Under conditions of starvation and disease, the gut barrier becomes impaired, and trophic feeding to prevent gut mucosal atrophy has become a standard treatment of critically ill patients. However, the mechanisms responsible for the beneficial effects of enteral nutrition have remained a mystery. Using in vitro and in vivo models, we demonstrate that the brush-border enzyme, intestinal alkaline phosphatase (IAP), has the ability to detoxify lipopolysaccharide and prevent bacterial invasion across the gut mucosal barrier. IAP expression and function are lost with starvation and maintained by enteral feeding. It is likely that the IAP silencing that occurs during starvation is a key component of the gut mucosal barrier dysfunction seen in critically ill patients.A mong the most critical functions of the mammalian gut mucosa is to provide a barrier to luminal microbes and toxins while simultaneously allowing for the necessary digestion and absorption of dietary nutrients. The molecular mechanisms that govern barrier function are incompletely understood, but it is clear that under conditions of starvation and disease, the gut barrier becomes impaired, leading to significant morbidity and mortality (1-8). Trophic enteral feeding to prevent gut mucosal atrophy and resultant barrier dysfunction has become part of the standard treatment of intensive care unit patients (9-14). The mechanism(s) responsible for the beneficial effects of trophic feeding are not understood.Intestinal alkaline phosphatase (IAP), a brush-border protein that hydrolyzes monophosphate esters, is expressed exclusively in villus-associated enterocytes and is considered an excellent marker for crypt-villus differentiation (15-18). Narisawa et al. (19) reported that compared with their wild-type (WT) littermates, mice lacking IAP gained more weight under conditions of a high-fat diet. In addition, several studies have shown that the IAP enzyme is capable of detoxifying LPS, likely through dephosphorylation of the lipid A moiety, the primary source of its endotoxic effects (20). Despite these few reports, the physiological role of IAP within the gut has not been elucidated. Results and DiscussionTo examine the functional role of IAP, we developed in vitro model systems using intestinal cell lines (T84, HT-29, and IEC-6) that express little or no IAP under basal conditions. Stable cell lines were created that overexpress IAP (Fig. 1A), and enzyme assays were used to determine the cellular localization of the ectopically expressed IAP protein. The results in Fig. 1B show that parent cells make little, if any, endogenous IAP. In contrast, large amounts of IAP enzyme are seen in the stably transfected cells. Importantly, the vast majority of the IAP activity is in the membrane fraction as opposed to the cytosol. Fig. 1C shows the results of the control experiment used to validate our separation of membranous and cytosolic fractions, confirming that the majority of the MAPK enzyme activity exists within the cytosol rather than the membrane. This membrane ...

Reperfusion injury of ischemic tissue represents an acute inflammatory response that can cause significant morbidity and mortality. The mechanism of injury is not fully elucidated, but recent studies indicate an important role for natural antibody and the classical pathway of complement. To test the hypothesis that injury is initiated by specific IgM, we have screened a panel of IgMproducing hybridomas prepared from peritoneal cells enriched in B-1 cells. One clone, CM22, was identified that could restore pathogenic injury in RAG-1 ؊/؊ mice in an intestinal model of ischemia͞reperfusion (I͞R). In situ activation of the classical pathway of complement was evident by deposition of IgM, complement C4, and C3 in damaged tissue after passive transfer of CM22 IgM. Sequence analysis of CM22 Ig heavy and light chains showed germ-line configurations with high homology to a V H sequence from the B-1 repertoire and a VK of a known polyreactive natural IgM. These data provide definitive evidence that I͞R injury can be initiated by clonally specific natural IgM that activates the classical pathway of complement. This finding opens an avenue for identification of I͞R-specific self-antigen(s) and early prevention of injury.