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Identification of macrophage phenotypes and evaluation of their role in the immunopathogenesis in animal model of Behçet’s disease

초록/요약

Although several immunological abnormalities have been demonstrated in Behçet’s disease (BD), the exact mechanism of the inflammatory changes occurring during the development of this disease remains unclear. Macrophage phenotypes are markedly heterogeneous and include classically activated (M1) and alternatively activated (M2) macrophages. In a herpes simplex virus (HSV)-induced BD mouse model, it has confirmed the abundant expression of the M1 phenotype (CD16/32) compared to the M2 phenotype (CD23). For M1/M2 phenotypic transformation in vivo normal mice, recombinant interferon-gamma (rIFN-g) significantly increased the M1/M2 ratio (1.74±0.42) when compared with recombinant interleukin-4 (rIL-4) (0.83±0.20) or the phosphate buffered saline (PBS) treated control (0.66±0.34). In BD mice, rIL-4 treatment decreased the M1/M2 ratio (1.2±0.3) compared to PBS treated (2.4±2.9) or rIFN-g treated groups (2.1±2.3). The M1/M2 ratio in rIL-4 treated BD mice was similar to the asymptomatic BD mice (BD normal, BDN) (1.0±0.9). In BDN mice, rIFN-g or rIL-4 treatment did not affect the M1/M2 ratio (1.2±0.4, 1.2±0.7, respectively). This amelioration of BD-like symptoms was correlated with a decrease in the M1/M2 ratio accompanied by down-regulation of IL-17 and IL-6, and up-regulation of IL-4. Furthermore, colchicine and pentoxifylline, which are frequently prescribed BD medications, decreased the M1/M2 ratio and improved BD-like symptoms in mice. This study clearly demonstrates the correlation of the M1 and M2 macrophage phenotypes and the M1/M2 ratio with HSV-induced BD-like symptoms. Therefore, a shift in the population of macrophages is a pathologic factor and modulation of macrophage phenotypes may have important implications for the treatment of BD. Key words: Behçet’s disease, CD16/32, CD23, colchicine, HSV-induced inflammation, M1/M2 macrophages, mouse model

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목차

ABSTRACT ……………………………………………………………………….i
TABLE OF CONTENTS ………………………………………………………….iii
LIST OF FIGURES ...…..………………………………………………………….v
LIST OF ABBREVIATIONS …………………………………………………….vi
I. INTRODUCTION ……………………………………………………………….1
A. Background of Behçet’s disease………………………………………….1
B. Macrophage activation and functional heterogeneity.2
C. Behçet’s disease mouse model……………………………………………… 4
D. Immune responses in Behçet’s disease…………………………….6
E. Effects of anti-inflammatory drugs in Behçet’s disease…………………….9
F. Aims of the study……………………………………………………….12
II. MATERIALS AND METHODS ……………………………………………….13
A. Animals …………………………………………………………………….13
B. Clarification of BD, BDN and normal mouse ………….13
C. Severity score of BD mouse ……………………………………………….13
D. Recombinant cytokines, biologically active substances and peritoneal macrophages culture.14
E. Reverse Transcriptase (RT)-PCR …………………………………….15
F. Flow cytometry …………………………………………………………….16
G. Transmission Electron Microscopy ……………………………….16
H. BD incidence ……………………………………………………………….17
I. Measurement of cytokines by ELISA ……………………………….17
J. Adoptive transfer of macrophages ………………………………….18
K. Treatment of BD-like symptoms ………………………………………. 18
L. Statistical analysis ………………………………………………………….18
III. RESULTS ……………………………………………………………………….19
A. Confirmation of macrophage phenotype in BD mice...19
B. Macrophage phenotypic transformation in vivo normal mice …………….23
C. Macrophage phenotypic transformation in vivo BD mice ….28
D. Confirmation of macrophage phenotypes in HSV inoculated mice at early infection.33
E. Changes of intracellular morphology in the presence of rIFN-g or rIL-4….36
F. Proliferation of macrophages by M1 and M2 stimulators in vitro………….39
G. Macrophage phenotypes can influence BD incidence....41
H. Cytokine levels after treatment of BD mice with M1 or M2 stimulators….43
I. Adoptive transfer of M1 and M2 macrophages into BD mice …………….45
J. Colchicine or pentoxifylline down-regulates M1/M2 ratio and improves the BD-like symptoms ……………………………….49
IV. DISCUSSION ………………………………………………………………….53
V. CONCLUSION ………………………………………………………………….59
REFERENCES…………………………………………………………………….60
ABSTRACT in Korean language………………………………………………….76

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