Kathryn Chen, MD, Madhu Malo, MD, Golam Mostafa, MD, Sayeda Alam, MD, Sundaram Ramasamy, MD, Elizabeth Hohmann, MD, Richard Hodin, MD
Massachusetts General Hospital- Department of Gastrointestinal Research; Boston, MA
PURPOSE OF STUDY
Intestinal alkaline phosphatase (IAP) is recognized as an antimicrobial gut defense factor, and we sought to examine the cellular mechanisms by which IAP functions.
METHODS USED:
Varying amounts of TLR ligands (LPS, CpG) and non-TLR ligand (TNF-alpha) were incubated +/- calf intestinal alkaline phosphatase (CIP) and free phosphate measured by malachite green assay. Ligands +/- CIP were applied to HT29 cells and IL-8 secretion measured by ELISA. HT29 cells stably transfected with an IAP expression plasmid (HT29-IAP), parent cells and TLR-deficient HEK-293 cells were incubated with Escherichia coli or Salmonella typhimurium and IL-8 secretion measured.
SUMMARY OF RESULTS:
HT29 cells showed dose-dependent Il-8 increases following exposure to LPS, TNF, and CpG. IL-8 production was blocked when LPS or CpG, but not TNF, were pre-incubated with CIP, and this inhibition was CIP dose-dependent. Increased free phosphate was detected when LPS and CpG, but not TNF, were incubated with CIP. Parent cells showed increased IL-8 production when incubated with E. coli and S. typhimurium as compared with HT29-IAP or HEK-293 cells.
CONCLUSIONS:
CIP inhibited IL-8 induction by TLR ligands LPS and CpG, but not by TNF-alpha, and concomitantly manifested increased free phosphate, suggesting that ligand dephosphorylation was responsible for inhibition of target cell effects. HT29-IAP and TLR-deficient HEK-293 cells, but not parent cells, were hypo-responsive in terms of IL-8 production when incubated with E. coli and S. typhimurium. These data indicate that the gut defense factor IAP may down-regulate TLR mediated inflammatory pathways by dephosphorylation of their cognate TLR ligands.