G-CSF (Granuocyte Colony Stimulating Factor) Granulocyte Colony Stimulating Factor (G-CSF) Attenuates 2,4,6-Trinitrobenzene Sulfonic Acid (TNBS)-induced Colitis in Mice Eun-Young Choi 1, Chang-Duk Jun 1, Jae-Min Oh 2, Yu-Rim Kim 2, Soo-Teik Lee 3 and Sang-Wook Kim 3 1 Department of Physiology, Kyungpook National University School of Medicine, Daegu, 2 Department of Anatomy, Wonkwang University School of Medicine, Iksan, 3 Department of Internal Medicine, Chonbuk National University School of Medicine, Jeonju, Korea ABSTRACT Background: Granulocyte colony stimulating factor (G-CSF) is known as a cytokine central to the hematopoiesis of blood cells and to modulate their cellular functions. Besides granulocytes and their precursors, monocytes/macrophages and endothelial cells are direct target cells of G-CSF action. G-CSF influences immune cells in an antiinflammatory way. Methods: To evaluate whether G-CSF has a potential for preventing or ameliorating diseases characterized by mucosal inflammation, we used a mouse model with trinitrobenzene sulfonic acid (TNBS)-induced inflammatory colitis. To the mice model G-CSF was administrated daily by intraperitoneal injection. Macroscopic evaluation and immunohistochemical analysis of colonic tissues were performed. Results: Recombinant human G-CSF significantly inhibited LPS-induced TNF- mrna expression in THP-1 cells. As for in vivo relevance, G-CSF dramatically reduced the weight loss of mice, colonic damage, and mucosal ulceration that characterize TNBS colitis. Moreover, G-CSF suppressed the expression of tumor necrosis factor-, interleukin-1, and intercellular adhesion molecule-1 in TNBS colitis. Conclusion: Current results demonstrate that G-CSF may be an effective agent for the treatment of diseases characterized by mucosal inflammation. (Immune Network 2006;6(1):13-19) Key Words: G-CSF, inflammatory bowel diseases, inflammation, TNBS Immune Network 13
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G-CSF Attenuates Inflammatory Bowel Diseases 15 LPS - - - - + + + + + + Time (h) 0 2 4 6 2 4 6 2 4 6 TNF-α GAPDH + G-CSF Figure 1. G-CSF inhibits the expression of TNF- induced by LPS. THP-1 cells (5 10 5 /ml) were pretreated with G-CSF (500 ng/ml) for 1 h, and then the cells were further incubated for 16 h with LPS (10 g/ml). Levels of TNF- mrna were determined by RT-PCR. This is a representative of three separate experiments. A Weight (g) 26 Vehicle TNBS G-CSF 24 G-CSF+TNBS 22 20 18 B Macroscopic Score 10 9 8 7 6 5 4 3 2 계열 1 16-5 0 5 10 Time (days) 1 0 TNBS - 1 + 2-3 4+ G-CSF - - + + Figure 2. G-CSF ameliorates the clinical and macroscopic features of TNBS-induced colitis. Colitis was induced by rectal administration of 2 doses (separated by a 7-day interval) of TNBS in 50% ethanol. Mice treated with 50% ethanol alone were used as controls. G-CSF (250 g/kg) was injected intraperitoneally everyday 1 day (-1) before and after TNBS administration. All mice were sacrificed on day 9 after the first TNBS administration, and the clinical evaluation and severity were monitored by mouse weight changes. Each point represents the mean±sd from 5 separate experiments (5 mice/group/experiment).
16 Eun-Young Choi, et al. A B Histologic Score 10 8 6 4 2 0 TNBS - + - + G-CSF - - + + Figure 3. G-CSF ameliorates the histopathologic features of TNBS-induced colitis. The histopathologic signs were further estimated with the mice described in Figure 4. Photomicrographs of colon sections after treatment with ethanol, TNBS, G-CSF, and G-CSF+TNBS, on day 9 after the induction of colitis with TNBS are shown (original magnification 100 ) (A). Histopathologic scoring is shown (B). Results are the mean±sd from 5 mice per group.
G-CSF Attenuates Inflammatory Bowel Diseases 17 Figure 4. G-CSF reduces the expression of TNF-α IL-1β, and ICAM1 in colonic mucosa of TNBS colitis. Tissue sections from experimental groups (control group, TNBS- and/ or G-CSF-treated groups) were incubated with primary antibodies (αtnf-α, α-il-1β, and α-icam- 1). The slides were then incubated with anti-rabbit biotinylated secondary antibody and colorized by ABS/hematoxylin (original magnification 100). 모델을 이용한 실험에서도 증명이 되었다. 예를 들면, 랫 트에서 LPS로 유도된 독성을 G-CSF가 전신성 TNF-α를 억제함에 따라 예방할 수 있음을 보고하였다(14). 또한 마우스에서 LPS로 유도된 IL-18 및 IFN-γ 생성을 G-CSF 전처리로 감소시켰다(22). 또 다른 보고에 따르면, 마우 스에서 G-CSF 전처리 시 LPS로 유도되는 IL-10 및 monocyte chemoattractant protein-1 의 분비가 증가되는 반면, IL-1β, IL-6 및 IL-8의 분비에는 영향을 주지 않았다(23).
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