Intestinal alkaline phosphatase inhibits the proinflammatory nucleotide uridine diphosphate.

Uridine diphosphate (UDP) is a proinflammatory nucleotide implicated in inflammatory bowel disease. Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor capable of inhibiting intestinal inflammation. We used the malachite green assay to show that IAP dephosphorylates UDP. To study the anti-inflammatory effect of IAP, UDP or other proinflammatory ligands (LPS, flagellin, Pam3Cys, or TNF-α) in the presence or absence of IAP were applied to cell cultures, and IL-8 was measured. UDP caused dose-dependent increase in IL-8 release by immune cells and two gut epithelial cell lines, and IAP treatment abrogated IL-8 release. Costimulation with UDP and other inflammatory ligands resulted in a synergistic increase in IL-8 release, which was prevented by IAP treatment. In vivo, UDP in the presence or absence of IAP was instilled into a small intestinal loop model in wild-type and IAP-knockout mice. Luminal contents were applied to cell culture, and cytokine levels were measured in culture supernatant and intestinal tissue. UDP-treated luminal contents induced more inflammation on target cells, with a greater inflammatory response to contents from IAP-KO mice treated with UDP than from WT mice. Additionally, UDP treatment increased TNF-α levels in intestinal tissue of IAP-KO mice, and cotreatment with IAP reduced inflammation to control levels. Taken together, these studies show that IAP prevents inflammation caused by UDP alone and in combination with other ligands, and the anti-inflammatory effect of IAP against UDP persists in mouse small intestine. The benefits of IAP in intestinal disease may be partly due to inhibition of the proinflammatory activity of UDP.

Local peritoneal irrigation with intestinal alkaline phosphatase is protective against peritonitis in mice.

BACKGROUND: The brush-border enzyme intestinal alkaline phosphatase (IAP) functions as a gut mucosal defense factor and detoxifies different toll-like receptor ligands. This study aimed to determine the therapeutic effects of locally administered calf IAP (cIAP) in a cecal ligation and puncture (CLP) model of polymicrobial sepsis. METHODS: C57BL/6 mice underwent CLP followed by intraperitoneal injection of cIAP or normal saline. Blood leukocyte counts, levels of cytokines and liver enzymes, and lung myeloperoxidase activity were determined. Peritoneal lavage fluid (PLF) was assayed for neutrophil infiltration and both aerobic and anaerobic bacterial counts. RESULTS: After intraperitoneal injection, cIAP activity in PLF decreased 50% within 15 min with minimal activity evident at 4 h. Compared with irrigation with normal saline, cIAP irrigation increased the 7-day survival rate in mice undergoing CLP, with maximal effects seen at 25 units of cIAP (0% vs. 46% survival rate, respectively; p < 0.001). cIAP treatment reduced lung inflammation, liver damage and levels of tumor necrosis factor alpha and interleukin-6. CONCLUSIONS: Peritoneal irrigation with cIAP significantly enhances survival in a mouse model of peritonitis, likely through reduction of local inflammation and remote organ damage. We suggest that intraperitoneal cIAP irrigation could be a novel therapy for intra-abdominal sepsis.

A role for intestinal alkaline phosphatase in the maintenance of local gut immunity.

BACKGROUND AND AIMS: Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor known to dephosphorylate lipopolysaccharide (LPS); however, the role of IAP in the gut response to luminal bacteria remains poorly defined. We investigated immune responses of wild-type (WT) and IAP-knockout (IAP-KO) mice to LPS and Salmonella typhimurium challenges. METHODS: Cryostat sectioning and standard indirect immunohistochemical staining for major histocompatibility complex (MHC) class II molecules were performed on liver tissue from WT and IAP-KO mice. WT and IAP-KO mice were orally gavaged with S. typhimurium; bacterial translocation to mesenteric nodes, liver, and spleen was determined by tissue homogenization and plating. In other experiments, WT and IAP-KO mice received intraperitoneal injections of LPS, with subsequent quantification of complete blood counts and serum interleukin (IL)-6 by enzyme-linked immunosorbent assay (ELISA). WT and IAP-KO whole blood were plated and stimulated with LPS and Pam-3-Cys, followed by cytokine assays. RESULTS: Immunohistologic liver examinations showed increased expression of MHC class II molecules in IAP-KO mice. Following S. typhimurium challenge, WT mice appeared moribund compared with IAP-KO mice, with increased bacterial translocation. WT mice had >50% decrease (P<.005) in platelets and 1.8-fold (P<.05) increased serum IL-6 compared with IAP-KO mice in response to LPS injections. IAP-KO whole-blood stimulation with LPS and Pam-3-Cys resulted in increased IL-6 and tumor necrosis factor (TNF)-alpha secretion compared with WT. CONCLUSIONS: IAP-KO mice exhibit characteristics consistent with local LPS tolerance. Whole-blood response of IAP-KO mice did not reflect systemic tolerance. These data suggest that IAP is a local immunomodulating factor, perhaps regulating LPS-toll-like receptor 4 (TLR4) interaction between commensal microflora and intestinal epithelium.

Intestinal alkaline phosphatase has beneficial effects in mouse models of chronic colitis.

BACKGROUND: The brush border enzyme intestinal alkaline phosphatase (IAP) functions as a gut mucosal defense factor and is protective against dextran sulfate sodium (DSS)-induced acute injury in rats. The present study evaluated the potential therapeutic role for orally administered calf IAP (cIAP) in two independent mouse models of chronic colitis: 1) DSS-induced chronic colitis, and 2) chronic spontaneous colitis in Wiskott-Aldrich Syndrome protein (WASP)-deficient (knockout) mice that is accelerated by irradiation. METHODS: The wildtype (WT) and IAP knockout (IAP-KO) mice received four cycles of 2% DSS ad libitum for 7 days. Each cycle was followed by a 7-day DSS-free interval during which mice received either cIAP or vehicle in the drinking water. The WASP-KO mice received either vehicle or cIAP for 6 weeks beginning on the day of irradiation. RESULTS: Microscopic colitis scores of DSS-treated IAP-KO mice were higher than DSS-treated WT mice (52±3.8 versus 28.8±6.6, respectively, P<0.0001). cIAP treatment attenuated the disease in both groups (KO=30.7±6.01, WT=18.7±5.0, P<0.05). In irradiated WASP-KO mice cIAP also attenuated colitis compared to control groups (3.3±0.52 versus 6.2±0.34, respectively, P<0.001). Tissue myeloperoxidase activity and proinflammatory cytokines were significantly decreased by cIAP treatment. CONCLUSIONS: Endogenous IAP appears to play a role in protecting the host against chronic colitis. Orally administered cIAP exerts a protective effect in two independent mouse models of chronic colitis and may represent a novel therapy for human IBD.

Identification of specific targets for the gut mucosal defense factor intestinal alkaline phosphatase.

Intestinal alkaline phosphatase (IAP) is a small intestinal brush border enzyme that has been shown to function as a gut mucosal defense factor, but its precise mechanism of action remains unclear. We investigated the effects of IAP on specific bacteria and bacterial components to determine its molecular targets. Purulent fluid from a cecal ligation and puncture model, specific live and heat-killed bacteria (Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes), and a variety of proinflammatory ligands (LPS, CpG DNA, Pam-3-Cys, flagellin, and TNF) were incubated with or without calf IAP (cIAP). Phosphate release was determined by using a malachite green assay. The various fluids were applied to target cells (THP-1, parent HT-29, and IAP-expressing HT-29 cells) and IL-8 secretion measured by ELISA. cIAP inhibited IL-8 induction by purulent fluid in THP-1 cells by >35% (P < 0.005). HT29-IAP cells had a reduced IL-8 response specifically to gram-negative bacteria; >90% reduction compared with parent cells (P < 0.005). cIAP had no effect on live bacteria but attenuated IL-8 induction by heat-killed bacteria by >40% (P < 0.005). cIAP exposure to LPS and CpG DNA caused phosphate release and reduced IL-8 in cell culture by >50% (P < 0.005). Flagellin exposure to cIAP also resulted in reduced IL-8 secretion by >40% (P < 0.005). In contrast, cIAP had no effect on TNF or Pam-3-Cys. The mechanism of IAP action appears to be through dephosphorylation of specific bacterial components, including LPS, CpG DNA, and flagellin, and not on live bacteria themselves. IAP likely targets these bacterially derived molecules in its role as a gut mucosal defense factor.