title: Development of a cytokine-producing immortalized murine Kupffer cell line.
abstract: Kupffer cells (KC) play a critical role in both liver physiology and the pathogenesis of various liver diseases. Isolated primary KC have a limited lifespan in culture, and due to the relatively low number obtained, limit their study in vitro. Here, a cytokine-producing immortalized KC (ImKC) line was established from transgenic mice that express the thermolabile mutant tsA58 of the Simian virus 40 large T antigen under the control of the H-2k(b) promoter. Primary KC were obtained using a three step procedure: liver perfusion, centrifugal elutriation, and sorting for F4/80⁺ cells. ImKC were identified within the small-intermediate population of KC that maintained stable expression of F4/80, and the surface antigens CD11b, CD14 and TLR4. ImKC grow at IFNγ-independent manner at 37°C and exhibited a doubling time of ∼24 h when cultured in RPMI 1640 with 5% FBS. Our observations indicate that both activation of telomerase and expression of P53 are markedly increased, suggesting that enhanced telomerase activity and P53 expression may contribute to the immortalization of this cell population. ImKC cells maintained a high capacity to phagocytose FITC-latex beads, and bind/phagocytose erythrocytes. In addition, similar to primary KC, ImKC responded to stimulation with lipopolysaccharide (LPS: 0.1-1μg/ml) by upregulating mRNA levels of TNFα (23-fold), IL-6 (28-fold), and IL-1β (1459-fold), as measured by qRT-PCR. Protein levels of TNFα and IL-6 were also increased, 10-fold and 12-fold, respectively. Reactive oxygen species (ROS) and nitric oxide (NO) production were significantly enhanced in ImKC following an LPS challenge. Furthermore, LPS elicited a marked increase in mitogen activated protein kinase (MAPK) phospho-(ERK1/2, JNK) and NF-κB p50 with decreased IκBα in ImKC, as assessed by Western blot. Collectively, these results demonstrate that the ImKC line retains critical characteristics of primary KC, and thus provides a useful tool to assess the role of KC in liver injury and chronic diseases.
authors.affiliation: Department of Surgery, Indiana University School of Medicine, MS B005, 635 Barnhill Dr., Indianapolis, IN 46202, United States. Electronic address: zywang@iupui.edu.
authors.forename: Zheng-Yu
authors.initials: ZY
authors.lastname: Wang
authors.affiliation: Department of Surgery, Indiana University School of Medicine, MS B005, 635 Barnhill Dr., Indianapolis, IN 46202, United States.
authors.forename: Christopher
authors.initials: C
authors.lastname: Burlak
authors.affiliation: Department of Environmental Health, Indiana University School of Public Health, 925 E 7th, Bloomington, IN 47405, United States.
authors.forename: James E
authors.initials: JE
authors.lastname: Klaunig
authors.affiliation: Department of Environmental Health, Indiana University School of Public Health, 925 E 7th, Bloomington, IN 47405, United States.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Kamendulis
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation; D016207:Cytokines / Q000096:biosynthesis*; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D018836:Inflammation Mediators / Q000378:metabolism; D007728:Kupffer Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D008810:Mice, Inbred C57BL; D008863:Microspheres; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis; D010587:Phagocytosis / Q000187:drug effects; D010641:Phenotype; D017382:Reactive Oxygen Species / Q000378:metabolism; D019098:Telomerase / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D016207:Cytokines; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D009569:Nitric Oxide; D020928:Mitogen-Activated Protein Kinases; D019098:Telomerase
keywords: Cell immortalization; Cytokine; Kupffer cell; Mouse