Journal of Pathology J Pathol 2008; 216: 183–192 Published online 17 June 2008 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/path.2394 Original Paper Phagocyte-specific S100 proteins are released from affected mucosa and promote immune responses during inflammatory bowel disease D Foell,1,2* H Wittkowski,1,2,3 Z Ren,4 J Turton,4 GPang,5 JDaebritz,1,2 J Ehrchen,3 J Heidemann,6 T Borody,7 J Roth1,2,3 and R Clancy4 1Department of Paediatrics, University of Munster,¨ Germany 2Interdisciplinary Centre of Clinical Research, University of Munster,¨ Germany 3Institute of Immunology, University of Munster,¨ Germany 4Hunter Immunology Unit, Hunter New England Area Health Service, John Hunter Hospital, Newcastle, NSW, Australia 5Discipline of Immunology and Microbiology, School of Biomedical Sciences, University of Newcastle, NSW, Australia 6Department of Medicine B, University of Munster,¨ Germany 7Centre for Digestive Diseases, Five Dock, NSW, Australia *Correspondence to: Abstract D Foell, Department of Paediatrics, University of Phagocyte-derived S100 proteins are endogenous activators of innate immune responses. Munster,¨ S100A12 binds to the receptor for advanced glycation end-products, while complexes of Albert-Schweitzer-Strasse 33, S100A8/S100A9 (myeloid-related proteins, MRP8/14; calprotectin) are ligands of toll-like D-48149 Munster,¨ Germany. receptor 4. These S100 proteins can be detected in stool. In the present study we analyse E-mail: [email protected] the release of S100A12 and MRP8/14 from intestinal tissue. Specimens from patients with Crohn’s disease (CD; n = 30), ulcerative colitis (UC; n = 30), irritable bowel syndrome No conflicts of interest were = = declared. (IBS; n 30) or without inflammation (n 30) were obtained during endoscopy. After 24 h culture, S100A12 and MRP8/14 were analysed in supernatants. Endoscopic, histological, laboratory and clinical disease activity measures were documented. We found an increased spontaneous release of S100A12 from tissue in inflammatory bowel disease (IBD). The release of S100A12 into the supernatants was 28-fold enhanced in inflamed tissue when compared to non-inflamed tissue (mean 46.9 vs. 1.7 ng/ml, p < 0.0001). In active CD, release of S100A12 and MRP8/14 was strongly dependent on localization, with little release from sites of active ileal inflammation compared to colonic inflammation. This difference was more pronounced for S100A12 than for MRP8/14. S100A12 and MRP8/14 provoked up-regulation of adhesion molecules and chemokines on human intestinal microvascular endothelial cells (HIMECs) isolated from normal colonic tissue. The direct release of phagocyte-derived S100 proteins from inflamed tissues may reflect secretion from infiltrating neutrophils (S100A12) and also monocytes or epithelial cells (MRP8/14). Via activation of pattern recognition receptors, these proteins promote inflammation in intestinal tissue. The enhanced mucosal release can explain the correlation of fecal markers with disease activity in IBD. Copyright 2008 Pathological Society of Great Britain and Ireland. Published by John Received: 28 March 2008 Wiley & Sons, Ltd. Revised: 5 May 2008 Keywords: S100A12; MRP8/14; IBD; IBS; calprotectin; mucosa; gut; colon; inflammatory Accepted: 4 June 2008 bowel disease Introduction neutrophils, production of pro-inflammatory mediators and, finally, tissue damage [1–3]. In recent years the Both Crohn’s disease (CD) and ulcerative colitis (UC) role of innate immunity in IBD has been emphasized, are the consequence of chronic inflammatory reac- as an example of the association of CD with abnor- tions in the gastrointestinal tissue and are collectively malities in pattern recognition receptors (PRRs) such defined as inflammatory bowel disease (IBD). IBD as NOD2, which play a key role in innate host defence results from a disturbed interplay of the environ- [4]. Therapies targeting cytokines mainly produced by ment — with various exogenous factors, such as nutri- phagocytes improve the clinical course of IBD. How- tion — and the intestinal microflora with the immune ever, there is a growing body of evidence that factors system of a genetically susceptible host. The result- other than classical cytokines contribute to the activa- ing inflammatory processes involve both adaptive and tion of innate immune responses, including responses innate immune mechanisms, such as the activation of towards self molecules. Recently, a novel group of Copyright 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk 184 DFoellet al important pro-inflammatory molecules has been intro- tissue specimens. The biological activity of these duced to the concept of innate immunity. In parallel DAMP molecules on primary intestinal microvascu- to pathogen-associated molecular patterns (PAMPs) as lar endothelial cells was investigated to confirm the exogenous factors initiating inflammation, the term relevance of their local release for the induction of ‘damage associated molecular pattern (DAMP) pro- inflammatory responses in bowel tissue. teins’, for endogenous molecules that are released by activated or damaged cells under conditions of cell stress, has been established. Examples of this group Methods are phagocytic S100 proteins, which mediate inflam- matory responses via interaction with PRRs [5,6]. Patients and controls DAMP molecules accumulating in the intesti- We included patients with CD and UC into the IBD nal mucosa represent excellent markers for mucosal group. The non-inflamed group consisted of patients inflammation. These proteins may also spill over into with irritable bowel syndrome (IBS) and individu- the gut lumen, and the additional release by inflam- als without inflammation. Subjects in screening pro- matory cells in crypt abscesses results in significant grammes for early detection of polyps and colon amounts that mix with other secretions and gut con- cancer and having normal colonoscopic finding were tents, which ultimately appear in the patients’ stool. chosen for normal controls. Random colonic biop- Therefore, assays which determine intestinal inflam- sies were collected in healthy subjects, and histolog- mation by detecting neutrophil-derived DAMPs in ical examination was normal in all these individuals. stool bear a great diagnostic potential [7]. All patients underwent endoscopic examination, and Among the factors released by infiltrating neu- biopsies were taken for histological investigations at trophils are DAMP proteins of the S100 family [8]. all colonoscopies. Each patient gave signed informed One example is a complex of two distinct S100 consent. The study was approved by the Centre for proteins, S100A8 (myeloid-related protein 8, MRP8) Digestive Diseases Human Research Ethics Commit- and S100A9 (MRP14) [9–11]. In recent years, the tee, Sydney, and Human Research Ethics Committee, MRP8/14 complex has been proposed as a fecal University of Newcastle, Australia. All patients with marker of gut inflammation, termed ‘fecal calpro- IBS fulfilled the ROME II criteria [27]. Endoscopic, tectin’ [7,12–18]. MRP8/14 is found in large amounts histological and clinical disease activity measures were in granulocytes as well as monocytes but it is also documented. inducible in epithelial cells, e.g. in lactose intolerance [13,14,19,20]. S100A12, as a marker of neutrophil Determination of disease activity activation, is more restricted to granulocytes and is To define inflammatory activity in the gut, macro- released during inflammatory conditions at the site of scopic signs of inflammation were recorded during inflammation and abundant in the intestinal mucosa colonoscopy by the experienced gastroenterologist, of patients with IBD [8,21,22]. Over-expression at using the simple endoscopic score for CD (SES-CD) the site of inflammation and correlation with disease or a similar index for UC [28,29]. In addition, biopsies activity in a variety of inflammatory disorders under- were taken from multiple sites, including the terminal score the role of this granulocytic protein as pro- ileum. Biopsy sections were encoded and analysed by inflammatory molecule [23]. The phagocyte specific independent observers who were blinded to diagnosis S100 proteins represent classical DAMPs which pro- and clinical data. As a further inclusion criterion, the mote inflammation by specific interactions with pattern histology score had to be in accordance with endo- recognition receptors, MRP8/14 via activation of the scopic signs of inflammation. Endoscopic and histo- Toll-like receptor 4 (TLR4) complex and S100A12 via logical appearance was used to categorize patients into binding to the receptor for advanced glycation end groups of disease severity (0 = no inflammation, 1 = products (RAGE) [24,25]. Data on murine models of mild inflammation; 2 = moderate to severe inflamma- colitis as well as human IBD point to an important tion), as previously described [22]. This inflamma- role of these DAMPs during the pathogenesis of IBD tion score served as the reference standard to deter- [8,25,26]. mine absence of inflammation or active IBD in our In previous studies, we have already demonstrated study cohort. Levels of C-reactive protein (CRP), ery- that S100A12 is over-expressed during chronic active throcyte sedimentation rate (ESR), white blood count IBD and serves as a useful serum and stool marker of (WBC), haemoglobin (Hb), platelets (Plt) and haema- inflammation [8,22]. In the present study we investi- tocrit (Hct) were determined in all patients. Mucosal gated