Establishment of a human in vitro model to investigate inflammatory immune reactions between Kupffer cell activation and hepatocyte damage – an approach to study DILI

Hepatic tissue damage can occur due to surgical interventions, infections or drug induced liver injury (DILI). Kupffer cells (KC) play an important role in initiating tissue damage but also in regenerative processes. KC activation by LPS, foreign proteins or endogenous proteins can lead to an activation of the NF-κB signaling pathway mediated by reactive oxygen intermediates (ROI). Once activated, KCs release different cytokine patterns that support inflammation or induce tolerance reactions. Aim of the present study was to establish a human in vitro model which enables the detection of cell-cell communication between KCs activated by damaged or stressed hepatocytes.

Primary human hepatocytes (PHH) and KCs were isolated from human liver resectates using a two-step collagenase perfusion technique. The isolated KC population was characterized by the initial intracellular reactive oxygen intermediates (ROI) level using the DCF-assay. Direct KC activation was investigated by treatment with LPS and subsequent determination of an increase in ROI. The cell viability was measured using the XTT-assay. In order to simulate the activation of KC following hepatocyte damage, KCs were incubated with supernatants of PHH previously incubated with diclofenac and acetaminophen, respectively. Then the KC reaction was determined by measurement of the cytokine profiles using a cytokine array.

The isolated KC yield was 1.1±0.8×106 cells per g liver tissue with a purity of >80% determined by counting CD68 and phagocytosis positive cells. In general, most of the KC isolations showed high initial ROI levels, which could be in some cases related to the different donor livers. The stimulation of KCs with LPS leads to a concentration-dependent increase in ROI formation. The incubation of KCs with supernatants from drug-treated PHH increased mitochondrial activity measured by the XXT-assay and increased the formation of ROIs compared to untreated KCs. Furthermore, KCs treated with supernatants from drug-treated PHH showed changes in their secreted cytokine profile.

In summary, most of the isolated KCs showed high basal ROI levels, which are most likely linked to the isolation process and to the quality of the donor liver tissue. However, LPS and drug-damaged hepatocytes led to an activation of KCs. The KC activation and reaction depends strongly on the donor liver tissue and the patients' original desease state. First attempts aiming the transfer of this method into a transwell coculture and the determination of the donor-dependent cytokine production are made for the development of a system biological approach.

This study was supported by the Virtual-Liver-Network, BMBF:0315741