The ability of rat hepatic sinusoidal endothelial cells (HSEC) to become activated in response to diverse inflammatory stimuli was analyzed. Whereas the classical macrophage activators, IFNγ and/or LPS upregulated expression of iNOS in HSEC, the alternative macrophage activators, IL-10 or IL-4+IL-13 upregulated arginase-1 and mannose receptor. Similar upregulation of iNOS and arginase-1 was observed in classically and alternatively activated Kupffer cells, respectively. Removal of inducing stimuli from the cells had no effect on expression of these markers, demonstrating that activation is persistent. Washing and incubation of IFNγ treated cells with IL-4+IL-13 resulted in decreased iNOS and increased arginase-1 expression, while washing and incubation of IL-4+IL-13 treated cells with IFNγ resulted in decreased arginase-1 and increased iNOS, indicating that classical and alternative activation of the cells is reversible. HSEC were more sensitive to phenotypic switching than Kupffer cells, suggesting greater functional plasticity. Hepatocyte viability and expression of PCNA, β-catenin and MMP-9 increased in the presence of alternatively activated HSEC. In contrast, the viability of hepatocytes pretreated for 2h with 5mM acetaminophen decreased in the presence of classically activated HSEC. These data demonstrate that activated HSEC can modulate hepatocyte responses following injury. The ability of hepatocytes to activate HSEC was also investigated. Co-culture of HSEC with acetaminophen-injured hepatocytes, but not control hepatocytes, increased the sensitivity of HSEC to classical and alternative activating stimuli. The capacity of HSEC to respond to phenotypic activators may represent an important mechanism by which they participate in inflammatory responses associated with hepatotoxicity.
[Show abstract][Hide abstract] ABSTRACT: Recently, Pfeiffer and colleagues have published protocols that allow the isolation of human hepatocytes and non-parenchymal liver cells from the same donor (Pfeiffer et al., 2014). Cell isolation is performed with resected liver tissue, usually obtained from hepatectomy because of metastasis from co-lon cancer. Human liver cells are initially iso-lated by the conventional two-step EDTA/ collagenase perfusion technique. Next, hepa-tocytes and non-parenchymal cells are sepa-rated by low-speed centrifugation. After puri-fication by Percoll density gradient centrifu-gation, Kupffer cells, sinusoidal endothelial cells and stellate cells are further separated by specific adhesion and by magnetic bead sorting. Typical yields of a single isolation are 1.9x10 6 Kupffer cells, 2.7x10 5 sinusoidal endothelial cells and 4.7x10 5 stellate cells. All cell types can be cultivated either as mono-or co-cultures. Co-cultivation of non-parenchymal cells and hepatocytes from the same donor may become an important approach of hepatotoxi-city testing in future. It is well known that non-parenchymal cells play a critical role in hepatotoxicity (Laskin, 1996; Kantari-Mimoun et al., 2014; Krell et al., 1987). Si-nusoidal endothelial cells have been shown to support liver regeneration, where they serve as 'guide-rails' for regenerating hepatocytes and guarantee the rapid re-establishment of functional liver tissue (Hoehme et al., 2010; Schliess et al., 2014). Moreover, sinusoidal endothelial cells have been shown to secrete HGF and Wnt factors during liver regenera-tion, which stimulate hepatocytes to prolifer-ate (Ding et al., 2010). They condition the vascular niche by angiocrine signals which can be disturbed after repeated administration of hepatotoxic compounds, leading to activa-tion of stellate cells and fibrosis (Ding et al., 2014). Currently, in vitro systems for hepato-toxicity testing are in the focus of toxicologi-cal research (Ghallab, 2013; Krell et al., 1987; Schyschka et al., 2013; Grinberg et al., 2014). Although the important role of non-parenchymal cells is out of question (Liu et al., 2013; Hammad et al., 2014; McCuskey et al., 2005; Yee et al., 2003), currently mostly hepatocyte monocultures are tested (Miszczuk et al., 2014; Rodrigues et al., 2013; Watzek et al., 2013; Vinken et al., 2013; Huang et al., 2013). An explanation probably is that cultivation of non-parenchymal cells is still challenging. For example stellate cells tend to be activated spontaneously in culture and sinusoidal endo-thelial cells may lose capacity to secrete cy-tokines. Although there is still a long way to go, the technique of Pfeiffer et al. (2014) will improve the availability of human non-parenchymal cells and may facilitate the de-velopment of in vitro systems that recapitu-late the communication between hepatocytes and non-parenchymal cells. REFERENCES
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