Background Previous methods for Kupffer cells (KCs) isolation require sophisticated skills and tedious procedures. Few studies have attempted to explore the self-renewal capacity of KCs in vitro. Therefore, the aim of this study was to establish a simple method for rat KCs isolation and further investigate the mitotic potential of KCs in vitro. Methods KCs were obtained by performing one-step perfusion, enzymatic tissue treatment, differential centrifugation and selective adherence. The proliferation ability of cultured KCs was determined by MTT assay and Propidium Iodide FACS analysis. Phagocytic assay and ED-1, ED-2 immunofluorescence were used to identify cell phenotype. After stimulation with LPS, the expression of surface antigens (MHCII, CD40, CD80, and CD86) and the production of cytokines (NF-κB, TNF-α, IL-6 and IL-10) were measured for cell function identification. Results KCs were isolated with certain numbers and reasonable purities. The KCs were able to survive until at least passage 5 (P5), and at P3 showed equally strong phagocytic activity as primary KCs (P0). After stimulation with LPS, the change in the expression of surface antigens and the production of cytokines for P3 cells was similar to that for P0 cells. Conclusions Our study provides a simple and efficient method for KCs isolation, and reveals that self-renewing KCs have the same phagocytic activity and functions as primary KCs.
References
[1]
Liaskou E, Wilson DV, Oo YH (2012) Innate immune cells in liver inflammation. Mediators Inflamm 2012: 949157.
[2]
Kolios G, Valatas V, Kouroumalis E (2006) Role of kupffer cells in the pathogenesis of liver disease. World J Gastroenterol 12: 7413–7420.
[3]
Diehl AM (2002) Nonalcoholic steatosis and steatohepatitis IV. Nonalcoholic fatty liver disease abnormalities in macrophage function and cytokines. Am J Physiol Gastrointest Liver Physiol 282: G1–5.
[4]
Murray PJ, Wynn TA (2011) Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 11: 723–737.
[5]
Zimmermann HW, Trautwein C, Tacke F (2012) Functional role of monocytes and macrophages for the inflammatory response in acute liver injury. Front Physiol 3: 56.
[6]
Mandal P, Pratt BT, Barnes M, McMullen MR, Nagy LE (2011) Molecular mechanism for adiponectin-dependent M2 macrophage polarization: link between the metabolic and innate immune activity of full-length adiponectin. J Biol Chem 286: 13460–13469.
[7]
Anthony BJ, Ramm GA, McManus DP (2012) Role of resident liver cells in the pathogenesis of schistosomiasis. Trends Parasitol 28: 572–579.
[8]
Reyes JL, Terrazas LI (2007) The divergent roles of alternatively activated macrophages in helminthic infections. Parasite Immunol 29: 609–619.
[9]
Kong X, Horiguchi N, Mori M, Gao B (2012) Cytokines and STATs in Liver Fibrosis. Front Physiol 3: 69.
[10]
Papackova Z, Palenickova E, Dankova H, Jana Z, Vojtech S, et al. (2012) Kupffer cells ameliorate hepatic insulin resistance induced by high-fat diet rich in monounsaturated fatty acids: the evidence for the involvement of alternatively activated macrophages. Nutr Metab (Lond) 9: 22.
[11]
Iwaisako K, Haimerl M, Paik YH, Taura K, Kodama Y, et al. (2012) Protection from liver fibrosis by a peroxisome proliferator-activated receptor delta agonist. Proc Natl Acad Sci U S A 109: E1369–1376.
[12]
Wojtalla A, Herweck F, Granzow M, Klein S, Trebicka J, et al. (2012) The endocannabinoid N-arachidonoyl dopamine (NADA) selectively induces oxidative stress-mediated cell death in hepatic stellate cells but not in hepatocytes. Am J Physiol Gastrointest Liver Physiol 302: G873–887.
[13]
Bouwens L, Baekeland M, Wisse E (1984) Importance of local proliferation in the expanding kupffer cell population of rat liver after zymosan stimulation and partial hepatectomy. Hepatology 4: 213–219.
[14]
Kitani H, Takenouchi T, Sato M, Yoshioka M, Yamanaka N (2011) A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells. J Vis Exp doi: 10.3791/2757.
[15]
Widmann JJ, Fahimi HD (1975) Proliferation of Mononuclear Phagocytes (Kupffer Cells) and Endothelial Cells in Regenerating Rat Liver. Am J Pathol 80: 349–366.
[16]
Naylor AJ, Azzam E, Smith S, Croft A, Poyser C, et al. (2012) The mesenchymal stem cell marker CD248 (endosialin) is a negative regulator of bone formation in mice. Arthritis Rheum 64: 3334–3343.
[17]
Nemeth K, Leelahavanichkul A, Yuen PS, Mayer B, Parmelee A, et al. (2009) Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med 15: 42–49.
[18]
Heil F, Ahmad-Nejad P, Hemmi H, Hochrein H, Ampenberger F, et al. (2003) The Toll-like receptor 7 (TLR7)-specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily. Eur J Immunol 33: 2987–2997.
[19]
Parker GA, Picut CA (2012) Immune functioning in non lymphoid organs: the liver. Toxicol Pathol 40: 237–247.
[20]
Alric L, Orfila C, Carrere N, Beraud M, Carrera G, et al. (2000) Reactive oxygen intermediates and eicosanoid production by Kupffer cells and infiltrated macrophages in acute and chronic liver injury induced in rats by CCl4. Inflamm Res 49: 700–707.
[21]
Tadbir AA, Pardis S, Ashkavandi ZJ, Najvani AD, Ashraf MJ, et al. (2012) Expression of Ki67 and CD105 as Proliferation and Angiogenesis Markers in Salivary Gland Tumors. Asian Pacific Journal of Cancer Prevention 13: 5155–5159.
[22]
Ye Q, Wang X, Wang Q, Xia M, Zhu Y, et al. (2013) Cytochrome P4502E1 Inhibitor, Chlormethiazole, decreases LPS-induced inflammation in rat Kupffer cells with ethanol treatment. Hepatology Res doi: 10.1111/hepr.12063.
[23]
Fan JH, Feng GG, Huang L, Tsunekawa K, Honda T, et al. (2012) Role of naofen in apoptosis of hepatocytes induced by lipopolysaccharide through mitochondrial signaling in rats. Hepatol Res 42: 696–705.
[24]
Ikejima K, Enomoto N, Seabra V, Ikejima A, David A, et al. (1999) Pronase destroys the lipopolysaccharide receptor CD14. Am J Physiol 276: G591–598.
[25]
Fukada H, Yamashina S, Izumi K, Komatsu M, Tanaka K, et al. (2012) Suppression of autophagy sensitizes Kupffer cells to endotoxin. Hepatol Res 42: 1112–1118.
[26]
Wen JW, Olsen AL, Perepelyuk M, Wells RG (2012) Isolation of rat portal fibroblasts by in situ liver perfusion. J Vis Exp doi: 10.3791/3669.
[27]
Golbar HM, Izawa T, Murai F, Kuwamura M, Yamate J (2012) Immunohistochemical analyses of the kinetics and distribution of macrophages, hepatic stellate cells and bile duct epithelia in the developing rat liver. Exp Toxicol Pathol 64: 1–8.
[28]
Kang L-I, Mars W, Michalopoulos G (2012) Signals and Cells Involved in Regulating Liver Regeneration. Cells 1: 1261–1292.
[29]
Xie G, Choi SS, Syn WK, Michelotti GA, Swiderska M, et al. (2013) Hedgehog signalling regulates liver sinusoidal endothelial cell capillarisation. Gut 62: 299–309.
[30]
Su LJ, Chang CC, Yang CH, Hsieh SJ, Wu YC, et al. (2013) Graptopetalum paraguayense ameliorates chemical-induced rat hepatic fibrosis in vivo and inactivates stellate cells and kupffer cells in vitro. PLoS One 8: e53988.
[31]
Watanabe A, Sohail MA, Gautam S, Gomes DA, Mehal WZ (2012) Adenine induces differentiation of rat hepatic stellate cells. Dig Dis Sci 57: 2371–2378.
[32]
Valatas V (2003) Isolation of rat Kupffer cells: a combined methodology for highly purified primary cultures. Cell Biology International 27: 67–73.
[33]
Klein I, Cornejo JC, Polakos NK, John B, Wuensch SA, et al. (2007) Kupffer cell heterogeneity: functional properties of bone marrow-derived and sessile hepatic macrophages. Blood 110: 4077–4085.
[34]
Choi SS, Diehl AM (2009) Epithelial-to-mesenchymal transitions in the liver. Hepatology 50: 2007–2013.
[35]
Wynn TA, Barron L (2010) Macrophages: master regulators of inflammation and fibrosis. Semin Liver Dis 30: 245–257.
