Purpose: Intestinal alkaline phosphatase (IAP), an intestinal brush border enzyme, is a newly recognized gut mucosal defense factor. We have shown that IAP confers protection from luminal bacteria, with inhibition of bacterial translocation into mesenteric nodes in a remote ischemia mouse model. However, the specific mechanism by which IAP exerts its effects has yet to be fully elucidated. Here we seek to define more precisely how IAP protects the host against bacterial injury.Materials and Methods: Escherichia coli, Salmonella typhimurium andStaphylococcus aureus bacteria were incubated +/- calf intestinal alkalinephosphatase (CIP), and bacterial viability estimated by enumeration of colony forming units (CFUs) by plating at various time points. Free phosphate from whole E. coli or free lipopolysacchardie (LPS) +/- CIP was measured by the malachite green assay; the bacteria or LPS +/- CIP were also applied to HT29cells and Il-8 response measured by ELISA. HT29 cells were previously stablytransfected with an IAP expression plasmid; enzyme activity was confirmedbiochemically. Parent and IAP cells were incubated with E. coli and S.typhimurium at varying incubation times and bacteria/cell ratios. Extracellular bacteria were killed by gentamicin application, intracellular bacteria enumerated by cell lysis and plating, and Il-8 secretion measured in the media.Results: CIP had no effect on the viability of E. coli, S. typhimurium orS. aureus. CIP did not liberate free phosphate from whole E coli, but was able to dephosphorlyate free LPS; furthermore, CIP did not inhibit the Il-8 response to E. coli, but did inhibit the Il-8 response to free LPS. E coli preferentially invaded parent cells compared to IAP cells, and concordantly, Il-8 secretion was repressed in IAP cells. Interestingly, although S. typhimurium exhibited greater invasiveness in IAP cells compared to parent cells, the IAP cells continued to inhibit Il-8 secretion.Conclusions: The free IAP enzyme is able to directly dephosphorylate and confer protection from LPS, but does not directly affect the growth or viability of bacteria, nor does it attenuate the induced inflammatory response in target cells. In contrast, cells that over-express membrane bound IAP were able to specifically inhibit E coli invasion and attenuate the Il-8 response induced by both E. coli and S. typhimurium. Taken together, it seems unlikely that IAP is, in itself, a bactericidal agent, but inhibits injury specific to commensal bacteria related to inflammatory signal transduction.