Expression and functions of purinergic receptor P2X7 in colonic macrophages and T lymphocytes from normal and inflammatory bowel disease mucosa

bacteria-relatedantigens, 78% of patients respondedto at least 1 but only 26% responded to all 3. 57% were double positive. Assessing the magnitude of response showed further heterogeneity. There was a high degree of variability in magnitude of response among CD patients who respondedto all 3 bacterial/bacteria-relatedantigens. Some patients had a high level responseto only a single antigen while others had high responsesto all 3. SUMMARY 1) IgA responseto OmpCis IBD specific (predominantlyCD). 2) The use of a panelof antigens to determineserological response identifies many more CD patients than any single antigenic responsealone. 3) Ratherthan global loss of toleranceto bacteria,there appearsto be subsets of CD patients with variable responses to selected bacterial antigens. CONCLUSIONThese results reflect findings in various rodent models of colitis that demonstratedifferent immune reactivityto and dependenceon selectsingle bacteriumfor diseaseexpression.The relationship of these patterns of immune responsesto human CD behavior will need to be determined. 2654 Expression and Functions of Purinergic Receptor P2X? in Colonic Macruphagu and T Lymphocytesfrom Normal and Inflammatory Bowel Disease Muce=a Chris K F Li, Nottingham Univ, Nottingham United Kingdom; Keith Bowers, AstraZeneca R&D Charnwood, Loughborough United Kingdom; Shri Pathamakanthan,Trevor Gray, Nottingham Univ, Nottingham United Kingdom; Mandy Lawson, Malbinder Fagura, AstraZeneca R&D Charnwood, Loughborough United Kingdom; Chris J. Hawkey, Nottingham Univ, Nottingham United Kingdom Background: P2X7 receptor is an ATP-gated ion channel which can form large membrane pores and has been implicated in cytokine releaseand apoptosis in inflammatory cells. We therefore investigatedits expression,activation and inhibition in normal and diseasedcolinic macrophages and T lymphocytes. Methods: Expressionof P2X7receptor mRNA was studied by RT-PCR. Concentration-dependentchanges in pore formation (ethidum bromide influx), apoptosis (annexin V staining) and IL-1,8 release (sandwich ELISA) were used as functional assays. Ultra-structural changes were studied by transmission electon microscopy. Results: P2X7 receptor mRNA was present in all colonic macrophages and T cells studied. Pore formation and apoptosis were induced by purinergic ligands, with a potency order typical for P2X7 receptor agonists (BzATP>ATP>2MeSATP).The concentration of ATP to induce 50% apoptosis was 84 /~M for macrophages and 20/~Vl for T cells respe~ey (n = 4). AlPinduced apoptosis could be specifically inhibited both by apyrase hydrolysis or by the P2XT receptor antagonists, oxidized ATP, KN-62 and P2 pan receptor antagonist PPADS, but not the P2Y receptor antagonist. Functionalevidenceof apoptosisand pore formation was associated with loss of cell membranecontinuity. In LPS-primedmacrophages,ATP causedcaspase1 activation and rapid concentration-dependentreleaseof mature IL-1/], which was inhibited by o-ATP (IC~ = 30 ,~M). The proportion of unstimulated macrophagesand T cells recovered from ulcerative colitis (72%, n=5) and Cruhn's disease (70%, n=4) tissues that were apoptotic was significantly higher than for normal (32%, n = 8), and not further increasedby BzATP.o-ATPreducedthe spontaneousIL-1,8releasein IBD samples.Conclusions:Expression and functional datasupport the presenceof P2X7receptoron colonic mucosal macrophapesand T lymphocytes.This may play a role in the immunopathologyof inflammatory bowel diseases. 2655 Deficiency Of Exlracellular Matrix Integrin , ~ Protects Mice Against 9exlrae Sulfate Sodium Induced Colitis Christian F. Krieglstein, Stephen F. Laroux, Wun L. Chang, Matthew B. Grisham, LSU Health Science Ctr, Shreveport, LA; Guido M Schuermann, Westfalian Wilhelms Univ Muenster, Muenster Germany;Anthony R. DefougeroUes,Biogan fnc, Cambridge, MA; Daniel Nell Granger, LSU Health Science Ctr, Shreveport, LA introduction: Recent studies suggest that interaction betweenthe leukocyte-associatedcollagen-binding integrin ~,81and the interstitial matrix may promote migration and/or activation of extravasatedleukocytes(e.g. T-ceUs,monocytes, PMNs) within the tissue interstitium. The objectiveof this study was to determinewhether m~-integrin contributes to the pathophysiology of mucosal injury and inflammation in experimentalcolitis in mice. Methods: Colitis was induced by feeding wild-type (WT) BALB/c mice and ~-deficient (a~-'-) mice 5 % dextran sulfate sodium (DSS) in drinking water for 7 days (n = 8/group). Healthy WT controls received millipore water (n = 5). Clinical diseaseactivity (DAI) was assesseddaily based on weight loss, stool consistency and presenceof blood in stools over 7 days. Mice were then sacrificed and additional measurements taken; including colon length, weight, WBC count and hematocrit. Granulocyte (e.g. PMN, monocyte) infiltration was assessed by quantifying colonic myeloperoxidase(MPO) activity. Data were analyzedfor significance (P < 0.05) by the ANOVA-Scheff~-testand are expressedas mean _+SE. Results:WT mice fed DSS exhibited weight loss, bloody stools, colonic shortening and granulocyte infiltration. DSS-treateda~-~ mice exhibited a significantly delayed onset and severity of disease (DAI day 7:3.6 _+ 0.1 vs 1.4 -+ 0.2 for DSS-WT and DSS-m-~ , respectively). While all DSS-treated WT mice developedrectal bleeding by day 7, no overt bleedingwas detected in DSS-treatedm-'-mice and their fecal occult blood tests were positive in only 37.5 %. Colon length was significantly decreased in DSS-treatedWT compared to ,~?-~-mice (10.4 ± 0.4 vs 6.5 _+ 0.3 vs 8.4 _+ 0.3 cm for WT control, DSS-WTand DSS-~-J-, respectively).MPO activity in colon samples correlated well with inflammatory tissue changes and diseaseactivity, and was significantly lower in DSS-treated~, ~ mice, when comparedto DSS-treatedWT mice (8.1 -+ 0.7 vs 17.7 -+ 2.2). Conclusions: Taken together, these data suggest that engagement of leukocyteassociated ~,8~receptors by interstitial collagen type IV may mediate mucosal injury and inflammation in this model of colitis by promoting leukocyte movement and/or activation within the inflamed interstitium Therapeutic strategies designed to disrupt the movement and/or activation of leukocytes outside of the vasculature may prove beneficial in treating chronic gut inflammation. (Supported by POl DK43785 & DK47663)

2656 Direct Effects of Serum from Children with Crohn's Disease on Bone Formation Samuel Varghese, Nancy Wyzga, Saint Francts Hosp & Medical Ctr, Hartford, CT; Francisco A. Sylvester, Univ of Connecticut Sch of Medicine, Farmington, CT Background/Objectives:Low bonemineral density is presentin about half of Crohn's disease(CD)patients. Bone loss can result from either low bone formation, increased resorption, or both. In earlier studies, a decrease in calcium content and decalcified bone weight were observed when bone explants were exposed to serum from newly diagnosed children with CD, while bone resorption was not increased. Abnormal bone histology was also seen. Interleukin (IL-6) antibodies protected the bone organ cultures from these effects. This suggested that the bone loss in CD is caused by a decrease in bone formation, and that IL6 plays a role. Thus, we hypothesizedthat the developmentand (or) function of bone-forming cells (octeoblasts) is impaired in CO patients and that this effect is mediated by IL-6. In this study, we examined if serum of patients with CD affected osteoblast function, and examined the role of IL-6. Methods: We isolated primary osteoblast-enriched(Ob) cells from calvariae of fetal rats and cultured them in DMEM containing 10% fetal bovine serum until reaching confluence. The confluent cells were then exposed to medium containing 10% serum from 9 healthy children(control group) or from 9 children newly diagnosedwith CD for 2 to 3 wks. The ability of Ob cells to form mineralized nodules in culture, an indicator of bone formation potential, was measured by von Kossa staining, mRNA from Ob cells was isolated and the expressionof bone cell markers, such as osteocalc=nand alkaline phosphatase,was assessed by Northern blotting. Serum levels of IL-6 were measured by ELISA (R&D Systems). IL-6 antibodies (IL-6 Ab) and recombinant human tL-6 (rhlL-6)were obtained from R&D Systems. Results: Nodule formation was markedly decreasedin cultures exposed to serum from 5/9 of CD patients relative to cultures exposed to serum from control patients. Mean serum IL6 was 19 pg/mL in CD patients and 1 pg/mL in controls. Neutralizing concentrations of an IL-6 Ab did not increasethe number of mineralized nodules. Addition of rhlL-6 in 3 different concentrations (2.5-25 pg/mL) did not decrease nodule formation. Expressionof osteoblast cell markers,osteocalcinand alkaline phosphatase,was reduced in cultures exposedto serum from 5/9 of CD patients. Conclusion: Factors present in the serum of CD patients cause a decreasein nodule formation and expressionof osteoblasticmarkers.The effects of CD serum in osteoblasts do not appear to me mediated by IL-6. Our data indicate that bone loss in CD may be caused partly by decreasedosteoblast ceil function. 2857 Offieruntlal Activity and Expression of Mitogen-aotivated Protein Kinases in iidlammato~ 8owel Oisease Georg H. Wactzig, Dirk Seegort, Susanna Nikolaus, Philip Rosenstiel, Nikolaos Sfikas, Stetan Schreiber, Chriatian-Albrechts Univ, Kiel Germany Background:Mifogen-activatedprotein (MAP) kinasepathwaysplay a key role in inflammatory signal transduction. The inflammatory bowel diseases Crohn's disease (CD) and ulcerative colitis (UC) are complexdisorderswhose etiologiesremain unknown but clearly involvegenetic, immunological, and er~vimnmentalfactors. Aim: To investigatethe extent of participation of I)38 kinase subtypes, c-Jun N-terminal kinases (JNKs), and extracellular signal-regulated idnase 1/2 (ERK1/2) in the inflammatory signal transduction in CD and UC. Methods: Protein expressionand astivity of p38 subtypes, JNKs, and ERK1/2were determined by western blot analysis of denatured extracts of colonic biopsy samples of untreated (5-ASA permissible) and glucocorticoid-treatedCD (n = 16) and UC (n = 13) patients as well as normal controls (n = 12). Kinase mRNA expression was investigated by RT-PCR. p38 activity is assessed by in vitro Idnaseassays. Results: p38-alpha,JNKs and ERK1/2were activatedup to threefold in the inflamed mucosa of IBD patients (P
A-522