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

Background. Sustained intestinal ischemic injury often leads to shock and m ultiorgan failure, mediated in part by a cytokine cascade. Animal models ha ve also identified a central role of Kupffer cells in amplification of cyto kines following intestinal ischemia, To better understand this gut-liver ax is, we developed an in vitro model. Materials and methods. Kupffer cells were isolated from rat livers by arabi nogalactan gradient ultracentrifugation and adherence purification, Cells w ere 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 al pha and interleukin-6 production by ELISA. Trypan blue exclusion was perfor med to assess cell viability. Results. Intestinal and Kupffer cells remained viable during the experiment al time. Production of both tumor necrosis factor alpha and interleukin-6 b y Kupffer cells increased with increasing ischemia time of the intestinal c ells. Conclusions, Consistent with animal studies of intestinal ischemia, this st udy found an increase in cytokine production by Kupffer cells following hyp oxia of intestinal cells. This in vitro model offers a new tool to study th e expression of cytokines, proteins, and messengers involved in the cascade of events that follow intestinal ischemia, (C) 2000 Academic Press.