MMP12缺失引起小鼠血液和白色脂肪中巨噬细胞的变化
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摘要
目的 MMP12(matrix metalloproteinase-12)又称巨噬细胞弹性蛋白酶,几乎能分解各种细胞外基质成分和血管成分,且和单核细胞募集有关。还发现MMP12参与肿瘤细胞的侵袭及转移,此外,MMP12还与脂肪组织密切相关。本研究旨在探讨MMP12敲除(MMP12~(-/-))小鼠血液和白色脂肪中巨噬细胞的变化。方法利用PCR对MMP12~(-/-)小鼠进行基因型鉴定。用同窝MMP12~(+/+)雄性小鼠做对照,检测两组小鼠的血常规并进行分析;进一步利用流式细胞术检测巨噬细胞标记物,了解巨噬细胞的变化;最后,利用免疫组化等方法来观察MMP12缺失后脂肪组织中巨噬细胞的变化。结果 (1)鉴定MMP12~(-/-)小鼠基因型并扩群;(2)与同窝MMP12~(+/+)雄性小鼠相比,MMP12~(-/-)雄性小鼠的红细胞计数、血红蛋白量、血小板计数、单核细胞数以及其百分数等参数均下降,具有显著差异(P<0.05);淋巴细胞百分数和嗜酸性细胞百分数,显著上升(P<0.05),其绝对计数无统计学差异;(3)流式结果提示:巨噬细胞的特异标记物CD11b与F4/80双阳性细胞的比例在血液中显著下降,其绝对计数也降低;(4)H&E染色和免疫组化的结果显示:与同窝MMP12~(+/+)雄性小鼠相比,MMP12~(-/-)雄性小鼠的白色脂肪组织中的CD68表达明显提高。结论提示巨噬细胞的变化可能与MMP12敲除有关。其意义在于发现MMP12可能调控了巨噬细胞的分化,血液中巨噬细胞减少,白色脂肪中的巨噬细胞增多。
Objective Matrix metalloproteinase-12(MMP12) also known as macrophage elastase, is capable of decomposing almost all extracellular matrix components and is required for monocyte recruitment. MMP12 has been found to be involved in cancer invasion and metastasis. In addition, MMP12 is also closely related to adipose tissue. This study investigated whether MMP12 regulates the macrophages in blood and white fat in MMP12 knockout(MMP12~(-/-)) mice. Methods MMP12~(-/-)mice were raised and genotyped, and littermate MMP12~(+/+) male mice were used as controls. The routine blood indexes of the two groups of mice were analyzed, and the macrophage markers were detected by flow cytometry. Pathdogy using HE staining and immunohistochemistry was performed to determine the macrophages in adipose tissue after MMP12 deletion. Results(1) The genotype of MMP12~(-/-)mice and expansion was identified.(2) Compared with those of littermate MMP12~(+/+) male mice, parameters including RBC count, HGB level, PLT count, and monocyte count and percentage were significantly reduced in MMP12~(-/-) male mice. However, the proportions of lymphocytes and eosinophils were significantly increased compared with those of control mice, but their absolute counts did not differ significantly.(3) Further result suggested that the proportion of macrophage-specific marker CD11 b and F4/80 double positive cells was significantly decreased in the blood, and their absolute count was also reduced.(4) The pathdogy using HE staining and immunohistochemistry confirmed that macrophage(CD68~+) expression was significantly increased in the white adipose tissue of MMP12~(-/-)male mice. Conclusions These results suggest that macrophage changes may be related to MMP12 knockout. The significance is that MMP12 may regulate the development of macrophages, the macrophages are reduced in the blood and increased in white fat.
Objective Matrix metalloproteinase-12(MMP12) also known as macrophage elastase, is capable of decomposing almost all extracellular matrix components and is required for monocyte recruitment. MMP12 has been found to be involved in cancer invasion and metastasis. In addition, MMP12 is also closely related to adipose tissue. This study investigated whether MMP12 regulates the macrophages in blood and white fat in MMP12 knockout(MMP12~(-/-)) mice. Methods MMP12~(-/-)mice were raised and genotyped, and littermate MMP12~(+/+) male mice were used as controls. The routine blood indexes of the two groups of mice were analyzed, and the macrophage markers were detected by flow cytometry. Pathdogy using HE staining and immunohistochemistry was performed to determine the macrophages in adipose tissue after MMP12 deletion. Results(1) The genotype of MMP12~(-/-)mice and expansion was identified.(2) Compared with those of littermate MMP12~(+/+) male mice, parameters including RBC count, HGB level, PLT count, and monocyte count and percentage were significantly reduced in MMP12~(-/-) male mice. However, the proportions of lymphocytes and eosinophils were significantly increased compared with those of control mice, but their absolute counts did not differ significantly.(3) Further result suggested that the proportion of macrophage-specific marker CD11 b and F4/80 double positive cells was significantly decreased in the blood, and their absolute count was also reduced.(4) The pathdogy using HE staining and immunohistochemistry confirmed that macrophage(CD68~+) expression was significantly increased in the white adipose tissue of MMP12~(-/-)male mice. Conclusions These results suggest that macrophage changes may be related to MMP12 knockout. The significance is that MMP12 may regulate the development of macrophages, the macrophages are reduced in the blood and increased in white fat.
引文
[1] Rodriguez-Pla A, Martinez-Murillo F, Savino P. J, et al. MMP-12, a novel matrix metalloproteinase associated with giant cell arteritis [J]. Rheumatology (Oxford), 2009, 48(11): 1460-1461.
[2] Liu M, Sun H,Wang X,et al. Association of increased expression of macrophage elastase (matrix metalloproteinase 12) with rheumatoid arthritis [J]. Arthritis Rheum, 2004, 50(10): 3112-3117.
[3] Hautamaki RD, Kobayashi DK, Senior RM, et al. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice [J]. Science, 1997, 277(5334): 2002-2004.
[4] England KA, Price AP, Tram KV, et al. Evidence for early fibrosis and increased airway resistance in bone marrow transplant recipient mice deficient in MMP12 [J]. Am J Physiol Lung Cell Mol Physiol, 2011, 301(4): L519-526.
[5] Houghton AM, Grisolano JL, Baumann ML, et al. Macrophage elastase (matrix metalloproteinase-12) suppresses growth of lung metastases [J]. Cancer Res, 2006, 66(12): 6149-6155.
[6] Wang X, Liang J, Koike T, et al. Overexpression of human matrix metalloproteinase-12 enhances the development of inflammatory arthritis in transgenic rabbits [J]. Am J Pathol, 2004, 165(4): 1375-1383.
[7] Italiani P, Boraschi D. From monocytes to M1/M2 macrophages: Phenotypical vs. functional differentiation [J]. Front Immunol, 2014, 5:514.
[8] Gordon S, Pluddemann A, Martinez Estrada F. Macrophage heterogeneity in tissues: phenotypic diversity and functions [J]. Immunol Rev, 2014, 262(1): 36-55.
[9] Den Breems N Y, Eftimie R. The re-polarisation of M2 and M1 macrophages and its role on cancer outcomes [J]. J Theor Biol, 2016, 390:23-39.
[10] Koh TJ, DiPietro LA. Inflammation and wound healing: the role of the macrophage [J]. Expert Rev Mol Med, 2011, 13:e23.
[11] Yeung OW, Lo CM, Ling CC, et al. Alternatively activated (M2) macrophages promote tumour growth and invasiveness in hepatocellular carcinoma [J]. J Hepatol, 2015, 62(3): 607-616.
[12] Sheng J, Ruedl C, Karjalainen K. Most tissue-resident macrophages except microglia are derived from fetal hematopoietic stem cells [J]. Immunity, 2015, 43(2): 382-393.
[13] Munro DAD, Hughes J, The origins and functions of tissue-resident macrophages in kidney development [J]. Front Physiol, 2017, 8:837.
[14] Tsuji T, Kelly NJ, Takahashi S, et al. Macrophage elastase suppresses white adipose tissue expansion with cigarette smoking [J]. Am J Respir Cell Mol Biol, 2014, 51(6): 822-829.
[15] Lee JT, Pamir N, Liu NC, et al. Macrophage metalloelastase (MMP12) regulates adipose tissue expansion, insulin sensitivity, and expression of inducible nitric oxide synthase [J]. Endocrinology, 2014, 155(9): 3409-3420.
[16] Li J, Zhang X, Liu Q, et al. Myeloid-derived suppressor cells accumulate among myeloid cells contributing to tumor growth in matrix metalloproteinase 12 knockout mice [J]. Cell Immunol, 2018, 327:1-12.
[17] Lumeng CN, DelProposto JB, Westcott DJ, et al. Phenotypic switching of adipose tissue macrophages with obesity is generated by spatiotemporal differences in macrophage subtypes [J]. Diabetes, 2008, 57(12): 3239-3246.
[18] Nesbit M, Schaider H, Miller TH, et al. Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in nontumorigenic melanoma cells [J]. J Immunol, 2001, 166(11): 6483-6490.
[19] Bauters D, Van Hul M, Lijnen HR. Macrophage elastase (MMP-12) in expanding murine adipose tissue [J]. Biochim Biophys Acta, 2013, 1830(4): 2954-2959.
[20] Mills CD. Anatomy of a discovery: M1 and M2 macrophages [J]. Front Immunol, 2015, 6:212.
[2] Liu M, Sun H,Wang X,et al. Association of increased expression of macrophage elastase (matrix metalloproteinase 12) with rheumatoid arthritis [J]. Arthritis Rheum, 2004, 50(10): 3112-3117.
[3] Hautamaki RD, Kobayashi DK, Senior RM, et al. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice [J]. Science, 1997, 277(5334): 2002-2004.
[4] England KA, Price AP, Tram KV, et al. Evidence for early fibrosis and increased airway resistance in bone marrow transplant recipient mice deficient in MMP12 [J]. Am J Physiol Lung Cell Mol Physiol, 2011, 301(4): L519-526.
[5] Houghton AM, Grisolano JL, Baumann ML, et al. Macrophage elastase (matrix metalloproteinase-12) suppresses growth of lung metastases [J]. Cancer Res, 2006, 66(12): 6149-6155.
[6] Wang X, Liang J, Koike T, et al. Overexpression of human matrix metalloproteinase-12 enhances the development of inflammatory arthritis in transgenic rabbits [J]. Am J Pathol, 2004, 165(4): 1375-1383.
[7] Italiani P, Boraschi D. From monocytes to M1/M2 macrophages: Phenotypical vs. functional differentiation [J]. Front Immunol, 2014, 5:514.
[8] Gordon S, Pluddemann A, Martinez Estrada F. Macrophage heterogeneity in tissues: phenotypic diversity and functions [J]. Immunol Rev, 2014, 262(1): 36-55.
[9] Den Breems N Y, Eftimie R. The re-polarisation of M2 and M1 macrophages and its role on cancer outcomes [J]. J Theor Biol, 2016, 390:23-39.
[10] Koh TJ, DiPietro LA. Inflammation and wound healing: the role of the macrophage [J]. Expert Rev Mol Med, 2011, 13:e23.
[11] Yeung OW, Lo CM, Ling CC, et al. Alternatively activated (M2) macrophages promote tumour growth and invasiveness in hepatocellular carcinoma [J]. J Hepatol, 2015, 62(3): 607-616.
[12] Sheng J, Ruedl C, Karjalainen K. Most tissue-resident macrophages except microglia are derived from fetal hematopoietic stem cells [J]. Immunity, 2015, 43(2): 382-393.
[13] Munro DAD, Hughes J, The origins and functions of tissue-resident macrophages in kidney development [J]. Front Physiol, 2017, 8:837.
[14] Tsuji T, Kelly NJ, Takahashi S, et al. Macrophage elastase suppresses white adipose tissue expansion with cigarette smoking [J]. Am J Respir Cell Mol Biol, 2014, 51(6): 822-829.
[15] Lee JT, Pamir N, Liu NC, et al. Macrophage metalloelastase (MMP12) regulates adipose tissue expansion, insulin sensitivity, and expression of inducible nitric oxide synthase [J]. Endocrinology, 2014, 155(9): 3409-3420.
[16] Li J, Zhang X, Liu Q, et al. Myeloid-derived suppressor cells accumulate among myeloid cells contributing to tumor growth in matrix metalloproteinase 12 knockout mice [J]. Cell Immunol, 2018, 327:1-12.
[17] Lumeng CN, DelProposto JB, Westcott DJ, et al. Phenotypic switching of adipose tissue macrophages with obesity is generated by spatiotemporal differences in macrophage subtypes [J]. Diabetes, 2008, 57(12): 3239-3246.
[18] Nesbit M, Schaider H, Miller TH, et al. Low-level monocyte chemoattractant protein-1 stimulation of monocytes leads to tumor formation in nontumorigenic melanoma cells [J]. J Immunol, 2001, 166(11): 6483-6490.
[19] Bauters D, Van Hul M, Lijnen HR. Macrophage elastase (MMP-12) in expanding murine adipose tissue [J]. Biochim Biophys Acta, 2013, 1830(4): 2954-2959.
[20] Mills CD. Anatomy of a discovery: M1 and M2 macrophages [J]. Front Immunol, 2015, 6:212.