene poisoning. Also, IL-l , IL-6 and TNF-0t administration have also been associated with anorexia and weight loss (Abbas et al., 1994; Schobitz et al., 1994). Thus, feed refusal and reduced weight gain observed in the trichothecene-exposed animals (Rotter et al. , 1996) might be mediated by superinduction of proinflammatory cytokines. Of the several known cytokines that mediate IgA response (McGhee and Kiyono, 1993), IL-6 is one of the most important (Akira et al., 1990; Bao et al., 1993). Macrophages are a primary producer of IL-6 (Bauer et al., 1988) and VanCott et al. (1996) demonstrated that IL-6 secreted by macrophages contributes critically to development of mucosal IgA responses. IL-6 drives IgA production when it is added to Peyer’s patch B cell cultures (Beagley et al., 1989; Yan et al., 1997) and mucosal IgA-producing cells are dramatically decreased in IL-6 knock-out mice (Ramsay et al., 1994). In previous studies, we found that acute oral VT exposure in mice can increase serum IL-6 (Zhou et al., 1997) as well as the capacity of PP and spleen cell cultures to secrete IL-6. Furthermore, specific neutralizing anti-IL-6 Ab inhibits IgA production in vitro in Peyer’s patch cultures obtained from mice exposed to single oral dose of VT (Y an et al., 1997). Thus, it is very likely that, by superinducing IL-6 expression during VT exposure, macrophages provide excessive help for B cell production of IgA, and contribute to the manifestations of IgA nephropathy. -67- The exact mechanisms for superinduction of specific cytokines in macrophages by VT are unclear. Gene expression is regulated at several levels, including transcriptional, translational and post-translational control. Protein synthesis inhibitors enhance and prolong the normally transient expression of specific gene transcripts via impaired synthesis of hi gh tum-over proteins that downregulate transcription or decrease half-life mRNA (Sun et al., 1 993; Cockfield et al, 1 993; Efrat and Kaempfer, 1984). Transcriptional control mechanisms have been suggested to be primary in regulation of macrophage activation (Y u at al, 1990). Previous studies have revealed that transcriptional control of genes in macrophages appears to involve several transcriptional factors such as those in the Rel homology family. For example, nuclear factor -KB (NF -KB) plays an important role in induction of various genes expression in macrophages, such as Macrophage colony stimulating factor (M-CSF), G- CSF (Granulocyte colony-stimulating factor), TNF-0t, IL—1, IL-6 and IL-2 Receptor alpha (Rat) chain and Nitric Oxide synthase (Akahane et al., 1994; Baeuerle and Henkel, 1994). An inhibitory protein, IKB—0t, is known to be able to regulate the gene expression by binding to the NF-KB dimer as an inactive complex in the cytoplasm (Baldwin, 1996). Upon stimulation with mitogen, IKB-a is degraded rapidly and NF-KB is activated and translocated into the nucleus for gene expression. Relatedly, the translational inhibitor CHX inhibits the resynthesis of degraded IKB-(1 in phorbol 12-myristate l3-acetate (PMA)- stimulated Jurkat T cells thereby increasing nuclear bound NF-KB and IL-2 mRNA expression (Sun et al. , 1993). It has similarly been demonstrated that VT inhibits resynthesis 0f degraded IKB-0t in EL-4 and CD4+ cells stimulated with PMA and ionomycin (Ouyang 91‘ al., 1996) and this increases nuclear-bound NF-KB thus leading to increased IL-2 -68- transcription. Therefore it is reasonable to suggest that NF-KB activation might similarly be involved in VT toxicity and contribute to cytokine superinduction and dysregulation in macrophages. We can conclude from the present study that VT is capable of superinducing cytokine production and mRN A expression in a clonal model of the murine macrophage. Increases in these cytokines might contribute to the toxicity and immunopathological effects associated with foodbome VT. Further studies of the mechanisms of cytokine superinduction in the RAW 264.7 model should facilitate our understanding of the effects of VT on regulation of the immune system in vivo and their contribution to the diverse toxic effects of trichothecenes. -69- CHAPTER III EFFECTS OF VOMITOXIN ON TRANSCRIPTION FACTOR BINDING IN MACROPHAGE RAW 264.7 CELLS -70- ABSTRACT Vomitoxin (VT, deoxynivalenol), a trichothecene mycotoxin, superinduces IL-l B, IL-6 and TNF-or gene expression in macrophage RAW 264.7 cells. Cytokine gene regulation in macrophages may be affected by VT at the transcriptional level. In this study, the effects of VT on the binding activity of AP-l , NF-KB and NF-IL6, transcription factors important to pro-inflammatory cytokine regulation, were assessed using electrophoretic mobility shift assay (EMSA) with RAW 264.7 cells. When cells were treated with VT in the absence of LPS (synchronous model) , DNA binding activity of AP-l was increased at 8 hr with 250 ng/ml of VT. When cells were incubated in medium alone for 2 hr prior to the addition of VT (delayed synchronous model), binding activity of AP-l was enhanced at 2 and 8 hr with VT treatment. Binding activities of AP-l were not enhanced when cells were treated with VT in the presence of LPS. Using specific antibodies in supershift EMSA, VT was found to preferentially induce phosphorylated c-Jun and JunB, c-Fos and Fra-2 binding activities of the AP-l family. VT was also found to alter binding activity of NF- KB/Rel complexes and this was verified by competition assays. A faster migrating band (lower complex) was constitutively expressed whereas a slower migrating band (upper complex) was induced by the stimulation of LPS. The binding activities of the upper complex were increased at 2 and 8 hr when cells were subj ected to synchronous and delayed synchronous VT-exposure in the presence of LPS. The p-50 and c-Rel subunits of the NF- KB/Rel were found to be specifically affected by a supershifi EMSA. NF-IL6 binding was increased at 2 hr with or without LPS in synchronous and delayed synchronous VT-exposure models, respectively. Here, C/EBPa and B subunits were involved in the enhancement of -71- NF-IL6 binding. Binding specificity for all three transcription factors was confirmed in EMSA competition studies with unlabeled and mutant probes. The capacity of VT to elevate binding of AP-l, NF-KB, and NF-IL6 may contribute to the VT-mediated cytokine superinduction in macrophages as well as other toxic manifestations of this mycotoxin. INTRODUCTION Vomitoxin (VT, deoxynivalenol), a trichothecene mycotoxin produced by F usarium graminearum, is frequently found in feed and grain supplies (Ueno, 1985; Vesonder and Hesseltine, 1980; Rotter et al., 1996) . Trichothecenes can inhibit protein synthesis and cause a wide range of toxicological and immunological effects (Ueno, 1983; Cundliffe et al., 1974; Bamburg, 1983; Forsell et al., 1987; Fitzpatrick et al., 1988; Thompson and Wannemacher, 1990; Bergrnann et al. , 1992). Trichothecenes are also able to elevate serum IgA, IgM and IgG levels in mice exposed with VT ( Forsell et al., 1987; Atroshi et al., 1994). It was demonstrated that dietary exposure of mice to VT increases serum IgA and IgA complexes as well as accumulation in the mesangial kidney (Dong et al., 1991; Dong and Pestka, 1993). These features are highly analogous to the glomerulonephritis human IgA nephropathy (D’Amico, 1987). IL-6 plays an important role in VT-induced IgA nephropathy based on ex vivo culture studies with Peyer’s patches (Y an et al., 1997). Recently, Yan et al. (1998) have also demonstrated that increased I gA and IL-6 production was observed in macrophage-depleted Peyer’s patches and spleen cells from VT-treated mice when cocultured with VT-treated peritoneal macrophages. Therefore, macrophages may be involved in the regulation of -72- superinduction of IL-6 and other cytokines by VT. Transient increases of mRN As for macrophage-associated cytokines, IL-1 B, IL-6 and TNF-01, have been shown upon exposure to VT in viva (Azcona-Olivera et al. , l995a; Zhou et al. , 1997). Miller and Atkinson (1986) demonstrated the superinduction of inflammatory cytokine IL-1 in peritoneal macrophages by VT. Recently, Wong et al.(1998) observed elevated expression of IL-6 and TNF -01 mRNA and proteins in macrophage RAW 264.7 cells. The superinductive effects by VT are quite consistent with the effects observed for protein synthesis inhibitors such as CHX (Efrat and Kaempfer, 1984; Cockfield et al. , 1993; Faggioli et al., 1997). The aforementioned macrophage cytokines may also be related to pathogenic manifestations associated with trichothecene exposure. Gene expression for these cytokines is regulated at the transcriptional, post-transcriptional and post-translational levels in macrophages. Transcriptional control has been suggested to be important in the regulation of macrophage activation (Y 11 et al., 1990; Paulnock, 1994). Proinflarnmatory cytokine promoters contain various binding sites for functional transcription proteins. Therefore, it is highly possible that VT can superinduce cytokines in macrophage cells by altering the transcriptional activity via their promoters. One important transcription factor for cytokine expression is the activator protein-1 (AP-1), which is a complex of proteins made up from members of the Jun and Fos families. The AP-l family contains four Fos proteins (c-Fos, Fos B, Fra-l, and Era-2) and three Jun proteins (c-Jun, Jun B and Jun-D) (Pennypacker, 1998). The Fos and Jun proteins can form dimeric complexes with one another and these complexes can bind to the AP-l consensus sequence. The Fos-Jun heterodimers form the most stable complexes -73- (Halazonetis, 1988; Liebermann et al., 1998). AP-l has been found to be involved in B- and T-cell activation, differentiation of Th1 and Th2 cytokines, regulation of immunoglobulin production, and monocytic differentiation and apoptosis ( Pennypacker, 1998; F oletta et al, 1998). Notably, AP-l regulates IL-4, -5, -6, -10, IFN-y, TNF-ct, IL-8 and CM-CSF expression (Foletta et al., 1998). Also, AP-l binding sites have been identified in the promoters of IL-3 and IL-9 genes. Recently, it has been shown that VT can enhance the binding activity of AP-l in the EL—4 cells (Li et al., 2000). Therefore, it is reasonable to suggest that VT similarly enhances AP-l binding activity in macrophages. A second important transcription factor in monocytes is the nuclear factor kappa B (NF -KB). The NF-KB/Rel family is known to regulate a variety of signal pathways (Lenardo and Baltimore, 1989; Legrand-Poels et al. , 1997). It is an inducible transcriptional activator which can respond to different stimulation agents, such as lipopolysaccharide (LPS), phorbol esters, IL-l , -2, TNF-01 and protein synthesis inhibitors (Grilli et al., 1993). NF-KB/Rel can also regulate a wide variety of genes involved in acute phase responses and inflammation, such as M-CSF, G-CSF, TNF-or, lL-lB, IL-6, IL-2, IL-8, IL-2R01 chain and NO synthase (Baeuerle and Henkel, 1994; Akahane et al. , 1994). Five members of the NF- KB/Rel family have been identified : c-Rel, NF-KB,(p50/p105), NF-KB2 (p52/p100), Rel A (p65), and Rel-B (Baeuerle, 1991; Baeuerle and Henkel, 1994). In the resting phase, dimers of these proteins are inactive in the cytoplasm, and are associated with an inhibitory factor, IKB. Upon stimulation, IKB is phosphorylated, and dissociates from NF-KB complexes. The NF-KB dimer translocates into the nucleus in an active form and activates different responsive genes (Li et a1, 1994; Finco and Baldwin, 1995; Baldwin, 1996). Previous -74- studies of Ouyang et al.(1996) revealed that the activities of the transcription factor NF-KB were increased concurrently with IL-2 gene expression in EL-4 cells treated with VT. Therefore, VT might also be able to increase the NF-KB activity in the macrophage cells. NF-IL6, another important monocytic transcription factor, is also known as CCAAT enhancer-binding proteins or C/EBP. It can be induced by various stimuli such as LPS, IL- 1, TNF-0t and IL-6 (Ohmori and Hamilton, 1994). It is also able to stimulate different target genes in macrophages such as IL-1 B, IL-6, IL-8, TNF-or, M-CSF and IP-10 (Ohmori and Hamilton, 1994; Koj, 1996). Expression of C/EBP protein is limited to the myeloid lineage (Scott et al., 1992; Haas et al., 1992; Valledor et al., 1998). C/EBPct is mainly expressed in undifferentiated myeloid cells. Expression of C/EBPB and C/EBP6 is observed during macrophage maturation. C/EBPB and 8 are phosphoproteins that translocate into the nucleus following phosphorylation by MAP kinase at their threonine residues (Y amanka et al., 1998) . Defects in C/EBPB(—/—)genes have been shown to increase the susceptibility of mice to infection by Listeria monocytogenes and impair tumor cytotoxicity of macrophages (Tanaka et al. , 1995). Disruption of C/EBPB genes also results in an increased production of IL-6 levels in viva (Screpanti et al., 1995). All three of the aforementioned transcription factors have been implicated in the regulation of macrophage cytokine gene expression. The specific objective of this study was to test the hypothesis that VT alters binding activities of AP-l, NF-KB and NF-IL6, in macrophage RAW 264.7 cells. MATERIALS AND METHODS -75- RAW 264.7 cell cultures. Raw 264.7 cells (TIB 77, American Type Culture Collection, Rockville, MD) (2.5 x 105 cells/ml) were cultured in lOO-mm tissue culture dishes (Corning, NY) in Dulbecco’s modified Eagle medium (DMEM) (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Gaithersburg, MD), 1% (v/v) NCTC 135, 1 mM sodium pyruvate (Gibco BRL), 100 U/ml penicillin (Sigma), and 100 pg/ml streptomycin (Sigma) in a 5-8% CO2 incubator at 37°C. For both temporal and dose-response studies, cells were stimulated with and without LPS (Salmonella typhimurium; Sigma, stimulation index 110.7) (0, 100, or 1000 ng/ml) and VT (Sigma) (0, 100 and 250 ng/ml) for 0, 2, 8, and 24 hr in both synchronous and delayed synchronous modes. In the synchronous model, cells were treated simultaneously with VT and either LPS or vehicle. In the delayed synchronous model, cells were pre-stimulated with LPS or vehicle for 2 hr prior to the addition of the VT. Nuclear Extract Preparation Cells were collected from cultures at time intervals and sub-cellular protein extracts were prepared according to Schreiber et al. (1989a). Briefly, supernatant was removed and Dulbecco’s Phosphate Buffer Saline (DPBS; Sigma) was added (10 ml/dish) to each dish. Using a cell lifter (Fisher Scientific, Springfield, NJ), cells were detached from the surface of dishes and transferred to a 50 ml conical centrifuge tube and centrifuged at 240 xg for 5 min. The pellet was resuspended in 400 pl of ice-cold buffer A [ 10 mM HEPES (Sigma) (pH 7.9) containing 10 mM KCl (Sigma), 0.1 mM EDTA (Boehringer Manheim, Indianapolis, IN), 0.1 mM EGTA (Sigma), 1 mM dithiothreitol (DTT; Sigma), 0.5 mM phenylmethysulfonyl fluoride (PMSF; Sigma), 1 %(v/v) aprotinin (Boehringer Manheim) , 1 pg/ml pepstatin A (Sigma), 2 11ng leupeptin (Sigma), 10 mM -76- sodium fluoride (Sigma) and 1 mM sodium orthovanadate (Sigma) ]. The resuspended cells were held on ice for 15 min and gently lysed by adjusting concentration to 0.7% IGEPAL (v/v) (Sigma Co., St. Louis, Mo) detergent. After a centrifugation at 4°C at 420 xg for 10 min, pelleted nuclei were resuspended in 100 pl of ice-cold buffer B [ 20 mM HEPES (pH 7.9) containing 0.4 M KCl, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 1 %(v/v) aprotinin, 10% (v/v) glycerol (Sigma), 1 pg/ml pepstatin A, 2 pg/ml leupeptin, 10 mM sodium fluoride and 1 mM sodium orthovanadate] and lysed by gently rocking for 1 hr at 4 °C. After centrifuging at 10,000 rpm for 10 min at 4°C, the supernatant was dialyzed in dialysis cassettes (10, 000 MW; Pierce Co.) for 2 hr in dialysis buffer contains 20 mM HEPES (pH7.9), 60 mM KCl, 1 mM EDTA, 0.5 mM DTT and 10% (v/v) glycerol, supplemented with 0.5 ng/ml leupeptin, 1% (v/v) aprotinin and 10 mM sodium fluoride, and stored at -80° C until analysis. Protein concentrations of the extracts were analyzed by using Bio—Rad Protein Assay kit ( Bio-Rad Laboratories Inc., Melville, NY) according to the method of Bradford (1976). Electrophoretic mobility gel shift assay (EMSA) Kinetics of the AP- 1 , NF-KB/Rel, and NF-IL6 binding activity in nuclear extracts were assessed by EMSA (Baeuerle and Baltimore, 1988). Nuclear extracts (5 pg) were added to DNA binding reaction buffer [ 20 mM HEPES (pH7.9) containing 60 mM KCl, 1 mM EDTA, 0.5 mM DTT and 10% (v/v) glycerol, and 2 pg poly (I-C)] to a total voliune of 30 pl and then pre—incubated on ice for at least 1 hour to block the non-specific binding. 32P-labeled consensus sequence of AP-l , NF-KB and NF-IL6 (Santa Cruz Biotechnology, Santa Cruz, CA) (Table 3.1) containing 30,000 cpm was added in a volume of 1 p1 and incubated at room temperature for 20 min to -77- Table 3.1. Oligonucleotides for Transcription factors in EMSA Transcription Factors Sequence for the Oligonucleotides AP-l consensus sequence mutant sequence NF-KB consensus sequence mutant sequence C/EBP consensus sequence mutant sequence 5'-CGC TTG ATG ACT CA6 CCG GAA-3' 5'-CGC TTG ATG ACT IQG CCG GAA-3' 5'-AGT TGA GGG GAC TTT CCC AGG C-3' 5'-AGT TGA GGQ GAC TTT CCC AGG C-3' 5'-TGC AGA TTG 95E A__AT CTG CA-3' 5'-TGC AGA GAC TAG IQT CTG CA-3' ~78- allow the formation of nucleoprotein. 32P-labeled probe was prepared using Ready To- GoT'“ T4 Polynucleotide Kinase (Pharmacia Biotech Inc., Piscataway, NJ) according to the method of Tabor ( 1 987). For competition assays, an excess of unlabeled probes of AP-l, NF-KB and NF-IL6 or mutants of those consensus sequences (Table 1) were added prior to the addition of labeled probe. For super-shift assays, 1- 2 pg of antibodies specific for NF-KB subunits (p65, p5 0, c-rel and Rel-B) (Santa Cruz), AP-l subunits(c-jun, JunB, JunD, c-Fos, Fra-l , and Fra-2) (Santa Cruz) , and NF-IL6 subunits (C/EBPa, C/EBPB, and C/EBPS) (Santa Cruz) or 2 pg rabbit IgG (Santa Cruz) control antibody were added and mixed after the addition of 32P-labeled probe and pre-incubated overnight at 4°C. Resultant nucleoprotein complexes were resolved on native 4 % (w/v) polyacrylamide gels, dried and visualized by autoradiography. RESULTS AP-l Binding activity The effects of VT on binding activities of AP-l was studied by EMSA in RAW 264.7 macrophage cells. Cells were incubated with different concentrations of VT with and without LPS for different time periods in both synchronous and delayed synchronous models. When cells were treated with VT alone in the synchronous model, effects of VT on AP-l binding activity were negligible at 2 hr (Fig. 3.1A). Whereas AP-l binding was increased in cells treated with 100 and 250 ng/ml of VT at 8 hr (Fig. 3.1A) . Upon simultaneous incubation with LPS (100 and 1000 ng/ml), the activity of AP-l binding was increased at 100 and 250 ng/ml of VT at 2 hr but not at 8 hr (Fig. 3.1 B and Fig. 3.1C). In the delayed synchronous model, cells were treated with and -79- Hr 0 2 8 VT C 0 100250 0 100 250 (A) VT C 0 100 250 0100250 (B) Hr 0 2 8 VT C 0 100 25010 100250 (C) Fig. 3.1. Dose and temporal effects, of VT on AP-l binding activity in a synchronous exposure model. RAW 264.7 cells were treated with VT in the absence (A) or presence of of (B) 100 11ng and (C) 1000 ng/ml of LPS for different time periods simultaneously. Nuclear protein extracts were prepared at the time indicated and 5 pg of nuclear protein of each sample was subjected to EMSA as described in the Materials and Methods. Data are representative of two separate experiments. -30- without LPS for 2 hr prior to addition of VT. AP-l binding activity was markedly increased with 100 and 250 ng/ml of VT in the absence of LPS after 2 and 8 hr (Fig. 3.2A). Upon addition of 1000 ng/ml of LPS, binding activities were inhibited by VT after 2 hr but unaffected after 8 hr (Fig. 3.2B). Thus, VT could differentially affect AP-l binding in RAW 264.7 cells. The specific involvement of AP-l in the aforementioned protein-DNA complexes was assessed by binding competition studies with unlabeled wild type Oligonucleotides. A decrease in the amount of bound complex was observed as the concentration of unlabeled AP-l consensus sequences increased in both synchronous and delayed synchronous models (Fig. 3.3A and 3.3B). Here, 100X excess of mutant AP-l Oligonucleotide did not inhibit the DNA-binding to the same extent as 100X excess of wild type. These results confirmed the specificity of the AP-l DNA-binding activity. Both c-Jun and c-Fos subunits are involved in AP-l transactivation effects. Supershifl EMSA was performed with nuclear extracts fiom control and VT-treated RAW cells to identify subunits altered by VT. Antibodies against the Jun and Fos family subunits were added prior to the addition of the labeled probe. The AP-l binding band was most markedly supershifted by antibodies against phosphorylated c-Jun, and J unB subunits of the Jun family, and by c-Fos, and Fra-2 of the Fos family (Fig. 3.4). A supershifi was not observed when IgG was added (data not shown). These results suggest that both homo- and hetero-dimers of c—jun and c—fos family might be involved in altered AP-l binding. NF-KB Binding activity. KB-binding of nuclear extracts was visualized in EMSA as multiple bands which is consistent with the existence of multiple monomers that form -31- Hr 0 2 8 VT C 0 100 250 0 100 250 (A) UHUHHUU* Hr 0 2 8 VT C 0 100 250 0 100 250 We Fig. 3.2. Dose and temporal effects of VT on AP-l binding activity in a delayed synchronous model. RAW 264.7 cells were stimulated in the absence (A) and (B) presence of 1000 ng/ml LPS for 2 hr prior to the addition of VT and incubated for another time periods. Nuclear protein extracts were prepared at the time indicated and 5 pg of nuclear protein of each samples were subjected to EMSA as described in Materials and Methods. Data are representative of two separate experiments. -32- M Competitor - 200x100x 25X 100X (A) M Competitor - 200x 100x 50X 25X 100X (B) Fig.3.3. Specificity of AP-l DNA binding activity in RAW 264.7 cells in (A) synchronous model and (B) delayed synchronous model. Cells were treated with LPS (1000 ng/ml) and VT (0, 100 rig/ml) for 2 hr in synchronous model. Cells were pre-stimulated with medium alone for 2 hr and then treated with VT (0, 100 ng/ml) for another 8 hr in the delayed model. EMSAs were per- formed by using protein nuclear extracts (5 pg/lane) from the RAW cell culture in the presence of 32P-labelcd AP-l probe alone or with 200, 100, 50 or 25 molar excess of unlabeled AP-1,and 100 molar excess of mutant AP-l probes. Data are representative of two separate experiments. .fiaofitonxo 88.38 92 mo 35.85830.- oaa Sun .9553 0% Co Gaza-60.- 05 no mun-B mafia-o. 8865 m30b< .Nécm we.» .Tfim .m mom don-..- d-SH .mfiq .570 use MO 02585 05 E can 863:5- .«o Seaman 05 E 8:5 38b 5% can 3 N .8.“ one? 8338 FEB @8335 803 £3 5ch 3.5“ .mzoo 5me >32 E 9:55 $558 :2 ofi :o 5:0 38b»- .wo 3A.. -34- dimeric NF-KB binding proteins (Baeuerle, 1991; Grilli et al, 1993). In the synchronous model, binding was not altered in a lower complex with VT in the absence of LPS at 2 hr, but increased with 250 ng/ml of VT at 8 hr (Fig. 3.5A). In the presence of 100 ng/ml of LPS, VT at 100 and 250 ng/ml enhanced binding of the upper band as early as 2 hr (Fig. 3.5B) whereas the effect at 1000 ng/ml LPS was less remarkable (Fig. 3.5C). In the delayed synchronous model, binding of the lower band was not enhanced by VT in the absence of LPS at 2 hr (Fig. 3.6A) but was at 8 hr. When cells were stimulated with 1000 ng/ml of LPS prior to the addition of VT, the upper band was increased slightly upon incubation with 100 and 250 ng/ml of VT for 2 and 8 hr (Fig.3.6 B). The specific involvement of NF-KB/Rel in the protein DNA complexes was determined by inhibition studies with unlabeled probe. When a nucleotide corresponding to the KB consensus site was added to the nuclear extracts of VT-treated RAW cells, two complexes (the upper and lower complexes) were found to be elevated. In the delayed synchronous model, 500X fold molar excess of cold KB oligonucleotidc decreased the binding of the lower band (Fig. 3.7A). A 200X concentration of the mutant inhibited the binding slightly in the delayed synchronous model. The slower migrating complex (upper complex) was decreased as the concentration of cold KB consensus sequences increased in the synchronous model (Fig.3.7B). Mutated KB sequence (100 X) failed to inhibit the binding of the upper complex in the synchronous model. These results suggested that the binding of the upper complex of NF-KB to DNA was specific. For the studies of the subunits of NF-KB/Rel family, antibodies against c-Rel, Rel-B, p65 and p50 subunits were used in a supershift EMSA (Fig. 3.8). The upper complexes of -35- Fig. 3.5. Dose and temporal effects of VT on NF-KB binding activity in a synchronous exposure model. Cells were treated with VT in the absence (A) or presence of (B) 100 ng/ml and (C) 1000 ng/ml of LPS for different time periods. Experiments were performed as described in the legend of Fig.3.]. Arrows indicate the upper complex (U) and lower complex (L). Data are representative of two separate experiments. -86- Hr’ 0 2 8 VT C 0100 250 0 100 250 *§'~'.O'.¢..v.0‘ (A) Hr 0 2 8 VT C 0 100250 0100250 o‘“'o~006"C-ttb U L Hr 0 2 8 VT C 0 100 250 0 100 250 «a no to a. ""9 9’ O. (C) U L -37- Hr 0 2 8 VT C 0 100 250 0 100 250 (A) Hr 0 2 8 VT C 0 100 250 0 100 250 n n o a . - Q Q a t O D r I (B) Fig.3.6. Dose and temporal effects of VT on NF-KB binding activity in a delayed synchronous model. Cells were stimulated with medium alone (A) and 1000 11ng of LPS (B) for 2 hr prior to the addition of VT and incubated for another time periods. Experiments were performed as described in the legend of Fig.3.2. Arrows indicate the upper complex (U) and lower complex (L). Data are representative of two separate experiments. -88- M Competitor - 200X 100X 50X 25X 100X (A) Competitor - 500x 200X 100X 50X100X WW.” W“ "in WW 1111- » e- y #1" ll (B) . F ig.3.7. Specificity of NF-KB DNA binding activity in RAW 264.7 cells in (A) delayed synchronous model and (B) synchronous model. Cells were incubated with medium alone for 2 hr and then treated with VT (0, 250 ng/ml) for another 8 hr in the delayed model. Cells were treated with LPS (100 ng/ml) and VT (0, 100 ng/ml) for 2 hr in synchronous model. Experiments were performed as described in the legend of Fig.3.3, except 200 molar excess of mutant NF-KB was used in the delayed synchronous model. Arrows indicated upper complex (U) and lower complex (L). Data are representative of two separate experiments. dung cog-Am 8 wag-B 2: mo momma-85 05 mo coco-62 3865 £5854 .mEoEtoqxo 2.238 95 mo 338-5358 0.8 San demo was men .03 relax “mam-«ma mammogram mo 3:805 was 828%. 05 05 E {warm mo gowB 05 E pontomou we cantor-on 2o? Beofitoaxm Eve:- Soaefi-sw a a :- E can: 8- .8 e> as can: 8: m5 £3 832- 22> £8 . £8 38 35- a mans-- ass-a seas-dz 2: 8 s> do 98% co 33 :3. :2 men - - 5. -90- the control and VT-treated group were supershified by anti-p50 and c-Rel. No detectable supershifted bands were observed with p65 and Rel-B antibodies. The lower complex was not supershifted by any of the antibodies. The results suggested that both p50 and c—Rel subunits might be involved in the VT-enhanced NF-KB binding. NF—IL6 Binding activity. The binding activity of NF-IL6 was also assessed in these studies with RAW 264.7 macrophage cells. In the synchronous model, the binding activity of NF-IL6 was increased with 250 ng/ml of VT at 8 hr in the absence of LPS (Fig. 3.9A). When cells were treated with 100 ng/ml of LPS, VT enhanced NF-IL6 binding at the concentrations of 100 and 250 ng/ml at 2 hr, but not at 8 hr (Fig. 3.9B). At a higher dose of LPS (1000 ng/ml), enhanced binding was found in cells treated with 100 and 250 ng/ml of VT at 2 hr (Fig. 3.9C). At 8 hr, no difference in the binding activity was found in either doses of VT. In the delayed synchronous model, 100 ng/ml of VT was able to enhance the binding at 8 hr; and 250 ng/ml at 2 and 8 hr in the absence of LPS (Fig.3.10A). A decrease in the binding activities were observed in LPS-stimulated cells treated with VT and 1000 ng/ml of LPS for 2 hr but no effect was seen at 8 hr (Fig. 3.10B). Nuclear extracts were further assessed for binding specificity. In both synchronous and delayed synchronous models, the increase in the concentration of cold C/EBP consensus sequences showed a dose-dependent inhibition in the DNA-binding (Fig. 3.11 A and B). Mutated C/EBP sequences (100 fold excess) were not effective in inhibiting binding in both models. These results suggested that a specific DNA-binding activity of NF-IL6. To identify NF-IL6 subunits affected by VT, antibodies against C/EBP-or, -B , and -6, were used in supershif’t studies. The C/EBP binding band was supershifled by anti- -91- Hr 0 2 8 VT C 0 100 250 0 100 250 .101. ’ ”In“, M M “in“ ”1011‘ .i‘ (A) Hr 0 2 8 VT C ‘ 0 100 250 0 100 250 (B) W M m m 1""? M m l 11 ., U ‘1 1’.” ,. .1111 «‘11. .‘1‘ ’1 , .. L . 2.111 1111111111112, 11111111.} .1 . Hr 0 2 8 VT C 0 100 250 0 100 250 11 11 m 111 m m ‘ M1" f 11:_ 111 L .1111 l“ 111111111111 Fig.3.9. Dose and temporal effects of VT on NF-IL6 binding activity in a synchronous model. RAW cells were treated with VT in the absence (A) or presence of (B) 100 ng/ml and (C) 1000 11ng of LPS for different time periods simultaneously. Experiments were performed as described in the legend of Fig.3. 1. Arrows indicate upper complex (U) and lower complex (L). Data are representative of two separate experiments. -92- Hr0 2 8 VT C 0 100 250 0 100 250 . .11 H ,.:" ‘ 1 . .1. 1‘" _ 11 1 ’ 31‘ '11 Hr 0 2 8 VT C 0 100 250 0 100 250 .1 .11 Ml 111111 11111 M 1111 W ‘ 1.. j .1. 111‘ _ l i w EC! Fig. 3.10. Dose and temporal effects of VT on NF-Il6 binding activity in a delayed synchronous model in the absence (A) or presence (B) of 1000 ng/ml of LPS. Ex- periments were performed as described in the legend of Fig.3.2. Arrows indicate upper complex (U) and lower complex (L). Data are representative of two separate experiments. -93- M Competitor - 200x100x 50x 25x 100x .. O '3 n O ’O O ‘C ”1 (A) Competitor - 200x100x 50X 25X 100X O’OOQOQQ- 010091 (B) F ig.3.1 1. Specificity of NF-IL6 DNA binding activity in RAW 264.7 cells in a (A) synchronous model and (B) delayed synchronous model. Cells were treated with LPS (1000 ng/ml) and VT (0, 100 ng/ml) for 2 hr in the synchronous model. Cells were incubated with medium alone for 2 hr and then treated with VT (0, 250 ng/ml) for another 2 hr in the delayed model. Experiments were performed as described in the legend of F ig.3.3. Data are representative of two separate experiments. -94- V —' - u .- C/EBPB in both control cells and 250 ng/ml VT-treated cells (Fig. 3.12). Anti- C/EBPor also had effects on reducing the binding of both control and treatment groups. This indicates that both C/EBPa and C/EBPB might be involved in increased NF-IL6 binding observed in cultures incubated with VT. DISCUSSION AP-l, NF-KB, and NF-IL6 have been implicated as important transcription factors in immune and inflammatory responses mediated by macrophages. Notably, the binding sites for these three factors have been found on the promoters of proinflammatory cytokines IL-1 B, IL-6 and TNF-01 (Fig. 3.13). In this study, we demonstrated that VT was able to alter binding activities of these transcription factors differentially in macrophage RAW 264.7 cells. Several major findings have been observed. First, VT alone induced the AP-l binding in both synchronous and delayed synchronous models. Secondly, the binding activity of upper complex of NF-KB was enhanced by VT in the presence of LPS of both synchronous and delayed synchronous models. Thirdly, NF-IL6 binding activity was enhanced by VT in the absence (synchronous and delayed synchronous models) and presence (synchronous model) of LPS. Finally, the results showed that VT specifically targeted the phosporylated c-Jun and J unB, c-Fos, and Fra-2 subunits of the AP-l components; p50 and c-Rel of NF-KB; and C/EBPa and C/EBPB of NF-IL6. AP-l regulates transcription of genes through its ability to bind specifically to the recognition site, 5'-TGANTCA-3', which is also known as the TPA(12-O-tetradecanoyl- phorbol-l3-acetate) response element (TRE) (Lee et al., 1987; Risse et al., 1989). AP-l -95- "‘A Bios-Comes 89398 23 mo 3385352 2w San .mp5: coo-«Em Lo 53an Rate 05 mo wax-=5 05 mo noun-62 on: 8865 mBoh< .wmmmxo EB .9580 .dmmmco «madame mom-3:5 Lo 3585 can 088% 05 E 6.0me we 28w”: 05 E canto-Moe we wogofioq 0.63 mansion-am 4268 308505 a a a N 5- sea-- o3 .8 e> as can-- so: was as, essa- eoa £8 38 35- a was-B €33 SE2 £- 8 e> do 98% do 3 hr at concentration of 100 and 250 ng/ml. The results suggest that the -132- post-transcriptional control via enhancement of mRNA stability is likely to contribute to cytokine superinduction in macrophages by VT and other trichothecenes. INTRODUCTION The trichothecene mycotoxin VT is a secondary metabolite produced by F usarium graminearum and F. culmarum during growth on agricultural staples such as wheat and corn (V esonder et al., 1973; Vesonder and Hesseltine, 1980). Trichothecenes can either be immunosuppressive or immunostimulatory depending on the dose and timing of exposure (Pestka and Bondy, 1994). Previous studies have shown that in viva trichothecene exposure inhibits DNA and protein synthesis in bone marrow, lymph nodes, spleen and thymus (Rosenstein and Larfarge-Frayssinet, 1983; Azcona-Olivera et al., l995a; Rotter et al., 1996). In vitro exposure to VT also depresses mitogen-induced proliferation of the murine lympohocytes (Robbana-Barnat et al., 1988; Tryphonas et al., 1986). In contrast, both single and multiple gavage of mice with trichothecene VT have been shown to increase resistance to the mastitis pathogen Mycabacterium and to Staphylococcus (Atroshi et al., 1994). Dong and Pestka(1993)demonstrated that the dietary exposure of mice to VT elevated serum IgA, circulating IgA immune complexes and mesangial IgA deposition in the kidney. These latter features are highly analogous to the human I gA nephropathy (D’Amico, 1987) VT may enhance immune function by upregulating cytokine production. Increased mRNAs of interleukin-2, -4, -5 and/or IL-6 and their secreted proteins have been observed upon the exposure to VT or cycloheximide (CHX) in T cells (Warner et al., 1994; Ouyang -133- et al., 1996). Superinduction of inflammatory cytokines such as IL-1 has been observed in peritoneal macrophages treated with VT (Miller and Atkinson, 1986). Increased IL-6 and TNF-or mRNA and proteins have also been found in RAW 264.7 macrophage cells treated with VT (Wong et al., 1998). Transient increases of mRNAs for cytokines IL-1 [3, IL-6, and TNF-or associated with macrophages have been found in mice exposed to VT orally within 2 hr (Azcona-Olivera et al. , 1995 a; Zhou et al. , 1997). Notably, because IL-6 drives IgA production when added to the Peyer’s patch B cell cultures (Yan et al., 1997), its upregulation by VT may provide help for B cells to over-produce IgA driving VT-induced I gA nephropathy. Although the exact mechanisms of the superinduction of macrophage cytokine gene expression by VT are not fully understood, transcriptional regulation may be contributory (Lu-Kuo et al. , 1996; Roger et al. , 1998a). In support of this contention, increased binding activities of transcription factors NF-KB and NF-IL6 by VT have been demonstrated in macrophage RAW 264.7 cells (Wong et al., chapter III). However, the results of transfection assays with a TNF promoter -CAT construct indicated that the transcription of the reporter was not increased dramatically. Interestingly, post-transcriptional control has also been previously implicated as an important in upregulation in the cytokine induction through protein synthesis inhibitors. For example, in the presence of a transcription inhibitor, actinomycin D (Act-D), CHX increases the half-life of TNF-or mRNA in macrophages (Celada et al., 1989; Taffet et al., 1989; Zuckerman et al., 1991 ). Upon the removal of this protein synthesis inhibitor, cytokine mRNA degradation seems to resume quickly (Fort et al., 1987). Roger et al. (1998a) has also shown that the increased mRNA -134- half-life contributes to IL-6 superinduction by CHX in lung epithelial cells. It was hypothesized that stabilization of cytokine mRNAs might play a key role in the proinflammatory cytokine superinduction by VT in macrophage cells. The objective of this study was to assess the effects of VT on cytokine mRNA half-life in the presence of a transcription inhibitor, 5,6-dichloro-1-beta-D-ribofuranosyl benzirrridazole (DRB). The results showed that VT can stabilize both TNF-or and IL-6 mRNA in macrophage RAW 264.7 cells. MATERIALS AND METHODS RAW 264.7 cell culture. RAW 264.7 cells (TIB 77, American Type Culture Collection, Rockveille, MD) (2.5 x 105 cells/ml) were plated on 100-mm diameter tissue culture dishes (Corning, NY) in Dulbecco’s modified Eagle medium (DMEM; Sigma Co., St. Louis, MO) supplemented with 10%(v/v) fetal bovine serum (FBS; Gibco BRL, Gaithersburg, MD), 1% (v/v) NCTC 135 (Gibco BRL), 1 mM sodium pyruvate (Gibco BRL) , 100 U/ml penicillin (Sigma), and 100 pg/ml streptomycin (Sigma) in a 5-8% CO2 incubator at 37 ° C. Two exposure models were used. An asynchronous model was employed in which cells were pre-stimulated with 1 pg/ml of LPS (Salmonella typhiumurium; Sigma; stimulation index: 110.7) for2 hr and the supernatant was removed. Then, VT (0 to 250 nglml) was added in the presence and absence of transcriptional inhibitor, 5,6-dichloro-1-beta-D-furanosyl benzirnidine (DRB, F luka Co., Madison, WI), for various time intervals. A delayed synchronous model was also used. Whereby, cells were preincubated with 1 pg/ml LPS for 2 hr and VT and DRB were added and incubated -l35- for appropriate time intervals. Cytokine assays. IL-6 and TNF-or concentrations were measured by ELISA as described by Dong et a1 (1994). Briefly, Irnmunolon IV Removawell nricrotiter strips (Dynatech Laboratories, Chantilly, VA) were coated with 50 pl/well of monoclonal rat anti- mouse IL-6 or TNF-or antibody (Pharmingen, San Diego, CA) diluted to 1 pg/ml in phosphate buffered saline (PBS; pH 7.2) and incubated at 4°C overnight. Plates were washed three times with PBS containing 0.05% (v/v) Tween 20 (Sigma) (PBS-T). To reduce non- specific protein binding, wells were blocked with 300 pl PBS containing 1% (w/v) bovine serum albumin (BSA), incubated for 30 min at 37°C and then washed four times with PBS- T. Reference IL-6 or TNF-or and samples were diluted in DMEM with 10% (v/v) FBS, and 50 pl was added to appropriate wells. Plates were incubated for 60 min at 37 °C and then washed four more times with PB S-T. Biotinylated IL-6 or TNF-or antibody (Pharmingen), diluted to 1 pg/ml in PBS containing 1% (w/v) BSA, were added to each well and the plate was incubated at room temperature for 60 min. Plates were then washed six times with PBS-T and 50 pl streptavidin hoseradish peroxidase (Sigma), diluted 1:1000 in PBS with 1% (w/v) BSA, was added to each well and incubated at room temperature for 60 rrrin. ELISA plates were washed eight times with PBS-T. Substrate (100 pl) consisting of 3', 3', 5', 5'- tetramethyl benzidine ( 0.1 mg/ml; F luka Chemical Corp., Ronkonkoma, NY ) in 0.1 M citric-phosphate buffer (pH 5.5) and 0.003% (w/v) hydrogen peroxide (H202) was added to each well and incubated for 20 minutes at room temperature. The reaction was terminated with 100 pl of 6 N sulfuric acid (HZSO4). Absorbance was measured at 450 nm with Vmax kinetic microplate Reader (Molecular Devices, Menlo Park, CA) and interleukin -136- concentration was quantified using the Sofimax software (Molecular Devices). cDNA probe preparation. Plasmids encoding IL-6, TNF-or and GAPDH were kindly supplied by Dr. Richard Schwartz (Michigan State University). The plasmids were transformed into E. cali DH-Sa strain by the heat shock method (Sambrook et al., 1989). Briefly, 5 pl of DNA were added to 100 pl DH-Sa competent cells and these were kept on ice for 30 min. Then the mixture was transferred to 42 °C for 2 min and 0.9 ml of the SOC medium were added. Bacterial cells were spread on agar plates for inoculation. The transfonnants were inoculated in flasks containing 250-ml LB(Laurie Bertani) medium and plasmids encoding the cDNAs were purified by using the Qiagen Maxi kit (Qiagen, Valencia, CA). After purification, plasmids were digested with restriction enzymes, Pst I and Bam HI (for 1.3 kb of TNF-a probe); Pst I (for 0.65 kb of IL-6 probe); and Pst I (for 1 kb of GAPDH probe) . The digested products were run on a 1% agarose gel and purified by using the Genelute spin column (Sigma). Collected cDNAs were used as probes in the further experiments. The housekeeping gene GAPDH cDNA was used as a positive control. Northern blotting and mRNA stability analysis. Total cellular RNA was extracted from RAW 264.7 cells by the modification of the single-step method (Chomczynski and Sacchi, 1987) using RNA-Stat—60 isolation reagent (Tel-test “B” inc., Friendswood, TX). Total RNA (10 pg) was separated by electrophoresis on a 1.5% (wt/vol) agarose, 0.6 M formaldehyde (Sigma), 0.02 M MOPS (Sigma) gel at 80 Volt for 3 hr. The gel was stained with ethidium bromide to verify for the integrity and equal RNA loading, and the RNA was transferred to a nitrocellulose membrane (Nytran, Schleicher & Schuell Inc., Keene, NH) with 20X SSC according to Sambrook et al. (1989). 3 2 P-labeled cDNA -137- probes were prepared by nick translation using the random priming kit (Boehringer Manheim, Indianapolis, IN). After auto-cross-linking for few minutes, the membrane was pre-hybridized at 42 °C in Denhardt’s hybridization buffer ( Kingston, 1997) for 2 hr. Blots were hybridized at the same temperature overnight in a hybridization incubator (Robbins Scientific, Sunnyvale, CA). The hybridized membrane then was washed with SSPE buffer and exposed to Amersham hyperfihn MP(Amersharn, Arlington Heights, IL) with an intensifying screen at -80 °C for variable time periods depending on the signal intensity. Blots were scanned with the Image Quantity (Molecular Dynamics) and the intensities of the signals were analyzed. Relative mRN A levels were determined by the normalization of the signal intensity of TNF-or and IL-6 mRNA against GAPDH mRNA intensity. Statistical analysis. A one way analysis of variance (ANOVA) using Student- Newman-Keuls tests and Dunnett’s test or Kruskal Wallis AN OVA on ranks was applied to parametric data and non-parametric data with application of Sigma-Stat Analysis System (J andel Scientific, San Rafael, CA). A p value of less than 0.05 was considered statistically significant. RESULTS Effects of DRB and VT an cytokine protein expression. An asynchronous model was used whereby RAW 264.7 cells were pre-stimulated with 1 pg/ml of LPS for 2 hr prior to the addition of the transcription inhibitor, DRB, and VT (0, 100 and 250 ng/ml) in fresh DMEM medium for 2, 4, 8 and 24 hr. Transcription inhibitors such as actinomycin D (Act- D) and immunosupressive drugs such as cyclosporin A (CsA) and dexamethasone effectively -138- inhibit both IL-6 and TNF-a expression in macrophages, fibroblasts, and mast cells (Williams and Coleman, 1995; Lu-Kuo et al., 1996; Witowski et al., 1996; Nguyen et al., 1990). DRB, an inhibitor for RNA polymerase during RNA synthesis (Chodosh et al., 1989), has been used frequently to inhibit transcription of cytokine mRN As including those for interleukin-2 (IL—2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Bickel et al. , 1990). In preliminaries study we found that neither Act-D or CsA profoundly inhibited transcription of TNF-0t or IL-6 proteins (data not shown). However, DRB effectively blocked cytokine transcription and was not as cytotoxic as Act-D. In the absence of DRB, a significant increase of TNF-a protein was observed with 100 ng/ml of VT at 2, 4, 8 and 24 hr. TNF-or protein production was completely suppressed by DRB (Fig. 5.1) at all the time points. In the absence of DRB, IL-6 protein expression of was also enhanced by addition of VT (Fig. 5.2). DRB significantly inhibited IL-6 protein production at all time points. DRB was subsequently used as the transcriptional inhibitor in studying the mRNA stability. Effects of Wm T NF-aand IL-6 mRNA stability in asynchronous model. In the asynchronous model, cells were treated with 1 pg/ml of LPS for 2 hr and the medium was removed. Cultures were washed by pipetting and fresh 15 ml DMEM medium containing 100 pM DRB and VT (100 and 250 nglml) or medium alone were added, cells were incubated for different time intervals to monitor the cytokine mRN A expression by Northern analysis. In the absence of VT, TNF-or mRNA levels were decreased by the addition of DRB at all time-points tested (30, 90, 180 and 300 min) (Fig. 5. 3A,B). Degradation of mRN A in the VT-treated cells was less than in untreated cells with mRN A levels remaining -139- 2 hr E DRB ‘ DRB + E 2 ~ 3 5 a 5’ a ‘5 FL FL I- 1 .- 0 ND ND ND 0 100 250 VT (nglml) 4 L DRB + 3 q A b E r v 2 _ V in z .— 1 - ND ND ND 0 r o 100 250 VT (nglml) :21 DRB- 4hr a DRB+ E 2 ‘ . i s i ? u z I- 1 1 0 ND ND ND 0 100 250 VT(nglml) 5 24 hr 8 [:1 DRB - DRB+ 4 .- ‘s‘ _ 3 3 5 .2 2 .4 E b 1 q o [-11 ND ND 7 1 ND 0 100 250 VT(nglml) Fig.5.1. Effect of VT on TNF-a production in LPS-treated RAW 264.7 cells. Cells were pre-treated with 1 pg/ml LPS for 2 hr prior to the addition of VT (0, 100 and 250 ng/ml) in the absence or presence of 100 pM DRB for further incubations. “a” denotes significant difference from control (V0) in the absence of DRB “b” denotes significant difference between each other of different VT treated group in the absence of DRB. Data are representative of two separate experiments. -140- IL-6 (nglml) 0.5 2.0 fl:6(nghn0 2 hr [:1 DRB - 4 hr ‘3 DRB - DRB + DRB {- 0.4 r 1.5 - A a 0.3 E _L j g... 0.2 - 3 7L 0.5 - 0.1 - C C °'° ' FND ND * 1 ND 0.0 ND % - VT (nglml) vr ("gm“) 5 14 8 hr E DRB; 24 hr I I IRB; a 12 ‘ a 4 ‘ F—L i _T__ 10 - .- j is; J— "' 5 - 2 ‘ Z _r_ 4 _ 1 4 o 100 250 o 100 250 F ig.5.2. Effect of VT on IL-6 protein production in LPS-treated RAW 264.7 cells. Experiments were performed as described in the legend of Fig.5. 1. Data are repre- sentative of two separate experiments. “a” denotes significant difference from control (V0) in the absence of DRB; “c” denotes significant difference between the DRB-treated group and the control group (DRB 0) in all VT concentrations. -l41- relatively constant until 180 min. After 300 min, a slight decrease in TNF-a mRNA level was evident in VT-treated cells but the mRNA level of the treatment group was still higher than the control group (0 ng/ml of VT). At the 300 time period, the GAPDH mRNA level was not constant but depleted immediately at all the samples. Therefore, the data could not be used for the calculation of half-life as the GAPDH expression at this time point seemed to be affected by the inhibitor to a broader range. The TNF-a half-life (t1/2) for the control group (0 ng/ml VT) was calculated as 25 min in the asynchronous model. The half-life was longer than 180 min (>180 min) with the addition of the 100 and 250 ng/ml of VT. In the asynchronous model, control IL-6 mRNA level increased over time upon the removal of LPS and in the absence of DRB (Fig. 5.3A, C). With the addition of DRB, the IL-6 mRNAs were depleted as early as 30 min in the absence of VT treatment. In contrast, the IL-6 mRN A levels were stabilized by the addition of VT at all the time periods (30 to 300 min). The GAPDH mRNA level in the asynchronous model remained constant at all VT concentrations except at 300 min. For the IL-6 half-life study, 100 and 250 ng/ml of VT was able to extend the mRNA half-life from 60 min to greater than 3 hr. Effects of VT on T NF -aand IL-6 mRNA stability in delayed synchronous model. In the delayed synchronous model, cells were pre-treated with 1 ug/ml of LPS for 2 hr prior to the addition of VT (100 and 250 ng/ml )or vehicle alone and 100 uM DRB. Cells were incubated and TNF-0t and IL-6 mRNA were monitored over 300 min. Again, DRB depressed TNF-0L mRNA expression at 30, 90, 180 and 300 min (Fig. 5. 4A, B). TNF-a mRNA levels in DRB-treated cells were higher in the presence of both 100 and 250 ng/ml VT compared to the controls at 30, 90, 180 and 300 min. TNF-oz half-life of the untreated -142- .mEoEr—onxo 2388 05 mo 03388292 Pa Sum 4888 3:85 an 8 new: 83 $920 anew mafioox 8:0: 23. .oSmm 05 E @2865 we wooing 2:: “:2th mo cotanzofi Esta com 3an mm? 921 8: man Shim GEE: omN can oofiov H> mfifificoo €3on 50E 60382 55 mm? 83on 05 98 E N com mm..— =Em1 H 5;» wouaoboa ob? £30 .260 Evom 35 coasting E 56855 3028:08an mo maooth/wv Wm .wE {anomalous-Ion.- :5 .ll...l.'.l...ll.. ea: . . . - ’. i , size :3 . p ‘ + + + - + + + - + + + - + + + - - mm: :32: a 0:32: a 0325:. o 32:: a u o b» 2:. a: all |% d 3.5 2:? g -143- .\° B a: 2 100 « g 75 < E «I 50 . E o n: < z 25 < a: E + WOng/ml $3 —0— VT 100 nglml % + VT 250 nglml h 10 r T t l T T I T I I O 20 40 60 80 100 120 140 160 180 200 Time after the addition of DRB (min) :5 C 8, 1001 m 75 4 .E .5 50 . a E o a: < 25 - E E + VT 0 nglml -O— VT 100 nglml 3 + VT 250 nglml - 10 I I T T T T W O 20 40 60 80 100 120 140 160 180 200 Time after the addition of DRB (min) Fig.5.3 (B) mRNA stability for TNF-oz in an asynchronous model; (C) mRNA stability for IL-6 in an asynchronous model. -l44- group was l.3 hr, and it was extended beyond 180 min by both 100 and 250 ng/ml of VT. DRB also depressed IL-6 mRNA expression(Fig. 5. 5A, B) . However, IL-6 mRNA levels were higher in the VT-treated cells than in the control group at 30, 90 and 180 min time periods with DRB. IL-6 mRNA half-life was 90 min in the absence of VT in this model but was extended to longer than 3 hr in the presence of 100 and 250 ng/ml of VT. DISCUSSION Superinduction of cytokines has been implicated to have a major contribution in toxicological and immunological pathogenesis caused by VT, such as IgA deposition in the kidneys of mice (Dong et al., 1994). Cytokine superinduction has previously been observed in macrophage RAW 264.7 cells treated with VT (Wong et al., 1998). In the present study, VT was found to increase both TNF-a and IL-6 mRNA half-life in this in vitro model. These data suggest that increased cytokine mRNA stability might be responsible for the superinduction of TNF-ct and IL-6 gene expression. The asynchronous model was employed here to study the stabilizing effects of VT without interference from continual stimulation of LPS. The results suggested that VT alone can stabilize both TNF-a and IL-6 mRNA level in this model. These result are consistent with my previous study (Wong et al., 1998), in which both TNF-a and IL-6 mRNA were markedly increased with VT in RAW 264.7 cells. To mimic in vivo experiments, a delayed synchronous model was also employed in this study. When LPS was retained in cell cultures during treatment of DRB and VT, stronger induction in the TNF-0t mRNA expression was observed compared to the asynchronous model. Nevertheless, VT -145- Fig.5.4 (A) Effects of delayed synchronous VT-exposure on TNF-oz mRNA expression in LPS-treated RAW 264.7 cells. Cells were pre-treated with 1 pg LPS for 2 hr prior to the addition of VT (0, 100 and 250 ng/ml) and/or DRB (100 uM). GAPDH gene was used as an internal control. (B) mRNA stability for TNF-oz mRNA in a delayed synchronous model. Data are representative of two separate experiments. -146- Es. man 3 .8228 2: sun 2:: SN 00— cow 0: our SF on 00 9 ON a bi O ’ .58 can 5 ll .55: 8F 5 IoI A6 Bigot/ll (% 5°l) 5U!U!3w93 VNHMI 7°"le -147- IIIIIIIIIIUIIIIII maze dumZH Fl +++-+++-+++-+++--mm: :32: c U amass c Ucmuca c UcmNccfic U U H> A v < can cw“ ca cm a AEEV 25H. F i g.5.5 (A) Effects of delayed synchronous VT-exposure on IL-6 mRNA expression in LPS- treated RAW 264.7 cells. (B) mRNA stability for IL-6 mRNA in a delayed synchronous model. Experiments were performed as described in the legend of Fig.5.4. Data are representative of three separate experiments. -148- 180 300 VT C C 0 100 250 C 0 100 250 C 0 100 250C 0 100 250 90 30 Time (min) 0 1201401W1w zoo «Bo Time after the addition of DRB (min) + W 0 nglml -—o— VT 100 nglml + W 250 nglml e 2 s a 9 (WM) Buwiewau vnuw 9-11 IL-6 GAPDH (A) -149- again prolonged the half-life of TNF—oz and IL-6 mRNA. CHX, another protein synthesis inhibitor, has been found to superinduce cytokine mRNA expression by modulating the mRNA degradation system (Zuckerman et al., 1991; Roger et al. , 1998a, b). Genes encoding inflammatory cytokines such as TNF-on and IL-6, contain several copies of an AU-rich sequence elements (ARE) in their 3'-untranslated region (UT R) (Caput et al., 1986; Chen and Shyu, 1995). The AU rich motif has been suggested to play an important role in destabilizing cytokine mRNA (Fig. 5.6A) (Shaw and Kamen, 1986; Pauli, 1994; Day and Tuite, 1998) and this is the cis determinants of cytoplasmic mRN A stability. Insertion of this sequence into the UTRs of genes with stable mRN As causes instability to the mRNAs (Shaw and Kamen, 1986). In contrast, deletion of this sequence from the UTRs of short-lived genes such as GM-CSF enhances their stability. Thus, in a similar mechanism, VT may prolong the half-lives of these cytokines by protecting the ARE motifs from degradation by endonucleases or exonucleases (Ross, 1996; Ross, 1997). It has been shown that CHX stabilizes mRNA by causing ribosomes to “freeze” or “cluster” on the mRNA, and ribosomes shield the mRNA from cleavage by ribonucleases in the cytoplasm (Fig.5.6B) (Vazquez, 1979; Ross, 1995). It is suspected that VT, which is also a translational inhibitor, may stabilize mRNA via a similar mechanism. VT could also stabilize cytokine mRNA via a “trans” effect. In the trans pathway, a labile protein which is required for degradation of mRNA is inactivated or its synthesis inhibited by the translational inhibitor (F i g. 5. 6C). Examples of proteins that recognize the ARE sequence include AU-A, AU-B, AU-C, and hnRNP (heterogeneous nuclear ribo- nucleoprotein)or also called as AUFl (Bohjanen et al., 1991; Chen and Shyu, 1995; Ross, ~150- F ig.5.6. (A) The AU-rich elements on the 3'-untranslated region of cytokine gene. The genes with this elements are targets of the ribonucleases; (B) With the addition of the translational inhibitors such as cycloheximide (CHX), ribosomes are freezed on the gene; (C) Many proteins recognize the AU-rich elements, they are called ARE-binding proteins. —151- Sesame 232 II I I I 3.28% 822.83% oz I I I I I £2882 magma 152.3 22328on A..." an» I I I 3 225 \ EEELSV . ; Tlm 20:38on 02 on , > A$231I£2V|=S22$ .m Sesame 25 I I I I I I I I a a. >. a II: :2 _e A2T|£222$ l .m CV @(fli2223i Tillam A”... H> E a: 55 A3 2.2 222 I|£2VT| £2223 .m £280.22 w2625-mm< mop—Smog?- . 823—95552 ® 1995). AU-A is mostly abundant in nuclear extracts and shuttles between nucleus and cytoplasm in the absence of RNA polymerase II transcription (Katz et al. , 1994). AU-A was found to induce stabilization of c-myc mRN A in vitro by binding to the AU-rich sequence (Ross, 1996). In contrast, the protein AUFl enhances mRNA degradation by binding to the ARE site in an in vitro assay (DeMaria and Brewer, 1996; Kiledjian et al., 1997). AU-B, another destabilization protein, is a cytoplasmic factor that binds ARE in the 3' UTR of cytokine mRNAs and causes degradation of their mRNAs (Bohjanen et al., 1991). The induction of this protein requires protein synthesis. With translational inhibitors, such as cycloheximide, the stabilization of the mRNAs can be achieved as it inactivates the translation of these critical degradation factors such as AU-B and AUFl. It is suspected that VT might act similarly in stabilizing the cytokine mRNA levels. Interestingly, both transcription and translation rates decline during mitosis (Ross, 1997). During this period, labile mRNAs are stabilized before entering into the Gl phase. These labile mRN As encode labile proteins which are essential for cell survival. From these observations, it seems that mRNA stabilization is a process important for cell viability or survival during mitosis. Therefore, mRNA stabilization in the cells treated with VT may be also vital for the cell survival and preventing them from apoptosis. Meanwhile, it has been shown that with the addition of transcriptional inhibitors, Act-D or DRB, the AU-rich binding proteins (AUBPs) may be transported from the nucleus to the cytoplasm and lead to and increase in the cytopalsmic level of these proteins (Fig. 5.6D) (Chen et al., 1995). This may result in a disturbance of other AUBPs (such as proteins for degradation) and change the equilibrium between these proteins and ARES. -153- Ultimately, this will lead to the disruption of the ARE-protein complexes needed for ARE- induced mRNA degradation (Chen et al., 1995; Sirenko et al., 1997). Thus, inhibition of transcription is also able to stabilize mRN A. Therefore, the choice of transcription inhibitors is critical in mRN A stability study. In this study, DRB seems to have more inhibition effect rather than the stabilization effect. Earlier in this thesis, it was shown that transcriptional regulation may contribute a small positive effect on the cytokine superinduction in macrophage cells (Wong et al., Chapter IV). In this study, stabilization of cytokine mRNA was found to possibly play a more major role in the cytokine superinduction by VT in RAW macrophage cells. These findings are consistent with the observations in EL-4 cells that VT not only induces NF-KB and AP-l binding activity but can stabilize the IL-2 mRNA (Ouyang et al., 1996; Li etal., 1997). The stabilization of these cytokine mRNAs might be related to the enhancement of the MAP kinase activities found during in vivo and in vitro studies recently (Zhou et al., personal communication; Yang et a1, 2000). INKS as well as other MAP kinases have been shown to be involved in the stabilization of cytokine and other targeted gene mRNAs (Chen et al., 1998; Miyazawa et al., 1998; Ming et al., 1998; Winzen et al., 1999; Wang et al., 1999). It has been implied that ARE binding activity is regulated by phosphorylation of these AUBPs by MAP kinases and protein tyrosine kinases (Sirenko et al. , 1997). The exact mechanisms by which these kinases contribute to the stabilization of cytokine mRN A levels are still unclear. Further research on these mechanisms will be necessary. In conclusion, VT was found to stabilize mRNA levels of pro-inflammatory cytokines in RAW 264.7 cells. Hence, stabilization may play a major role in the -154- mfiouoa $2225- 3 22. LA2V1|£222$| chi; fl. 1! ! smut“ £22< .350 I u ve .mcomwoe D< 2: 8 $5533 eeeee D< Ho 2:882 2% 8.2935 £83.35 233221853 .550 can mya .D o.m.wE nee—232383 2.2:... 0 .382 25 283—2523: 2-2.233 Ema—aegmu em 3 £222.: @0 ~155- superinduction of these cytokines and subsequent pathologic sequelae. -156- CHAPTER VI SUMMARY AND CONCLUSIONS -157- This study suggests that trichothecenes can superinduce IL-1 [3, IL-6 and TNF-a gene expression in murine macrophage RAW 264.7 cells. VT could differentially superinduce cytokines by either increasing numbers of cytokine secreting cells or increasing cytokine output per cell. Further investigations into the underlying mechanism of cytokine superinduction showed that the binding activities of transcription factors AP-l , NF-KB and NF-IL6 (C/EBP) were differentially enhanced; and VT specifically targeted the phosporylated c-Jun, JunB, c-Fos, and Fra-Z subunits of the AP-l components; p50 and c-Rel of NF-KB; and C/EBPoc and C/EBPB of NF-IL6. However, fiu'ther studies into the cis-acting elements of the promoter of the TNF-a showed that CAT mRNA expression of the TNF-a-CAT reporter construct was increased only to a small extent by VT in transient transfection assays. Since transcriptional activation might not be the primary mechanism for VT-induced cytokine superinduction in macrophage cells, post-transcriptional regulation was also assessed. In these studies, VT was shown to markedly increase mRNA half-life of both TNF-a and IL-6 in both asynchronous and delayed synchronous models. VT markedly extended the half-life of the TNF-on mRNAs by more than 2-fold in both experimental models. Therefore, post-transcriptional control might be critical to the the VT-mediated TNF-on superinduction in macrophages. In the case of IL-6, its half-life were also extended by at least 10-fold in the asynchronous model; and 2- and 4-fold in the delayed synchronous model. Though IL-6 promoter activity has not yet been investigated, I believe that the stabilization effects of VT might also play an important role in the IL-6 superinduction in macrophages. Since these cytokines play pivotal roles in regulating the VT-induced IgA -158- hyperelevation and its related toxicity, further assessment of the mechanisms are needed to be addressed. First, the effects of VT on the promoter activity of IL-6 could be assessed by using a plasmid containing the IL-6 promoter linked to a reporter gene transfected in the macrophage RAW 264.7 cells. This set of experiments will further determine the role of transcription in the IL-6 superinduction. Secondly, MAP kinases have been suggested to play a role in the stabilization of mRNA half-life in many cell models, but there is not yet direct evidence how these VT-induced MAP kinases stabilize these cytokines. 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