돼지 신장세포(PK15)를 이용한 이종이식 모델에서 마우스 인터루킨-18 및 인간 인터루킨-32에 의한 이식 거부반응의 조절 : Regulation of Xenograft Rejection by Recombinant Mouse Interleukin-18 and Human Interleukin-32 in Xenografting Model using Porcine Kidney PK15 Cells

국문 초록 (Abstract)

장기이식을 필요로 하는 말기 장기부전환자에 비해 공여 장기의 수는 절대적으로 부족해 이식 장기에 대한 수요를 충족시킬 수 없는 한계를 나타내고 있다. 이에 대한 대안으로 동물의 장기를 인간에게 이식하는 이종이식에 대한 많은 연구가 현재 수행되고 있다. 그러나 이러한 이종이식은 공여 장기와 수여자간의 유전적 차이로 인해 많은 제한이 따르고 있다. 지난 수십년간 이종이식시 수반되는 거부반응을 줄이기 위해 많은 연구가 수행되어 왔으며 그 결과, 현재는 이종이식 거부반응을 어느 정도 억제할 수 있는 면역억제제가 생산되고 있다.
싸이토카인은 세포와 세포간의 신호전달을 매개하는 조절단백질이다. 이들 싸이토카인은 일반적으로 백혈구에 의해 생성되어 주로 면역기능을 조절하는 역할을 수행한다. 인터루킨-18은 전염증성 싸이토카인으로서 조직손상시 염증을 유발하는 인터페론-감마의 생성을 촉진하며 또한 인터루킨-1베타와 인터루킨-8의 생성을 촉진한다. 인터루킨-32는 최근 발견되었으며 주로 T세포와 자연살해세포 및 상피세포에서 생성되는 전염증성 매개인자로서 종양괴사인자-알파와 인터루킨-8의 생성을 촉진시킨다.
본 연구에서는 C57BL/6 마우스의 비장세포에 대한 재조합 마우스 인터루킨-18 및 재조합 인간 인터루킨-32알파와 인터루킨-32베타의 영향을 조사하였으며, 또한 돼지 신장세포 (PK15) 를 마우스에 transfer 한 후 재조합 마우스 인터루킨-18 및 재조합 인간 인터루킨-32알파와 인터루킨-32베타를 처리하여 Th1 및 Th2 싸이토카인들의 발현 수준을 조사하였다. 부가적으로 cDNA microarray 를 이용하여 재조합 마우스 인터루킨-18 및 재조합 인간 인터루킨-32알파와 인터루킨-32베타의 처리에 따른 유전자 발현 양상을 비교하였고 이종이식 거부반응에 있어서 중요한 역할을 수행하는 면역관련 유전자를 확인하였다.
본 실험의 생체 내 및 생체 외 실험에서 이들 싸이토카인의 처리는 PBS 처리 그룹에 비해 세포의 생존율을 증가시키는 것으로서 확인되었다.
유세포분석 결과, CD4^(+) T 세포 및 CD8^(+) T 세포는 PBS 처리 그룹에 비해 재조합 마우스 인터루킨-18 및 재조합 인간 인터루킨-32알파와 인터루킨-32 베타 처리 그룹들에서 전반적으로 낮은 발현 양상을 보였다. 따라서 돼지 신장세포 (PK15) 를 마우스에 transfer 후 이들 싸이토카인을 처리하므로서 T세포의 활성을 억제하여 면역거부반응을 억제 시킬 수 있는 것으로 사료된다.
마우스의 혈청에서 Th1 및 Th2 싸이토카인들의 농도는 싸이토카인 처리 3일에는 PBS 처리 그룹에 비해 싸이토카인 처리 그룹에서 발현 수준이 높게 나타난 반면 싸이토카인 처리 6일과 9일에는 Th1 및 Th2 싸이토카인 모두 발현 수준이 낮았다. 마우스의 조직에서 Th1 및 Th2 싸이토카인의 발현 수준은 PBS 처리 그룹에 비해 재조합 마우스 인터루킨-18 및 재조합 인간 인터루킨-32알파와 인터루킨-32베타 처리그룹에서 높게 나타났으며 전반적으로 Th1 싸이토카인의 발현 수준이 높은 것으로 확인되었다.
cDNA microarray 결과, PK15/pIL-18 세포 및 재조합 인간 인터루킨- 32알파와 인터루킨-32베타를 처리한 그룹에서 정상 그룹에 비해 면역관련 유전자들 및 기타 생물학적 기능을 가진 다양한 유전자들이 2배 이상 발현이 증가 또는 감소된 것으로 확인되었다.
결론적으로 돼지 신장세포 (PK15) 를 마우스에 transfer 후 재조합 마우스 인터루킨-18 및 재조합 인간 인터루킨-32알파와 인터루킨- 32베타의 처리는 CD8^(+) T 세포 및 Th2 싸이토카인의 발현 수준을 감소시키므로서 이종이식 거부반응의 초기 단계에서 이종이식편에 대한 숙주 면역세포의 인식을 회피하도록 하여 면역거부반응을 조절하는 것으로 사료된다.

다국어 초록 (Multilingual Abstract)

The enormous disparity between the supply of donor organs and the number of patients requiring transplantation for end stage organ failure has resulted in research into the possibility of using donor organs from non-human sources. Xenotransplantation is a potential solution to organ shortage, but it is constrained by the large genetic difference between pigs, the potential donors, and humans. For successful xenotransplantation, many new immunosuppressive medica-tions have been developed in the past two decades for the treatment of graft rejection in xenotransplantation.
Cytokines are secreted regulatory proteins that mediate cell-to-cell communication. They are normally produced by leukocytes and principally serve to regulate the functions of the immune system. Interleukin 18 (IL-18) was first isolated as interferon-γ inducing factor, a mediator of inflammatory tissue damage. IL-18 is also a pro-inflammatory cytokine which enhances IL-1 β and IL-8 production. IL-32 is a recently described cytokine produced mainly by T, natural killer, and epithelial cells that has the properties of a proinflammatory mediator. IL-32 stimulates TNF-α and IL-8 production.
In this study, the influence of rmIL-18, rhIL-32α or rhIL-32β on the growth of C57BL/6 mouse spleen cells was investigated, and Th1 and Th2 cytokine profiles were examined in cytokine treated mice after PK15 cell transfer. Also, the gene expression profiles in acutely rejected mouse kidney cellular xenografts were investigated by means of a cDNA microarray, and examined that immune-related gene played a pivotal role during cellular xenograft rejection.
In vitro and in vivo experiments, cell viability of rmIL-18, rhIL-32α, and rhIL-32β treated group were higher than in the PBS treated group.
CD4+ T cells and CD8+ T cells were low in the cytokine treated group compared to PBS treated group. Therefore, the treatment of cytokine after PK15 cell xenografting seems to induce immune tolerance of recipient’s cells by attenuated the T cell activation. CD3+ T cells and B220+ cells, also, showed similar results. Therefore, these data shows that cytokine treatment reduced the cytolytic activity of splenocytes after xenograft.
In mouse serum, levels of Th1 (IL-2, IL-12, TNF-β, and IFN-g) and Th2 (IL-4, IL-5, IL-6, and IL-10) cytokines were high in the all cytokine treated groups than those of the PBS treated group at 3 days, but Th1 and Th2 cytokine levels at 6, and 9 days were low in the cytokine treated groups. Whereas, in mouse tissues, levels of Th1 and Th2 cytokines were high in the all cytokine treated groups than those of the PBS treated group at all time points. But, cytokines expression levels of cytokines were higher in the Th1 cytokines than those of in the Th2 cytokines.
In mouse spleen tissues, various genes were up- or downregulated more than 2-fold in the PK15/pIL-18 cells, rhIL-32α or rhIL-32β treated group compared to the normal control group. The expression profiles of immune related genes in response to cytokine administration after PK15 cell transfer shown highly expressed in the PK15/pIL-18 cells, rhIL-32α or rhIL-32β treated group compared with the PBS treated group.
In conclusion, the treatment of IL-18, IL-32α or IL-32β after PK15 cell transfer regulated xenograft cellular rejection by escape recognizing host immune cells of xenotransplantation during the early phases of xenograft rejection via downreulation of CD8+ T cells and Th2 cytokines.

목차 (Table of Contents)

• I. INTRODUCTION 1
• 1. Manipulation of cytokines to overcome xenotransplantation rejection 1
• 2. Role of interleukin-18 in the regulation of immune system 10
• 3. Role of interleukin-32 in immune system 11
• 4. Microarray gene expression analysis of nonhuman transplant models 14
• Ⅱ. MATERIALS AND METHODS 16
• 1. Materials 16
• 1.1. Mice 16
• 1.2. Cell lines and culture 16
• 1.3. Reagents 16
• 2. Methods 17
• 2.1. Effects of cytokines on cell viability 17
• 2.1.1. Mouse spleen cell preparation 17
• 2.1.2. Treatment of cytokines 18
• 2.1.3. Cell viability assay 18
• 2.1.4. Single cell cytotoxicity assay 19
• 2.2. Effects of cytokine treatment in pig-to-mouse PK15 cells xenografting model 20
• 2.2.1. PK15 cell transfer and cytokine administration 20
• 2.2.2. Mixed lymphocyte reaction (MLR) 21
• 2.2.3. Flow cytometric analysis 22
• 2.2.4. Cell-mediated cytotoxicity assay 22
• 2.2.5. Cytokine production in serum: multiplex bead-based immunoassay 24
• 2.2.6. RNA Isolation for semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) 26
• 2.2.7. Analysis of cytokine mRNA levels by semi-quantitative RT-PCR 26
• 2.2.8. RNA isolation and quality control for cDNA microarray 29
• 2.2.9. In Vitro transcription and RNA amplification for cDNA microarray 29
• 2.2.10. cDNA microarray data analysis 31
• 2.3. Establishment of stable PK15/pIL-18 cell line and gene expression profiles in PK15/pIL-18 cell xenografting model 32
• 2.3.1. Plasmid construct of porcine interleukin-18 32
• 2.3.2. Cell transfection 33
• 2.3.3. Transfer of PK15 cell and PK15/pIL-18 transfectants 33
• 2.3.4. Microarray analysis in PK15/pIL-18 xenografting model 33
• 2.4. Transfection of primarily cultured porcine embryonic fibroblasts and the production of cloned transgenic pigs 34
• 2.4.1. Cell lines and cell culture 34
• 2.4.2. Plasmid construction of human interleukin-32B 35
• 2.4.3. Stable transfection and selection of target cells 36
• 2.4.4. Identification of stable transfectants using reverse transcription polymerase chain reaction (RT-PCR) 37
• 2.4.5. Confocal microscopy analysis of stable transfectants 37
• 2.4.6. Transfer of PEF/C2 and PEF/IL-32B transfectants 38
• 2.4.7. Preparation of lymph node cells and flow cytometric analysis 38
• 2.4.8. Cloning of the transgenic pigs expressing human IL-32B 39
• Ⅲ.RESULTS 41
• 1. Effects of cytokine treatment on mouse splenocyte growth 41
• 2. Proliferation effect of responder T cells on xenogeneic stimulator cells after cytokine administration 44
• 3. Phenotype analysis of recipient’s cells in response to cytokine administration after PK15 cell transfer 46
• 3.1. CD4+ T cells and CD8+ T cells 46
• 3.2. CD3+ T cells and B220+ cells 50
• 4. Effect of cytokine treatment on cytolytic activities of donor cells after PK15 cell transfer 53
• 5. Cytokine production in mouse serum by cytokine treatment after PK15 cell transfer 56
• 6. Cytokine mRNA expression in mouse tissues by cytokine treatment after PK15 cell transfer 59
• 6.1. The sizes of amplified cytokine products by RT-PCR 59
• 6.2. Th1 cytokine expression 59
• 6.3. Th2 and IL-18 cytokine expression 63
• 7. Gene expression profiles in spleen tissues of mouse administered with cytokines after PK15 cell transfer 70
• 7.1. Identification of RNA purity for microarray analysis 70
• 7.2. Internal validation for microarray analysis 73
• 7.3. Global gene expression profiling in response to cytokine administration after PK15 cell transfer 75
• 7.4. Functional classification of differentially expressed genes in response to cytokine administration after PK15 cell transfer 78
• 7.5. Expression profiling of immune related genes in response to cytokine administration after PK15 cell transfer 84
• 8. Gene expression profile during hyperacute rejection in pig-to-mouse disconcordant kidney cellular xenograft by means of cDNA microarray 88
• 9. Construct of IL-32B-GFP expression transfectants and the production of cloned transgenic pigs 92
• 9.1. Specific expression of IL-32B in PEF cells transfected with IL-32B 92
• 9.2. Identification of IL-32B transfectants using confocal microscopy analysis 93
• 9.3. Comparison of the patterns between phenotypes of recipient's cells in response to xenogenic PEF vs PEF/IL-32B 94
• 9.4 Distribution of activated T cells 95
• Ⅳ.DISCUSSION 96
• Ⅴ.CONCLUSION 102
• Ⅵ. REFERENCES 105
• KOREAN ABSTRACT 120