Prothrombin-gene mutation and factor V leiden in inflammatory bowel disease complicated by venous thrombosis

A354 AGA ABSTRACTS

1897 CHEMOTACTIC PROPERTIES OF ICAM·l AND PECAM·l ON NEUTROPHILS IN ULCERATIVE COLITIS: INFLUENCE OF PREDNISOLONE. Ben Vainer, Ole H. Nielsen, Glostrup Hosp, Univ of Copenhagen, Glostrup, Denmark. Background and aim: Recent observations have suggested, that ICAM-I exhibit other effects in addition to attachment neutrophils (PMNs) to endothelial cells. Prednisolone, which together with mesalazine (5-ASA) are the prevaling treatment principles of ulcerative colitis (UC), inhibit the transendothelial migration of PMN. The molecular basis of this remains obscure. In this respect, it was investigated, whether ICAM-I act chemotactic on PMNs, and whether prednisolone interferes with this cell attraction. PECAM-I, P-selectin and mesalazine were included for comparison reasons. Material and methods: PMNs isolated from blood (II UC, 15 controls) were used in microchemotaxis chambers (NeuroProbe, MD, USA) with 5 /Lm pore size nitrocellulose filters. Physiologically relevant concentrations of recombinant human ICAM-I (0.05-500 pM), PECAM-I (0.01-100 nM), and P-selectin (0.01-100 nM) were added to the bottom wells, while 106 PMNs/ml were placed in the upper chambers. fMLP was used as positive chemoattractant, while Gey s buffer was applied to assess the spontaneous locomotion. For the drug assays, PMNs were preincubated with prednisolone (10-8-10-4 M) or mesalazine (0.65-10.4 nM). The leading front technique, which assess the depth of cell migration into the filter (um), was applied, and all tests were run in duplicate. Results: The migration of PMNs towards ICAM-I showed a bell-shaped curve with a maximum at 5 pM (relative migration; 144.6 % of the spontaneous locomotion, p
1898 HUMAN BETA DEFENSIN 2 IS INDUCED IN ULCERATIVE CO· LITIS BUT NOT IN CROHN'S DISEASE. Jan Wehkamp, Klaus Fellennann, Klaus Robert Herrlinger, Steffi Baxmann, Klaus Schmidt, Burkhard Rudolph, Eduard Friedrich Stange, Med Univ of Luebeck, Luebeck, Germany; Univ of Luebeck, Luebeck, Germany. Background: Chronic inflammatory bowel diseases (IBD) are characterized by a pronounced immune reaction against the luminal bacterial flora. This may be a consequence of dysregulated immune response or an impaired mucosal defense allowing bacterial invasion, the inflammatory state being secondary. Antimicrobial peptides, such as defensins, playa major role in mucosal barrier function. Therefore we compared defensins transcripts and protein expression from controls (C), patients with Crohn's disease (CD) and ulcerative colitis (UC). Methods:Up to four colonic biopsies were taken from each of 42 individuals (16 C. 13 CD, 13 UC). Total RNA was extracted and RT-PCR was performed with intron spanning primer pairs for human beta defensin I (HBD-I) and 2 (HBD-2) as well as human defensin 5 (HD-5) and 6 (HD-6). Additionally, immunohistochemistry was performed with polyclonal antibodies against HBD-I, HBD-2 and HD-5 (supplied by Dr. Ganz, UCLA) on resected specimens. Results: The most conspicuous finding was the deficiency of HBD-2 mRNA in 88% of CD biopsies vs. 42% (p
1899 Bl.INTEGRINS MEDIATE CONTRA·APOPTOTIC SIGNALS IN CD4+ LAMINA PROPRIA LYMPHOCYTES. Bianca M. Wittig, Martin J. Humphries, Stefan Meuer, Martin Zeitz, Andreas Stallmach, Dept of Internal Medicine II, Homburg, Germany; Welcome Trust Ctr Cell Matrix Research, Manchester, United Kingdom; Institute of Immunology, Heidelberg, Germany, T cell activation is an important feature of chronic inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis. Recent data

GASTROENTEROLOGY Vol. 118, No.4

indicate that in IBD the program of activation induced cell death (AICD) of T cells is disturbed and may result in inappropriate T cell accumulation contributing to chronic inflammation. Since activation of J3J-integrin influences apoptotic pathways in PBL and activated J31-integrins are expressed on LPL in inflammatory bowel disease, we examined the integrintriggered cell survival signal and associated pathways in CD4-positive lamina propria cells. Methods: LPL were isolated by density gradients from normal colonic specimens (n> 10) and separated using CD4-positive magnetic microbeads (purification> 95%). Cells were stimulated by crosslinking of anti-CD2 or anti-CD3 antibodies alone or in combination with a stimulating i31-integrin antibody (l2GIO) in the presence of a mouse Ltk-cell line stably expressing human CDw321FC')'-RII (J Exp Med 175: 671, 1992). Apoptosis was measured by flow cytometry after staining with propidium iodide. Results: anti-CD2 and anti-CD3 activation resulted in an increase of AICD in CD4-positive LPL (+63% and + 32%, respectively). Interestingly, costimulation with 12GI0 antibodies decreased AICD rates (-58% and -69%, respectively). Further, activation with anti-CD2 or antiCD3 increased the BcI-xLlBax-transcript ratio but did not influence the CD95IFas-ligand-ratio. BcI-xLlBax-transcript ratio were significantly higher in CD3/12GlO or CD2/12G10 costimulated LPL compared to CD2 or CD3 activation alone. Discussion: These data indicate that J31-integrin stimulation rescues LPL from undergoing apoptosis after TCRlCD3 activation. i3,-integrin activation may perpetuate the inflammatory process in inflammatory bowel disease.

1900 LAMINA PROPRIA MONONUCLEAR CELLS OF PATIENTS WITH INFLAMMATORY BOWEL DISEASE UNDERGO APO· PTOSIS UPON BLOCKADE OF CD44V7. Bianca M. Wittig, Martin Zeitz, Andreas Stallmach, Dept of Internal Medicine II, Homburg, Germany, Recently, we have demonstrated that expression of CD44v7 on hematopoetic cells is essentiell for experimental colitis in mice. Moreover, CD44v7, functioning as a costimulatory molecule, is increased in patients with chronic inflammatory bowel disease (IBD). Recent studies suggest a defective activation induced cell death of lymphocytes as major pathogenic mechanism in chronic inflammatory bowel disease. We therefore analyzed whether CD44v7 expression on lamina propria mononuclear cells provides a contra-apoptotic signal in patients with IBD. Methods: Lamina propria mononuclear cells (LPMC) were isolated from freshly resected uninflamed and inflamed mucosa of patients with IBD (n> 10) and control patients (n> 12) and cultured with monoclonal antibodies against CD2, Fas, CD44v7, or CD44 standard. Apoptosis was measured by flow cytometry after propidium iodide staining in a hypotonic solution. Results: As described before, LPMC of inflamed mucosa are resistant to CD2 activation induced cell death (mean value: 38.5% apoptotic cells in anti-CD2 vs 46.5% in untreated cells). In inflamed mucosa there also was a decreased rate of apoptosis upon Fas ligation. Opposed to this, blockade of CD44v7 significantly induced apoptosis in LPMC of inflamed mucosa (mean value: 67% apoptotic cells) but not in uninflamed lesions of patients with IBD (mean value: 26% in anti-CD44v7 vs 21% in untreated cells) or normal mucosa (mean value: 40% in anti-CD44v7 vs 35% in untreated cells). Interestingly, in inflamed mucosa these apoptotic cells were mainly CD33 positive and to a small extent CD4 positive cells. Conclusion: In inflamed mucosa blockade of CD44v7 induces apotosis in LPMC, and here mainly in macrophages. Thus, specific upregulation of the costimulatory molecule CD44v7 appears to endow LPMC with resistance towards apoptosis leading to sustenance of the chronic inflammation.

1901 PROTHROMBIN·GENE MUTATION AND FACTOR V LEIDEN IN INFLAMMATORY BOWEL DISEASE COMPLICATED BY VE· NOUS THROMBOSIS. Terence Wong, Jo Nightingale, Azhar Ansari, Jeremey Sanderson, Mark Winter, Andrew F. Muller, Kent and Canterbury Hosp, Canterbury, United Kingdom; Guys and St Thomas' Hosp, London, United Kingdom. Introduction Patients with inflammatory bowel disease (lBD) have an increased frequency of venous thromboembolism (VTE), and microvascular thrombosis has been postulated in the pathogenesis of IBD. Recently two new genetic mutations have been discovered which increase the risk of venous thrombosis- the G20210A mutation in the prothrombin (PT) gene and the Gl691A mutation in the factor V gene (Factor V Leiden[FVL]). The aims of the present study were to determine the prevalence of these genes in IBD patients with, and without VTE. Patients, Materials, and Methods 42 patients with IBD and VTE (25 Ulcerative Colitis [uq, 17 Crohns disease [CD]), 83 patients with IBD and no history of VTE (51 UC, 32 CD), and 95 normal controls were studied. Genomic analysis for G2021OA,and GI961A was performed using standard techniques for PCR amplification and subsequent restriction using HIND III or Mnl I respectively. Patient groups were compared using Fisher's exact test. Results Of the 42 patients with IBD and VTE I (2%) was homozygous, and I (2%) heterozygous for G2021OA; 6 (14%) were heterozygous for G1961A. Of the 83 patients with IBD and no history of VTE 7 (8%) were heterozygous for G2021OA,and 6 (7%) were heterozygous for G1961A. In the 95 normal controls 2 (2%) were heterozygous for G2021OA, and 4 (4%) were heterozygous for G1961A. FVL was significantly increased in patients with IBD and VTE when compared with normal controls (p<0.05). The PT mutation was increased in patients with IBD without VTE compared with

April 2000

normal controls (8% vs 2%, p=0.05), however did not increase the risk of VTE. Conclusions. FVL increases the risk of VTE complicating IBD, and the PT mutation may increase the risk of IBD. The PT mutation does not increase the risk of VTE in IBD. 1902 HLA-DPB1 0901 ALLELE, GENOTYPE OF DPW9. IS ASSOCIATED WITH SUSCEPTmILITY TO ULCERATIVE COLITIS AND AUTOANTmODY PRODUCTION AGAISNT TROPOMYOSIN PEPTIDE. Yoshiro Niitsu, Naohito Yoshizaki, Sumio Sakamaki, Norihiro Takayanagi, Shuya Hayashi, Yutaka Kawano, Sapporo Med Univ, Sapporo, Japan. Background; Although it is postulated that the autoimmune mechanism is involved in the pathogenesis of ulcerative colitis (DC), there are a few evidences that the autoantibodies are ascribed to the pathogenesis of Uc. To prove this issue, we have investigated autoantibody against tropomyosin peptide (HIAEDADRK) in the sera of UC and found that the epitope could be expressed on the surface of cells. Interestingly, it could bind to the surface of the cells that had HLA-DPw9 but not HLA-DR2 or DQ6. These data indicated that HLA-DPw9 bound to the tropomyosin peptide (HIAEDADRK), and the expression of tropomyosin peptide with HLA-DPw9 might cause the disease progression in the patients with UC (Gastroenterology 110; A 1007 1997). Aim; In this study, therefore, we determined HLA genotype of UC patients to confirm the importance of HLA-DPw9 in UC patients. Additionally, we analyzed the relation of the autoantibody production to HLA-DPw9 expression in the patients of UC. Patients and Methods; 1) Twenty patients of UC were subjected in this study. Informed consent was taken from all the patients. 2) Titer of anti-tropomyosin peptide autoantibodies in the sera from UC patients was measured by peptide ELISA method as previously reported. 3) Genotype of HLA-DP was determined by SMAI method based on PCR-RFLP method. Results; 1) Eleven out of 20 UC patients had HLA-DPw9; DPBl 0901 allele. The prevalence of the allele in UC patients (55%) is significantly higher than that of normal subjects (19%). 2) All sera of II patients with HLA-DPBI 090 I showed higher titer of anti-tropomyosin peptide antibodies than normal subjects did. 3) In HLA-DPBl 0901 positive patients, active cases showed higher titer of anti-tropomyosin antibodies than inactive cases. Conclusion; We showed here that HLA DPw9 is the key molecule for the surface expression of tropomyosin peptide. These data leads us to the conclusion that HLA DPw9 is clearly involved in the pathogenesis of UC in regard to the binding to the tropomyosin peptide as the autoantigen.

AGAA355

1904 CHEMOKINE RECEPTOR EXPRESSION AND FUNCTION IN LAMINA PROPRIA MONONUCLEAR CELLS OF THE HUMAN COLON. Susan J. Connor, Zeenath Hassim, Rosie Cardillo, Andrew R. Lloyd, Michael C. Grimm, St George Clin Sch, UNSW, Kogarah, Australia; Sch of Pathology, UNSW, Sydney, Australia; St George Clin Sch, Kogarah, Australia. Chemokines are chemotactic cytokines, important for normal leucocyte trafficking and for migration to sites of inflammation. Chemokine receptors are crucial for determining the nature of inflammatory and immune responses. The gastrointestinal tract forms a major arm of the immune system, but little is known of chemokine receptor expression and function in the intestine. This study sought to analyse the expression patteru of chemokine receptors and their function in normal human colon. Lamina propria mononuclear cells (LPMCs) were isolated from normal colons and analysed by flow cytometry. LPMCs were assessed for chemokine responsiveness using standard chemotaxis assays. A novel whole tissue chemotaxis assay was used to analyse LPMCs induced to migrate directly out of mucosal strips. Chemokine receptor expression by peripheral blood mononuclear cells (PBMCs) was determined by flow cytometry and results compared with LPMCs from the same patients. Analysis of the lymphocyte gate of LPMCs demonstrated that 80% were CD3+ of which 64% were CD4+ and 36% were CD8+. CXCR3 and CXCR4 were the predominant chemokine receptors expressed on dominantly CD45RO+ cells, as outlined in the table below. Peripheral blood T cells, like LPMCs, strongly expressed CXCR3 (92%). 36% of T cells expressed CCR5 although expression was heterogeneous. LPMCs responded chemotactically to the CXCR4 ligand SDF-la, in a biphasic pattern reflecting differential responses ofT cells and macrophages. IP-IO and SDF-la induced migration of both lymphocytes and macrophages from whole mucosal tissues, indicating the functioning state of chemokine receptors in human colon in vivo. These studies are the first to identify the phenotypic characteristics of chemokine receptor-expressing leucocytes in human colon and to delineate the functional chemotactic responsiveness of these cells. These findings have implications for the understanding of mucosal leucocyte trafficking and the treatment of gut inflammation. Chemokine receptor expression inLPMCs as determined byflow cytometry

'!oIDIaI '!oCD45RO

CXCR1+

CXCR2+

CXCR3+

CXCR4+

CCR2+

CCR5+

10.02±95 888±3.7

o87±1.2

9O.5±7.9 92.1±0.9

72.7±7.0 94.0±1.4

67±9.0 96.4±1.9

4.4±73 85.5±4.8

94.1±2.2

1903

1905

ENTERIC BACTERIAL ANTIGEN-SPECIFIC T-REGULATORY-1 (TRl) CELLS ARE PRESENT IN THE MURINE INTESTINAL LAMINA PROPRIA. Yingzi Cong, Arman Saparov, Casey T. Weaver, Charles O. Elson, Univ of Alabama at Birmingham, Birmingham, AL.

COLON EPITHELIAL PROTEIN(S)CAN INDUCE ORAL TOLERANCE WITH ALTERED CYTOKINE RESPONSES IN THE 2,4,6TRINITROBENZENE SULFONIC ACID (TNBS) INDUCED MODEL OF EXPERIMENTAL COLITIS. Arunansu Dasgupta, Kumaraswamy K. Ramaswamy, Masato Taniguchi, Peter S. Amenta, Kiron M. Das, UMDNJlRobert Wood Johnson Med Sch, New Brunswick, NJ. Rectal (PR) administration of TNBS produces chronic colitis in experimental animals that can be tolerized by total colon extract. It is unknown if such oral tolerance is associated with colon epithelial cell proteins. Three groups (Gr) of rats (Gr D-F), (n=5 each Gr) were fed with tissue extracts (E) from syngenic colon (CE), small intestine (SIE) and LS-180 human colon cancer cells (LSE). Gr-A received no treatment, Gr-B received PR-TNBS in ethanol (ETOH), Gr-C PR-ETOH only. Tissue extracts were fed to the rats X 5 on alternate days followed by PR-TNBS on the tenth day. Rats were sacrificed fifteen days later and the following measured: Disease score including histopathology (0-4 +), weight & thickness of proximal colon (PC) & distal 8 cm of colon (DC), Myeloperoxidase (MPO) values for PC & DC; and levels of TGFf3 and -yIFN in blood. Results: See Table. Conclusion: Human Colon epithelial cell extracted protein (s) can induce oral tolerance in the experimental Colitis. Rat total colon extract but not small intestinal extracts showed similar protection. This tolerization is associated with increase in TGFf3 and decrease in -yIFN levels. Further characterization of the tolerizing colon epithelial protein (s) may have important therapeutic value.

Purpose. Lamina propria lymphocytes (LPL) are known to respond poorly to anti-CD3 or antigen stimulation in vitro despite an activated/memory phenotype. Little is known about LPL responses to specific antigens, particularly responses to enteric bacterial antigens. The purpose of this study was to study LPL responses to cecal bacterial antigens (CBA) and to characterize the phenotype of the cells responding. Methods. Cecal bacterial antigens were prepared as previously described (J Exp Med 187;855, 1998). Intestinal LPL from C3H1HeJ mice were isolated by protease digestion, washed, and stimulated with CBA-pulsed APC. T cell proliferation was measured by 3H-TdR incorporation. Cytokine production in the culture supernatant was measured by ELISA. In situ hybridization (ISH) for cytokine mRNA was performed as previously described (PNAS 92: 7565, 1995). Results. LPL proliferated poorly to CBA-pulsed APCs as well as to anti-CD3 stimulation. When cytokine production was measured in CBA stimulated LPL, only IL-lO was detectable but not IL-2, IL-4, or IFNy. When anti-IL-lO mAb was added to LPL cultured with CBA-pulsed APCs, IFN'J'Was detectable but not IL-2 or IL-4. These results were supported by analysis of CBA-stimulated LPL using cytokine-specific riboprobes, which found that cells producing IL-lO and cells producing IFN'J'Were the dominant phenotypes. LPL inhibited a pathogenic Thl cell line in the presence of CBA-pulsed APC, but not in the presence of anti-CD3 mAb, indicating the inhibition was CBA specific. The inhibition was abrogated by depletion of LPL CD4+ cells, but not LPL CD8+ cells, indicating that CD4+ T cells were responsible for the inhibition. Addition of anti-IL-lO or anti-IL-lOR mAb partially reversed the inhibition. Conclusion. Enteric bacterial antigen-specific CD4+Trl cells are present in the murine lamina propria. These can inhibit pathogenic CD4+ Thl cells, at least partially through production of high amounts of IL-lO.

Results: GROUPS

A.CONTROL B.PR.TNBS C.PR·ETOH D.CE-TNBS E.SIE·TNBS F.LSE·TNBS

DISEASE THICKNESS MPo-OC MPO·SI SCORE (Unitslmg) IUnitslmg) OClf1lll) 0 3+-4+ 0 0-2+ 3.4+ 1-2

520±54 3156±483 1251±131 1222±18oa 3116±579 536±48'

18.4±4 149161 19.4±9 50.3±7' 107±21 4.915'

11±2 7.2±1.9 8.7±2.1 12.7±5 2.6±1.7

TGFp (pGlml)

ylFN 1f1!!lm!)

167±42 166±60 351±2 404±43' 674±386 7891306'

4.4±1 21.8±9 4.9±.6 4.3±1.5 16417 3.1+1 h

Pvalues are against PR-TNBS. DC-Thickness: a-p<0.01, b-p<0.005 (Gr-B); MPO-DC: c-p<0.02,dp<0.006; TGFP: e-p<0.OO01, f-p<0.009. y-IFN: g.p< 0.02, h-p