Granuloma consisting cells masquerading mature dendritic cells induce antigen presentation and T cell stimulation in Crohn's disease

J Pathol.),and the proliferatingactivity of surrounding CD4+ lymphocytes.[Methods] Surgical specimens, obtained from patients with CrD and ulcerative colitis (UC), were stained using monoclonal antibodies against costimulatory molecules, leukocyte integrins, CD68, CD83 (mature DC marker), CD4 and Ki67 (proliteratlon-associatedantigen).Toquantity the proliferative activity of CD4÷ T cells in the representative lesions of CrD, including Gra, double immunohistochemistry for CD4 and Ki-67 was performed (modified from Saiki et al. Lab Invest, 1996). [Results] Gra cells were positive for CD68, B7-1, B7-2, CD40, ICAM-1, CD58 (partly), HLA-DR,CDlf a, CDllb, CD11c, and also CD83 (partly). The phenotypesof Gra cells were very similar to that of interdigitatlng cells, one of the representativemature DCc in lymph nodes (LNs). 67-2(CD86)+ macrophages, expressing the same phenotypes as Gra cells, were also localized in VEGFR3÷ lymphatic vessels. Furthermore they were prominently increased in number in active CrD, compared with UC and the controls, suggesting these cells might be in migrating process to mesenteric LNs through lymphatic vessels. Labeling index of Ki-67 was 4.5% in CD4÷ lymphocytes within granulomas, which was s~tistically equal level to that of CD4* lymphocytes in T cell zone of the mesenteric LNs. In CrD, Gra cells masqueradingmature DC phenotypesmay play important roles in activating CD4÷ cells around granuloma respondingto certain diseaserelatedantigens invading through lymphatic system. [Conclusions] Our data indicated that Gra cells in CrD could function as APCs, and induce antigen-specific CD4+ lymphocyte proliferation in both intestinal tissues and regional mesenteric LNs via lymphatic vessels.

(371 _+132), and controls (297 _+171)(p-<0.05). Staining intensity of ETS-1 was increased in active UC compared to other groups, but no significant difference was observed in the staining intensity of MMP-I. (3) Expression of ETS-1 protein was increased in active UC compared to CD and controls. [Conclusion] Overexpressionsof angiogenetic factors were observedin the colonic lesions of IBD,especiallyin UC, suggestingthat abnormalangiogenetic reactions occur in the colonic lesions of IBD. 2659 The Crohn's Disease Lesion Derived Gene, 12, Encodesthe Core Paptide of a Transcriptional Regulator of Pse#domona$ fluore$cen$, and the Functional Domain of a Novel T Ceil Superantigen Be Wei, Tiffany T Y Huang, Harnisha Delwedi, Jonathan Braun, Univ of CA, Los Angeles, Los Angeles, CA BACKGROUND:Commensal bacteria are an important pathogenicfactor for Crohn's disease (CD). We previously isolated a CD-associatedgene called 12 (Gastroenterology119:23, 2000), based on its lesional localization and disease-specificantibody titers. We recently identified Pseudomonas fluorescensasthe microbial origin of this sequence,and that the proteinactivates CD4 + T cells by a superantigen-like mechanism. This study addressesthe structural basis of 12 bioactlvity. METHODS:The fuIHength 12 gene (PF-12) was isolated by genome walking (RACE). Recombinantproteins were constructed and expressedfor PF-12and P. aeruginosahomologue (PA2885), including the full-length, N-terminal, C-terminal, and central segments of the proteins.Transcriptional regulationwas tested using a plasmid-basedbacterialtransactlvation assay, Superantigenactivity was tested by CD4+ T cell proliferation. RESULTS:PF-12 and PA2885 are homologous proteins, with 85% identity and 93% similarity at the amino acid level. Heterologous expression of each protein stimulated transcriptional activity of a plasmid target gene bearing the bacterial phage promoter. Deletion studies implicated the Nterminal and central segmentsas required component of this regulatory activity. 12was found to express T cell superantigen activity. A comparison of structura-function activity in PF-12 and PA2885 will be presented. CONCLUSION:The CD-associated 12 sequence originates in P. fluorescens,and shares a close homologue, PA2885, in P. aeruginosa. The proteins display regulatorybacterialbioactivity (transcriptionalregulation)and CD4+ T cell immunostimulatory activity. These findings reveal mechanisms pertinent to the potential pathogenic role of PF12 and its host bacterium in Crohn's disease.

2662 Characterization of Helicobacter-inducedInflammatory Bowel Disease in I L - I O - / and T Cell Deficient Mice Andrew Burich, Univ of Washington, Seattle, WA; Robert Hershberg, Corixa Corp, Seattle, WA; Kim Waggle, ZymoGenetlcs, Inc, Seattle, WA; Weiping Zeng, Univ of Washington, Seattle, WA; Jeanne L. Viney, Immunex Corp, Seattle, WA; Lillian Maggie-Price, Univ of Washington, Seattle, WA A proposed factor in the developmentof inflammatory bowel disease(IBD) is a dysregulated immune responseto intestinal bacterialantigens,with T cells mediatingthe intestinalpathology. Previous studies have shown that both natural and experimentalinfections with Helicobacter spp. in immunodeficientmice, some of which lack T cells, can causesignificant IBD.Therefore, we wanted to further understand the role of T cells in Helicobacter-induced IBD. Three to five week old, female IL-lO-/-, Rag-1- / - , TCR/3-/-, and wild type mice were given H. hepaticus or H. bills or broth by oral gavage.Diseasewas evaluatedby colonic histopathologic analysis and expression of pro-inflammatory cytokines in diseasedtissue. IL-IO-/mice infected with either H. hepaticus or H. bills developed IBD; however, disease induced by H. bills was more severe, particularly in the middle and distal colon. RT-PCRand Rnase Protection Assay analysesshowed increased levels of the pro-inflammatory cytokines IFN-,y, IL-I~, and IL-1/~ in the diseasedcolons of IL-lO-/- mice. Interestingly, mice deficient in mature T cells (Rag-1 - / - and TCR/3-/-) did not develop IBD when infected with Helicobacter. These results indicate that different species of Helicobacter may vary in their ability to induce IBD in the same animal model and T cells appear to play an important role in initiating He/icobacter-induced IBD. The immunological basis of He/icobactsr-induced IBD is currently being explored. Further, Helicobacter spp. are likely to be a useful tool to decipher the mechanisms of microbial-induced IBD in a variety of mouse models.

2660 Intestinal Epithelial Transport In IBD: Alterations Of Both Apical Membrane And Microvillus Cytoskeleton Matthias Bruewer, Sabine Kersting, Axel Klotz, Klaus P. Zimmer, Norhert Senninger, Guido Schuermann, Westfalian Wilhelms Univ Muenster, Muenster Germany BACKGROUND:Structural alterationsof the apicalcell membraneand the microvillus cytoskelston may contribute to increasedtranscellular permeabilityin Crohn's disease(CD) and ulcerative colitis (UC). The aim of this study was to investigatethe distribution of sucrase-isomaltase (S-I), a disacchaddaseof the apical brush border, and villin (V), a protein of the microvillus cytoskeleton, in normal enterocytes (NE) and enterocytes with rapid antigen uptake into the cytosol (RACE) from healthy controls (HC) or patients with CD or UC. METHODS:Labeling densities of S-I- and V-binding antibodies were determined by immunoelectron microscopy in CD (ileum (il), n=5) and UC (colon (co), n=5) and was compared to HC (il: n=5; co: n=5). Wilcoxon U-test was used for statistical analysis with p
2663

Heteragenous Infiltration of Surface Thl Marker (CCR5) and Th2 Marker (CRTH2) Cells in Colonic Mueosa of Patients with Crahn's Disease. Yukiko Yeshida, Ryota Hokafi, Koji Matsuzaki, Shingo Kate, Atsuhiro Iwai, Chie Kurihara, Atsushi Kawaguchi, Shigeaki Nagao, Yoshikiyo Okada, Kazuro itoh, Soichiro Miura, National Defense Medical Coil, Saitama Japan; Kinya Nagata, BML research and development, Saitama Japan Background:Crohn's diseaseis characterizedby skip lesions,and these lesions are considered to be induced by excessive release of Th 1 cytokines. However, Thl/Th2 cell balance has beenstudied by comparing the cytokine profiles using stimulated lymphocytesfrom peripheral blood and its balance in inflammatory mucosa without stimulation of lymphocytes has not been elucidated. We investigated the expression of chemokine receptor 5 (CCR5) as Thf marker and chemoattractantrecegtor-horoologuecmolecule expressedon Th2 ceils (CRTH2, J Immunol 162: 1278, 1999) as Th2 marker on T cells in colonic mucosa of CD patients. Methods: Tissue samples were obtained from colonoscopic biopsies or surgical specimens from patients with CD with informed consent. As controls specimens were obtained from patients with colonic polyps. Immunohistochemical analysis was performed on cryostat sections using LSAB method. Monoclonal antibodies against CD4,/37-integrin, CCR5 and CRTH2were usedfor the primary antibodies.Thesesections were observedunderfluorescence microscope or confocal laser scanning microscope. Results: There were few CCR5 positive cells in the colonic mucosa of control subjects, while CRTH2 were expressed in the lamina propdal lymphocytes.The expressionof CCR5significantly increased in the inflamed mucosa and submucosa of CD, and the degree of CCR5 appearedto be well correlated to the extent of inflammation. In CD patients, CRTH2 positive cells almost disappeared in the inflamed mucosa, but there was a significant infiltration of CRTH2cells in the unifiamed mucosa even near the inflammatory lesion. There was no cell which shows simultaneous expression of both CCR5and CRTH2.Most of the CCR5positive cells simultaneouslyexpressed/37integrin. Conclusions: We demonstratedthat strong polarized Thl response occurred in the inflamed colonic mucosa of CD patients. However,there is also significant infiltration of Th2 dominant cells in the uninflamed colonic mucosa of the same patients, suggesting the heterogenous infiltration of Thl and Th2 cells in the CD mucosa.

Labeling density

S-I (particles per pn~) V (pertides per

HC-il

CD.NE

57.6 +_5.7 25.5 +_7.2

*40.5 +_11.2 "21.8 +5.1

CO-PACE H C - c o _*'16.8 +_7.7 *'7.1 ±3.0

0.5 ±0.6 29.9 +_fl.0

UC-NE

DC-RACE

*46.6 +_6.9 "19.4 +5.2

"15.2 ~.4 "9.4 +3.5

Data are means+ standarddeviation 2661 Granuloma Consisting Ceils Masquerading Mature Dendritic Cells Induce Antigen Presentation and T Cell Stimulation in Crahn's Disease Shire Nakamura, Takayuki Matsumoto, Yoshio Jinno, Yoshinori Sawa, Junichi Hara, Nobuhide Oshitani, Osaka City Univ, OsakaJapan; Haroo Otani, Hiroshi Nagura, Tohoku Univ, Sendal Japan; Yukio Nishiguchi, Kousei Hirakawa,Tetsuo Arakawa, Osaka City Univ, Osaka Japan [Background] Non-caseatinggranuloma (Gra) is one of the most characteristic pathological features of Crohns disease (CrD). However,its immunological functions are still unclear. To demonstrate the functions of Gra cells, such as epithelioid cells and multinucleated giant cells, as antigen presenting cells (APCs), we examinedphenotypicalfeatures and similarities between Gra cells and mature dendritic cells (DCs), the relationship with lymphatic vessels expressing vascular endothelial growth factor receptor-3 (VEGFR3) (Lymboussaki et al. Am

A-523