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Title Page

SUMMARY

Contents

LIST OF ABBREVIATION 14

GENERAL INTRODUCTION 15

CHAPTER 1. LITERATURE REVIEW 18

1. Cytokines in general 19

2. Types of cytokines in relation to immune responses 20

3. Cytokines with noncanonical features 28

CHAPTER 2. YEAST TWO-HYBRID SCREENING FOR IL-32-INTERACTING PROTEINS AND RT-PCR ANALYSIS FOR IL-32-MODULATED GENES. 30

1. Abstract 31

2. Introduction 33

3. Materials and methods 35

4. Results 43

5. Discussion 79

CHAPTER 3. A PROINFLAMMATORY CYTOKINE IL-32β PROMOTES THE PRODUCTION OF AN ANTI-INFLAMMATORY CYTOKINE IL-10 86

1. Abstract 87

2. Introduction 88

3. Materials and methods 90

4. Results 94

5. Discussion 104

GENERAL DISCUSSION 109

REFERENCES 113

SUMMARY IN KOREAN 124

List of Tables

CHAPTER 1 13

Table 1. Main types of cytokines 22

Table 2. Cytokines involved in inflammatory reactions 26

CHAPTER 2 13

Table 1. Identificaton of IL-32α interactors. Each positive clone was identified by sequencing and NCBI BLAST search. 47

Table 2. IL-32β-interacting molecules. Each candidate was identified by... 48

List of Figures

CHAPTER 2 10

Fig. 1. Schematic illustration of the yeast two-hybrid system. IL-32α or IL-32β was cloned into a pHybLex/ZT bait vector, then... 45

Fig. 2. Selected clones for IL-32α- (A) or IL-32β-interacting (B) candidates. 46

Fig. 3. Confirmation of constitutive expression of IL-32 in K562 and KG-1a stable... 52

Fig. 4. Investigation of the effect of IL-32 on hemoglobin genes. (A) Hemoglobin... 53

Fig. 5. Cytokines modulated by IL-32. Total RNAs were purified from non-... 54

Fig. 6. Investigation of the effect of IL-32 on cell surface molecules. Non-treated... 55

Fig. 7. PMA-treated K562 stable cell lines were tested for IL-32 modulation of... 56

Fig. 8. The effect of IL-32 on K562 cell differentiation. K562 vector-alone, IL-32α,... 57

Fig. 9. Isoforms of IL-32. Six isoforms, IL-32α-ζ, have been reported. Two... 59

Fig. 10. Sequences of the newly identified IL-32 isoforms. First DDX motif is located in exon 3 and second in exon 6. RGD motif is... 61

Fig. 11. The effect of IL-32 on surface marker expression in K562 cells. IL-... 65

Fig. 12. Cell-surface markers on monocytes, macrophages, and DCs. CD14 was... 66

Fig. 13. The effect of IL-32 on DC differentiation was investigated by silencing IL-... 67

Fig. 14. The effect of IL-32 on DC maturation. Monocytes were transfected with... 69

Fig. 15. Analysis of IL-32-incuded production of soluble factors in monocyte-... 70

Fig. 16. Analysis of the effect of soluble IL-32 on DC maturation. K562... 72

Fig. 17. NK cells were purified and examined for the surface molecules CD56 and... 74

Fig. 18. Analysis of the effect of IL-32 on the co-culture of DC and NK cells. The... 75

Fig. 19. Investigation of the effect of IL-32 on T-cell polarization. The culture supernatants of non-treated K562 stable cell lines... 78

Fig. 20. Negative controls of confocal analysis of IL-32 localization. Mock vector-... 80

Fig. 21. Confocal microscopy study of K562-IL-32α or K562-IL-32β cells. PMA-treated cells were compared with non-treated... 81

Fig. 22. Confocal analysis of IL-32 localization in U937 cells. Endogenously IL-... 84

CHAPTER 3 11

Fig. 1. Construction of IL-32α or IL-32β stable cell lines in K562 cells, and IL-10 induction by IL-32β upon PMA treatment. 95

Fig. 2. IL-10 expression by IL-32β in U937 cells. U937 cells were treated with... 97

Fig. 3. IL-32β induction in monocyte-derived macrophages or monocyte-derived... 100

Fig. 4. Time-course expression patterns of IL-10 and proinflammatory cytokines. 102

Fig. 5. IL-10 induction by IL-32β upon LPS stimulation in monocyte-derived DCs. 105

Fig. 6. The effect of IL-10 neutralization on proinflammatory cytokine production. 106

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