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% CO(2). Rat intestinal epithelial cells, IEC-6, were cultured under normoxic or anoxic (90% N(2), 10% CO(2)) 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. Copyright 2000 Academic Press.
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% CO(2). Rat intestinal epithelial cells, IEC-6, were cultured under normoxic or anoxic (90% N(2), 10% CO(2)) 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. Copyright 2000 Academic Press.
Authors: Jong Hwan Sung; Ying I Wang; Narasimhan Narasimhan Sriram; Max Jackson; Christopher Long; James J Hickman; Michael L Shuler Journal: Anal Chem Date: 2018-12-11 Impact factor: 6.986