Intestinal ischemia and the gut-liver axis:: An in vitro model

Background. Sustained intestinal ischemic injury often leads to shock and multiorgan failure, mediated in part by a cytokine cascade. Animal models have also identified a central role of Kupffer cells in amplification of cytokines following intestinal ischemia, To better understand this gut-liver axis, we developed an in vitro model. Materials and methods. Kupffer cells were isolated from rat livers by arabinogalactan gradient ultracentrifugation and adherence purification, Cells were grown in RPMI medium in 5% CO2. Rat intestinal epithelial cells, IEC-6, were cultured under normoxic or anoxic (90% N-2, 10% CO2) conditions for 2, 12, and 24 h, Kupffer cells were then grown in the conditioned medium of the IEC-6 cultures. After 24 h, the medium was replaced with fresh medium. This final Kupffer cell supernatant was tested for tumor necrosis factor alpha and interleukin-6 production by ELISA. Trypan blue exclusion was performed to assess cell viability. Results. Intestinal and Kupffer cells remained viable during the experimental time. Production of both tumor necrosis factor alpha and interleukin-6 by Kupffer cells increased with increasing ischemia time of the intestinal cells. Conclusions, Consistent with animal studies of intestinal ischemia, this study found an increase in cytokine production by Kupffer cells following hypoxia of intestinal cells. This in vitro model offers a new tool to study the expression of cytokines, proteins, and messengers involved in the cascade of events that follow intestinal ischemia, (C) 2000 Academic Press.