A STABLE LONG-TERM HEPATOCYTE CULTURE SYSTEM FOR STUDIES OF PHYSIOLOGICAL PROCESSES - CYTOKINE STIMULATION OF THE ACUTE PHASE RESPONSE IN RAT AND HUMAN HEPATOCYTES

Prior studies on the in vitro hepatic acute phase response have involved either hepatoma cell lines or conventional short-term cultures of primary hepatocytes. No data are available on the response of primary hepatocytes in stable long-term culture systems. In this study, the acute phase response of rat and human hepatocytes in a new long-term culture system was examined in response to interleukin-6 (IL-6), interleukin-1-beta (IL-1-beta), and tumor necrosis factor-alpha (TNF-alpha). The cultured cells were sandwiched between two layers of collagen in a (double-gel) configuration which has been shown to preserve both hepatocyte function and morphology over prolonged periods of time. The stability of this culture configuration enabled us to investigate, for the first time, the temporal aspects of the response in addition to the effects of the mediators on protein secretion. Exposure of rat hepatocytes to IL-6 after culture for 16 days resulted in a 2-fold reduction of albumin secretion and a 15-fold increase in the secretion rates of fibrinogen and alpha(2)-macroglobulin. In all instances, the peak response occurred at 48 h after IL-6 exposure, and all protein secretion rates returned to pretreatment values within 5 days post-treatment. Changes in the mRNA levels of these proteins in response to IL-6 corresponded with those changes seen with the secreted products, indicating pretranslational regulation. Administration of IL-1-beta to rat hepatocytes produced a similar decline of albumin secretion and a 5-fold increase of fibrinogen secretion, whereas alpha(2)-macroglobulin secretion remained undisturbed. In contrast, TNF-alpha did not affect the secretion of any protein examined. Human hepatocytes in a double-gel culture configuration reacted to IL-6 and IL-1-beta with an approximate 40-fold increase in serum amyloid A secretion, peaking at day 3 posttreatment. Both the secretion pattern and temporal response of these cultured hepatocytes to cytokines appear to closely mimic the in vivo hepatocellular response. The double-gel culture system is a stable, attractive tool for further investigation of the acute phase and other hepatocellular responses to physiologic stimuli.