IκBα- serine-phosphorylation is increased in the intestinal lamina propria of patients with IBD

April 1998

Immunology, Microbiology, mad Inflmmmatory Disorders A1015

three- to fourfold increase of M ceils (p<0.001). The rate of M cell induction strongly depended on the histological degree of inflammation. EM-studies showed that most M ceils changed their morphology compared to noninflamed mucosa with a strong increase of vacuoles in the cytoplasm and a complete lack of microfolds. The M cell surface often appeared desintegrated while the cytoplasm was usually well preserved. The number of mononuclear cells below the FAE was significantly increased. Discussion: The strong increase of M ceils during indomethacin induced ileitis in rats suggests an important role of M cells during chronic intestinal inflammation, e.g. in inflammatory bowel disease. G4156

LECTIN BINDING SITES IN HUMAN INTESTINAL M CELLS. T. Kucharzik, N. LUgering, *M.A. Schmidt, R. Stoll, W. Domschke Department of Medicine B, *Center for Molecular Biology of Inflammation, University of MiJnster, D-48129 Mtinster, Germany. Introduction: Although a preferential adherence and transcytosis of microorganisms and macromolecules to intestinal M cells has been shown, the molecular mechanisms of this process are largely unknown. Recently, we have shown that the intermediate filament cytoskeleton of M cells is not different from that of adjacent enterocytes (Gut, 1997, 41). As the specific adherence of microorganisms to M cells may be mediated by lectin-sugar bindings, we now compared glycoconjugates of human intestinal M cells with those of adjacent enterocytes. Methods: We examined the binding of 28 different lectins and the corresponding sugars on FAE of 16 human noninflamed appendices by using fluorescence microscopy and immunoelectronomicroscopy. M cells were identified by their typical morphology in electron microscopy or by missing staining for alkaline phosphatase using light microscopy. Results: In 5 individuals, we could find selective binding of different FITC-labeled lectins specific for N-acetyl° galactosamine like VVA, WFA and SBA in the apical cytoplasm of M cells. Preincubation of the lectins with the corresponding sugar resulted in a complete loss of their binding capacity on M cells. In two patients M cell cytoplasm could be selectively stained with PEA, M cells of another patient could be marked with RCA-I. However, the M cell specific staining was restricted only to few patients and showed strong inter-individual variations. In most of the appendices no differences between the lectin-binding pattern of M cells and enterocytes were found. Comparing different patients, we found strong inter-individual differences in lectin-binding sites in both cells. Discussion: Differences in lectin binding sites between M cells and enterocytes in some patients suggest different ways of adherence of microorganisms on these cells. However, we could not find a general specific lectin-binding pattern for human M cells. The inter-individual differences between lectin-binding sites of M cells and enterocytes suggest that glycoconjugate expression is not cell type specific and depends on the individual intestinal antigens and the microbial milieu. • G4157 IL-12 RECEPTOR 132 CHAIN UPREGULATION IN MUCOSAL TCELLS IN EARLY BUT NOT LATE CROHN'S DISEASE (CD): IMPLICATIONS FOR VARIABLE IFN-T PRODUCTION DURING DISEASE PROGRESSION. S. Kugathasan. J. Itoh, D. Kou, J.T. Boyle, A.D. Levine, C. Fiocchi. Rainbow Babies & Children's Hospital, Case Western Reserve University, Cleveland, OH. IL-12 is a cytokine produced primarily by antigen-presenting cells which induces T-cells to differentiate into IFN-y-secreting Thl cells. We have previously shown that IL-12 induces mucosal T-ceil clones from early but not late stages of CD to strongly respond to IL-12 induction of IFN-y (GE 112:A1021,1997). Recent evidence indicates that the selective expression of the [~2 chain of the IL-12 receptor (R) is critical for IFN-T production and clearly differentiates Thl from Th2 cells (JEM 185:825, 1997). Therefore, we investigated IL-12R~2 expression in mucosal T-cell clones from children with early and late colonic CD. Early CD was defined as the first clinical presentation, and late as disease established for at least 5 years. Children with functional abdominal pain served as normal controls. T-cell clones were derived from mucosal biopsies, and clonality confirmed by VJ3 region analysis of the T-cell receptor. Clones were generated in the presence of IL-2 (20 U/ml). IL-12RJ32 mRNA was detected by RT-PCR using GAPDH as an internal standard, and level of expression assessed as a ratio of IL-12R~2/GAPDH signal intensity. In T-cell clones from normal mucosa the IL-12R~2/GAPDH ratio was 0.175. In clones from early CD the ratio was 0.608, representing a 3.5 fold increase in expression compared to that of normal mucosa clones. More strikingly, the expression of IL-12RI32 in these early CD clones was 6 times greater than that of late CD clones, whose ratio was 0.148.

but lower in the late stages of the disease. The relative loss of IL-12R[32 expression by mucosal T-cells in established CD correlates well with the emergence of clones resistant to IL-12 immunomodulation, and explains the subsequent decrease in IFN-y production in the late stages of CD. G4158

ANTAGONISTIC REGULATION OF PMN APOPTOSIS BY G-CSF AND IL-10/IL-4. T. KUhbacher, B. Ebert, H. Lochs, S.Schreiber. Mucosal Immunology Group, 4th. Med. Dep., University Hospital Charit6, Berlin, Germany. Background: The high rate of spontaneous apoptosis of PMN is important for the self-limitation of inflammatory reactions. G-CSF (Granulocyte-ColonyStimulating-Growth-Factor), is a hematopoetic factor, which is important for proliferation, activation and differentiation in cells of the myelocyte lineage. G-CSF is upregulated in inflammation and it is known that G-CSF inhibits PMN apoptosis. Aim: To investigate the regulatory effects of the contrainflammatory cytokines IL-10 and IL-4 on G-CSF inhibited PMN apoptosis. Methods: PMN were isolated from peripheral blood and cultured for 24h. Apoptosis was detected by agarose gel electrophoresis, diphenylamine reaction and flow cytometry analysis (FACS). Results: Stimulation of PMN with G-CSF shows a reduction of the spontaneous DNA fragmentation rate(43.8 ± 5.5%, n=6) by up to 40 % in a dose dependent manner(500U/ml G-CSF: 25.2±5.2%, n=6, p<0.005). A preincubation of G-CSF is fully antagonized by 50U/ml of IL-10 (37.7±3.3%, n=6, p=0.009, against G-CSF(500U/ml) without IL-10). Stimulation with IL-4 also antagonizes G-CSF in a dose-dependent manner although less effectively than IL-10 (5 experiments). IL-10 alone does not produce an increase of apoptosis in unstimulated PMN. Results of DNA fragmentation were confirmed by FACS analysis, which demonstrated that apoptosis rates were decreased from 86% to 53.3 _+2.4% by G-CSF but reverted to 64.3 ± 2.1% by addition of IL-10 (5 experiments, p=0.003) Conclusions: The contrainflammatory cytokine IL-10 seems to play an important antagonistic role in the G-CSF regulation of PMN apoptosis. Because of the importance of apoptosis in the downregulation of inflammatory reactions the IL-10 regulatory effect may be an important mechanism in the initiation of inflammation. Supported by public grants from DFG (SCHR512/1-2) and by MFG. G4159 It:Ba- SERINE-PHOSPHORYLATION IS INCREASED IN THE INTESTINAL LAMINA PROPRIA OF PATIENTS WITH IBD. T. Ktihbacher. S. Nikolaus S. Wedel, H. Lochs, S. Schreiber. Mucosal Immunology Group, 4th Med. Dep. University Hospital CharitY, Berlin. Background: Serine-Phosphorylation of h:Bet at aminoacids 32 and 36 results in the release and nuclear translocation of NFt:B, a transcription factor of pivotal importance in the regulation of inflammation genes. The l~:Bet phosphorylation pathway is activated by inflammatory cytokines and chemokines. The mechanism is not completely understood. It appears that lt:B kinases, NIK-kinase and IL-1R associated kinase play a role. In IBD intestinal biopsies high expression of inflammatory cytokines and activation of NFt:B has been found. Aim: To investigate the degree of phosphorylation of 1KBet in the intestinal mucosa of patients with IBD. Methods: Biopsies from I0 patients with CD, 10 patients with UC and 6 normal controls were obtained during routine colonoscopies from different areas and snap frozen in liquid nitrogen. Diagnosis of IBD was established by histologic and endoscopic criteria. Protein extracts were prepared and immunoprecipitated against It:Bet. The immunoprecipitate was analyzed by Western Blotting using a Ser32 phosphospecific antibody as well as a polyclonal h:Bet antibody. Densitometry was used for semiquantitative assessment of protein bands in comparison with an l~:Bet / phospho-lt:Bct standard. Results: Total 1KBet levels are not different between Crohn's disease, ulcerative colitis and normal control colonic biopsies. A clear association between the histologic intensity (mild, moderate, severe) of intestinal inflammation and status of h:Bet phosphorylation is seen (Fig. 1). Intestinal inflammation in CD / UC is associated with a high degree of 1KBet serine-32phosphorylation (Figl). h:Bet phosphorylation levels appear to be higher in CD than in UC (data not shown). Fig 1: WesternBlot of lxBct immunoprecipitates using a phosphospeci.fic lxBct

antibody, a: severely inflamed b: moderately inflamed

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These results show a remarkable variation in the level of IL-12R~2 expression by mucosal T-cells during the clinical progression of CD: higher in the early

CD a

CD b

UC b

CD a

UC b

NC

Conclusions: Crohn's disease and ulcerative colitis show a high degree of phosphorylation of l•Bet. This data suggest a mechanism for the high level of NFKB activation seen in IBD. The further exploration of mechanisms in lt:B(z phosphorylation is important to understand the complex dysregulation of the lt:B / NF~B system in intestinal inflammation. Supported by public grants from DFG (SCHR512/1-2) and by MFG