Role of alkaline phosphatase in colitis in man and rats A Tuin, 1 K Poelstra, 1 A de Jager-Krikken, 1 L Bok, 2 W Raaben, 3 M P Velders, 3 G Dijkstra 2 c Competing interests: Declared (the declaration can be viewed on the Gut website at http://www.gut.bmj.com/ supplemental). 1 Department of Pharmacokinetics and Drug Delivery, University Centre for Pharmacy, University of Groningen, The Netherlands; 2 Department of Gastroenterology and Hepatology, University Medical Centre Groningen, The Netherlands; 3 AM-Pharma, Bunnik, The Netherlands Correspondence to: Dr A Tuin, Department of Pharmacokinetics and Drug Delivery, University Centre for Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands; a. tuin@rug.nl KP and GD contributed equally to this work. Revised 11 September 2008 Accepted 12 September 2008 Published Online First 13 October 2008 ABSTRACT Background and aims: Crohn’s disease (CD) and ulcerative colitis (UC) are chronic multifactorial inflam- matory bowel diseases (IBDs) with unknown aetiology, but a deregulated mucosal immune response to gut- derived bacterial antigens is thought to be involved. Toll- like receptor ligands, especially lipopolysaccharide (LPS), contribute to the maintenance of the disease. It has previously been shown that the enzyme alkaline phosphatase (AP) is able to detoxify LPS, and the aim of this study was to examine a possible role in IBDs. Methods: Intestinal AP (iAP) mRNA expression and LPS dephosphorylation in intestinal biopsies of control subjects and patients with IBD were examined, and the effect of orally administered iAP tablets on the progression of dextran sodium sulfate-induced colitis in rats was subsequently studied. Results: In healthy persons, iAP mRNA and enzyme activity was high in the ileum relative to the colon. In patients with UC and CD, iAP mRNA expression was found to be markedly reduced when inflamed tissue was compared with non-inflamed tissue. Oral administration of iAP tablets to colitic rats resulted in a significant attenuation of colonic inflammation as reflected by reduced mRNA levels for tumour necrosis factor a, interleukin 1b, interleukin 6 and inducible nitric oxide synthase NOS (iNOS), a reduced iNOS staining and inflammatory cell influx, and a significantly improved morphology of the intestinal wall. Conclusions: The present study shows that epithelial iAP mRNA expression is reduced in patients with UC and CD. The rat model demonstrates that oral administration of active iAP enzymes in the intestinal tract results in a significant reduction of inflammation. This provides new insight on IBD pathology and a novel treatment approach to this severe inflammatory disease. Crohn’s disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBDs) of the digestive tract that are the result of an inappropriate and ongoing activation of the mucosal immune system driven by the presence of the normal luminal flora. 1 The exact causes of IBD are still unclear, but environmental factors, genetic predisposition and immunological disorders are suggested to be involved. Studies have shown that mutations in several genes, such as NOD2/CARD15 in CD 2 and Toll-like receptor 4 (TLR4) in CD and UC, 34 are associated with the occurrence of IBD. The intracel- lular protein encoded by the NOD2 gene interacts with bacterial products such as peptidoglycans, 56 and TLR4 is the signalling receptor for lipopolysac- charide (LPS). So, deficiencies in response mechan- isms against bacterial products, in particular LPS, are an important factor in IBD. We demonstrated previously that alkaline phos- phatase (AP) dephosphorylates LPS, 7–10 which was also confirmed by others, 11 resulting in the forma- tion of a non-toxic lipid A group within the LPS molecule. In general, the lipid A group of LPS contains two phosphate groups that are respon- sible for the toxicity of LPS, and AP removes at least one of these phosphate groups. This enzyme is abundantly present along the microvilli in the small intestine of all species, 12 indicating a possible role in the protection of the host against LPS. As AP is able to detoxify LPS and response mechanisms against LPS are changed during IBD, we wondered whether the levels of AP are changed in the intestines of IBD patients. Therefore, iAP mRNA expression and LPS dephosphorylation in intestinal biopsies of control subjects and patients with IBD were determined. Furthermore, we studied the efficacy of orally administrated acid- resistant iAP tablets on dextran sodium sufate (DSS)-induced colitis in rats. In this study, we show that epithelial iAP expression is decreased in patients with UC and CD, and that oral iAP administration ameliorates inflammation in colitic rats. These observations provide novel insights and a rationale for new therapeutic strategies against IBD through augmentation of AP in the intestinal lumen. MATERIALS AND METHODS Patient characteristics/specimen collection Intestinal mucosal biopsy specimens were obtained during endoscopy following informed consent (approved by the Ethics Committee of the University Medical Centre Groningen) from patients with CD, UC and control subjects. Patient characteristics are described in table 1. Diagnosis of IBD was established by endoscopic and histopathological examination. The control subjects were referred to our endoscopy centre because of polyp surveillance or changed stool frequency. In control subjects, biopsies were obtained from four different intestinal areas (ileum, ascending colon, transverse colon and rectum). Biopsies from patients with CD were obtained from the edge of ulceration’s or aphtoid lesions if present, and from macroscopic non- inflamed areas using a standard biopsy forceps. Only patients with CD in whom the disease had affected the colon were included in our study— that is, only colonic biopsies were used. In patients with UC, biopsies were not taken from the proximal upper level (transition zone) but from the inflamed and non-inflamed part of the colon. Intestinal specimens were immediately snap-fro- zen in liquid nitrogen for mRNA and protein Inflammatory bowel disease Gut 2009;58:379–387. doi:10.1136/gut.2007.128868 379