Summary

在小鼠体内的巨噬细胞的枯竭和重建

Published: August 01, 2012
doi:

Summary

巨噬细胞发挥核心作用的动态平衡,并在许多组织的病理。这里介绍的协议描述消耗巨噬细胞的方法<em>在体内</em>,来自偏光巨噬细胞,骨髓穿刺,继转移到小鼠巨噬细胞。这些技术允许极化的巨噬细胞在健康和疾病中发挥的作用的决心。

Abstract

巨噬细胞是重要的球员,在传染病的挑战或损伤的先天免疫反应,发起的先天免疫反应和指挥后天免疫反应。巨噬细胞功能障碍可能导致无法安装适当的免疫反应,因此,已在许多疾病的进程,包括炎症性肠病牵连。巨噬细胞显示极化表型大致分为两类。经典活化的巨噬细胞,与干扰素γ或LPS刺激激活,发挥了至关重要的作用,在细菌的挑战,而另外激活的巨噬细胞,IL-4和IL-13激活,参与清除杂物和组织重塑和已在该决议牵连阶段的炎症。在体内的炎症反应,巨噬细胞的免疫细胞浸润的复杂混合物中发现,并可能加剧或resolvin参加Ğ炎症。巨噬细胞的作用,在整个动物模型的定义在原地 ,这是必要的检查消耗从复杂的环境中的巨噬细胞的影响。极化的巨噬细胞表型定义问原位巨噬细胞表型的作用的问题,可以导出体外 ,从骨髓穿刺,并加回或没有事先枯竭的巨噬细胞,小鼠。在这里提出的协议中含有氯膦酸二钠,脂质体,与PBS注射控件,用来消耗结肠巨噬细胞在葡聚糖硫​​酸钠(DSS)诱导小鼠结肠炎。此外,巨噬细胞的极化,推导出体外,并转移到小鼠静脉注射。这种方法的一个需要注意的是,氯膦酸二钠含脂质体耗尽所有专业的吞噬细胞,包括树突状细胞和巨噬细胞,所以,以确保枯竭所观察到的效果是巨噬细胞特异性,重组ØF的巨噬细胞过继转移表型是必要的。全身巨噬细胞耗竭小鼠也可以通过回交小鼠实现到细胞CD11b-DTR背景,这是一个很好的补充办法。含有氯膦酸二钠脂质体介导的枯竭的优势是,它不参与回交小鼠需要的时间和费用,它可以在小鼠小鼠(C57BL / 6小鼠,BALB / c小鼠,或混合背景的背景下,无论使用)。

Protocol

1。使用含有氯磷酸二钠,脂质体的消耗巨噬细胞脂质体储存在4°C注射前两个小时,从冰箱中取出氯膦酸二钠含脂质体,PBS含脂质体,或无菌PBS(喷射控制),使他们能够适应新环境室温(18℃)。 反转管含脂质体的8-10倍,以确保均匀分布,然后装入1 ml注射器200μL。将26号针头注射器顶端。 用你的左手,浮渣鼠标,略低于持有足够的皮肤以固定其头部和四肢的耳朵。 …

Discussion

巨噬细胞的吞噬细胞在免疫系统中发挥重要作用。他们是负责发起的先天免疫反应和指挥后天免疫反应。通常情况下,经典激活巨噬细胞被激活IFNγ的或LPS,并有责任为消除病原体和安装的炎症反应1。相反,IL-4和IL-13激活或者激活巨噬细胞和炎症的决议过程中发挥在碎片中的作用,清除和组织重塑。专门的巨噬细胞在肠道保留其杀菌活性,但不装入一个亲炎症反应2。这种有益菌?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了1“的研究资助津贴”的克隆氏病的和结肠炎加拿大基金会的LMS,谁是1​​消化科/加拿大协会健康研究/克罗恩的小号和结肠炎加拿大新研究者奖基金会加拿大协会的支持。

Materials

Name of the reagent Company Catalogue number
Clodronate-containing liposomes Clodronateliposomes.org  
PBS-containing liposomes Clodronateliposomes.org  
Sterile PBS pH7.4 Invitrogen 14190
IMDM Invitrogen 12440
Fetal Calf Serum (FCS) Invitrogen 12483
acetic acid BDH Aristar BDH3092-500MLP
Monothioglycerol (MTG) Sigma 96-275
Recombinant murine MCSF Stemcell Technologies 02951
Recombinant murine GM-CSF Stemcell Technologies 02935
Recombinant murine IL-3 Stemcell Technologies 02903
Recombinant murine IFNγ Stemcell Technologies 02746
Recombinant murine IL-4 Stemcell Technologies 02714
Cell Dissociation Buffer Invitrogen 13150-016
Rat anti-F4/80 Antibody AbD Serotec MCA497GA
Mouse anti-ArgI Antibody BD Transduction Laboratories 610708

Table 1. Specific reagents used in this protocol.

References

  1. Gordon, S. Alternative activation of macrophages. Nat. Rev. Immunol. 3, 23-35 (2003).
  2. Rescigno, M., Lopatin, U., Chieppa, M. Interactions among dendritic cells, macrophages, and epithelial cells in the gut: implications for immune tolerance. Curr. Opin. Immunol. 20, 669-675 (2008).
  3. Heinsbroek, S. E., Gordon, S. The role of macrophages in inflammatory bowel diseases. Expert Rev. Mol. Med. 11, e14 (2009).
  4. Collins, S. M., Piche, T., Rampal, P. The putative role of inflammation in the irritable bowel syndrome. Gut. 49, 743-745 (2001).
  5. Maeda, S. Colon cancer-derived factors activate NF-kappaB in myeloid cells via TLR2 to link inflammation and tumorigenesis. Mol. Med. Report. 4, 1083-1088 (2011).
  6. van Rooijen, N., van Kesteren-Hendrikx, E. Clodronate liposomes: perspectives in research and therapeutics. J. Liposome Res. 12, 81-94 (2002).
  7. Hunter, M. M. In vitro-derived alternatively activated macrophages reduce colonic inflammation in mice. Gastroenterology. 138, 1395-1405 (2010).
  8. Weisser, S. B. SHIP-deficient, alternatively activated macrophages protect mice during DSS-induced colitis. J. Leukoc. Biol. 90, 483-492 (2011).
  9. Qualls, J. E., Kaplan, A. M., van Rooijen, N., Cohen, D. A. Suppression of experimental colitis by intestinal mononuclear phagocytes. J. Leukoc. Biol. 80, 802-815 (2006).
  10. Van Rooijen, N., Sanders, A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J. Immunol. Methods. 174, 83-93 (1994).
  11. Smith, P. Infection with a helminth parasite prevents experimental colitis via a macrophage-mediated mechanism. J. Immunol. 178, 4557-4566 (2007).
  12. van Rooijen, N., Hendrikx, E. Liposomes for specific depletion of macrophages from organs and tissues. Methods Mol. Biol. 605, 189-203 (2010).
  13. Kataoka, K. The roles of vitreal macrophages and circulating leukocytes in retinal neovascularization. Invest. Ophthalmol. Vis. Sci. 52, 1431-1438 (2011).
  14. Weisser, S. B. Alternative activation of macrophages by IL-4 requires SHIP degradation. Eur. J. Immunol. 41, 1742-1753 (2011).
  15. Cailhier, J. F. Conditional macrophage ablation demonstrates that resident macrophages initiate acute peritoneal inflammation. J. Immunol. 174, 2336-2342 (2005).

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Cite This Article
Weisser, S. B., van Rooijen, N., Sly, L. M. Depletion and Reconstitution of Macrophages in Mice. J. Vis. Exp. (66), e4105, doi:10.3791/4105 (2012).

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