The nucleotide receptor antagonist reactive blue inhibited IL-8 production induced by UDP by 77% but did not affect IL-8 release stimulated by IL-1,8. IL-8 production induced by UDP was almost abrogated by the intraceliular calcium chelator BAPTA or by the phospholipase C inhibitor U-73122, supporting the involvement o1 a G protein-coupled receptor. Finally, UOP strongly activated the ERK pathway but not p38 or JNK1 MAP kinases. The MEK inhibitor PD 98059 (which prevents ERK activation) inhibited UDP-induced IL-8 production by 50% whereas the p38 inhibitor SB203580 was inactive. Conclusions: In human monocytic cells, UDP activates IL-8 gene expression via a G protein-coupled P2Y receptor (possibly P2Y6) leading to MEK/ERK-dependent IL-8 gene transcription. UDP appears to be a potent monocytic cell activator that may play an important role in modulating intestinal inflammation in IBD and other enteropathies.

969

The IL-4 Dependent Priming of Human Intestinal Mast Cells to Produce Preferentially Th2 Cytokines Instead of Proinflammatory Cytokinos Is Reversible Axel Lorentz, Mikosch Wilke, Rudolf Raab, Michael P. Manns, Stephan C. Bischoff, Medical Sch of Hannover, Hannover Germany

BACKGROUND: Recently, we reported that human intestinal mast cells cultured in the presence of stem cell factor (SCF) produce proinflammatory cytokines such as IL-6 and TNF-alpha upon stimulation. Supplementation of the cultures with IL-4 induces the production of 1112- type cytokines such as IL-3, IL-5, and IL-13, but suppresses the expression of IL-6. Here, we analyzed whether this IL-4-dependent mast cell priming for Th2 cytokine production is reversible. METHODS: Mast cells isolated from intestinal tissue and purified by magnetic cell separation were cultured for 14 d in the presence or absence of IL-4 (in addition to SCF), harvested, washed and then cultured for additional 14 d in the presence or absence of IL..4. Cytokines were measured at day 14 and 28 of culture. RESULTS: If mast cells have been challenged with IL-4 during the 14 d before the time of cytokine measurement Th2 cytoldnes were detectable whereas IL-6 was not. If the cells were cultured without IL-4 prior to cytoldne measurement, IL-6, but no Th2 cytokines, were found. If the cells were first cultured with IL-4 for 14 d and then without IL-4 for another 14 d mast cells lost their capacity of producing Th2 cytokines but started again to produce IL-6. We showed previously that IL-4 promotes the development of mast cells containing tryptase but not cbymase (MCT subtype). When IL-4 was added to the culture medium the percentage of MCT increased from 40-50 % (day O) to 60-80 % (day 14) and 90-95 % (day 28). If mast cells were cultured first with and then without IL-4 the increase of MCT was discontinued when IL-4 was withdrawn but the MCT/ MCTC ratio was not reversible within 14 d. CONCLUSION: The data show that the IL-4 induced alteration of cytokine profile in mast cells and possibly also the mast cell phenotype defined by protease content are reversible if IL-4 is withdrawn. These observations emphasize that mast cell functions are subjected to a considerable diversity and plasticity depending on the cytokine milieu within the tissue.

970

Virus-Induced Mucosal Smooth Muscle Cell (M-SMC) Hyaluronan (HA) Binds Leukocytes Via A Versinan-Facilitated Mechanism Carol A. De La Mofte, Judith Drazba, Vincent C. Hascall, Scott A. Strong, Cleveland Clin Fdn, Cleveland, OH

Inflammatory bowel disease (IBD) affects genetically susceptible persons in whom triggering factors, including viral agents, initiate interactions between mucosal immune cells, non- immune cells, and extracellular matrix. We have reported that mononuclear leukocytes bind to virus-infected human colon M-SMCs via a novel CD44:HA pathway. HA is produced by activated M-SMCs and integrated into the matrix through HA-binding proteins. Purpose: The aim of the study was to identify the mechanism by which HA is incorporated into the matrix structures responsible for virus-induced M-SMC binding of mononuclear leukocytes. Methods: M-SMCs were derived by enzymatic digestion of colonic mucosa from fresh surgical specimens resected for IBD or non-inflammatory conditions. Concomitantly, adjacent tissue was fixed, paraffin-embedded, and micro-sectioned. Confluent M-SMC cultures were treated with viral mimic (poly I:C), exposed to S~Cr-labeled U937 cells, and assayed for binding. Or, the poly I:C-treated cultures were exposed to U937 cells and fixed. The fixed co-cultures and tissue sections were prepared for immunohistochemical observation, viewed with a confocal micro- scope, and digitally imaged. Results: Confocal microscopy reveals that poly I:C-induced HA is arrayed in small patches on the M-SMC surface or lengtby cahles extending above the surface; the cable structures are the primary leukocyte binding site. Staining also shows that two HA-binding proteins, I~1 and versican, are localized in both patch and cable structures. Pre-treatment of poly I:C-induced M-SMC cultures with blocking antibody specific for lal reduces leukocyte binding by ~35%. However, pre-treatment of the activated M-SMCs with versican monoclonal antibody, or U937 cells with blocking antibody to versican's leukocyte receptor (L-selectin), almost completely (~85%) abrogates poly I:C-induced cell-cell adhesion. Lastly, confocal imaging of IBD tissue sections confirms in vivo up-regulation of HA as well as versican in inflamed mucosa, but not non-inflamed tissue from the same patient. Conclu- sions: Poly I:C-treated M-SMCs express increased amounts of HA with cables of HA emanating from the M-SMC surface responsible for up-regulated leukocyte binding through a dual receptor-ligand interaction involving CD44:HA and L-selectin:versican. A similar phenomenon is seen in vivo that is specific for inflamed IBD mucosa, suggesting these interactions play an important role in the pathogenesis of IBD.

971

The Role of Ca+* in PKC-Medinted Injury to IEC-18 Cells Barry L. Tepperman, Brian D. Soper, Oing Chang, Univ of Western Ontario, London Canada

Background: Protein kinase C (PKC) has been implicated in many cellular signalling pathways. PKC activation has also been associated with tissue injury including colonic inflammation and can compromise the integrity of a number of epithelial cell types including cells of the intestinal mucosa (Am J Physiol 276: G583, 1999; Gastroenterology 116: A674, 1999). In addition,

sustained increases in intracellular calcium (Ca "-) have been shown to disrupt the integrity of gastric mucosal and colonic epithelial cells (Am J Physiol 278: Gl19, 1991; Dig Dis Sci 44: 697,1999). It has also been demonstrated, in a number of cell systems that PKC activation can alter cellular Ca t - homeostasis and increase intracellular Ca* * levels (J Physiol 529: 159, 2000; J Biol Chem 271:21522, 1996). Therefore, in the present study, we have examined whether PKC activation can affect intestinal cellular integrity via alterations in cellular Ca + ÷ levels. Methods: The rat intestinal epithelial cell line, IEC-18, was used in these studies. Cells were incubated for I hr in the presence of the PKC activator, phorboJ myristate acetate (PMA; 0.1-1.0 p.M). In some experiments cells were incubated in medium depleted of Ca ++. Ceil integrity was assessed by trypan blue uptake and intracellular enzyme release. Results: PMA treatment resulted in a concentration-dependent increase in the extent of cell injury and PKC activity in IEC-18 cells. These responses were significantly attenuated by addition of the PKC inhibitor GF 109203X or if Ca'" was removed from the incubation medium. Furthermore, these responses were reduced if cells were incubated in the presence of the intracellular calcium chelator, BAPTA-AM (20 p.M) or the channel blocker, verapamil (1 ~M). Western blot analysis revealed the presence of PKC-a, ,~, ~ and ~ in unstimulated cells, In response to PMA challenge, protein levels for PKC-a and PKC-~ only were increased. In the absence of extracellular Ca • ", only PKC-a protein was reduced. Conclusions: These data suggest that PKC activation via PMA challenge affects the integrity of rat intestinal epithelial cells via changes in calcium homeostasis. PKC-a appears to be the predominant isoform of the enzyme associated with these responses.

972

Mucosal Repair By Growth Factors And Anti-inflammatory Cytokines in Inflammatory Bowel Disease. Kenji ina, Yuji Kuoo, Nagoya Univ, Nagoya Japan; Tshihiro Matsuura, Kazuhiro Kyokane, National Chubu Hosp, Ohbu Japan; Akira Imada, Maseaki Shimeda, Tsuyoshi Nishiwaki, Takafumi Ando, Kazuo Kusugami, Nagoya Univ, Nagoya Japan

Background & Aims: The pathogenesis of inflammatory bowel disease (IBD) may involve mucosal injury and repair, in which epithelial cell restitution and proliferation may be potentially important in the disease process. We investigated the role of soluble mediators on epithelial cell restitution and proliferation in the intestinal mucosa of patients with IBD. Method: Colonic mucosal biopsy tissues were obtained from 37 patients with ulcerative colitis (UC), 21 with Crohn's disease (CD), and 28 controls, and they were cultured on a culture insert for 24 hours. The contents of growth factors (HGF, KGF. bFGF, and VEGF) and anti-inflammatory cytoldoes (IL-4 and IL-IO) in the organ culture supernatant (OCsup) were measured by ELISA. The effects of growth factors, anti-inflammatory cytokines, and OCsup on epithelial cell restitution were analyzed using in vitro wound model of intestinal epithelial cell line (IEC-6), where restitution was assessed by the residual uncovered area at 8 hr after wounding. Cell proliferation of IEC-6 was measured by 3H-thymidine incorporation. Results: Measurable levels of growth factor activity were detected in OCsup, and the inflamed mucosa of both UC and CD patients showed significantly higher contents of HGF, KGF, and bFGF. Although IL-4 activity was totally absent in OCsup, IL-IO activity was detected with an increase in the inflamed mucosa with CD. HGE and IL-IO upregulated epithelial cell restitution to a significant degree, but KGF, bFGF, and VEGF showed less significant or no effects. All these growth factors, but not IL-IO, increased epithelial cell proliferation in a dose-dependent manner. OCsup from the inflamed mucosa of IBD patients showed stimulatory effects on epithelial cell restitution. Inflamed UC OCsup rather suppressed epithelial cell proliferation, compared to control and inflamed CD OCsup. Conclusion: Soluble factors, such as HGF and IL-IO, may contribute to mucosal repair in IBD patients. Furthermore, unknown soluble factor(s) capable of suppressing epithelial cell proliferation, but not restitution, may be involved in the pathogenesis of UC.

Effects of organ culture supernatants on IEC-6 cells

Medium Control Sup CD Sup UC Sup Uncovered area (xlO 3 2 2 . 5 + 0 . 9 18.9±1.8 17.7+1.9 14.7 ± 1.0" Proliferation (cpm) 531 _+ 195 949 ± 84 983 ± 165 240 ± 31"

*p< 0.05

973

Adenosine Induces Polarized Secretion of Interleukin-6 in Intestinal Epithelial Cells: Bidirectional Epithelial/Nentrophil Parecrine Regulation in Model Crypt Abscess. Shanthi V. Sitaraman, Didier Merlin, Lixin Wang, Michelle Wong, Emory University, Atlanta, GA; Andrew T. Gewirtz, Mustapha Si-Tahar, James L. Madara

Background and Aims: Adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5 AMP. In this study we investigated the effect of adenosine on secretion of IL-6, a pro-inflammatory cytokine that is upregulated in patients with inflammatory bowel disease (IBD) and it may play a role in the pathogenesis of IBD. Methods: The model intestinal epithelial cell line, T84, was used to the study the effect of adenosine and other agents on the secretion of IL-6. Results: Stimulation of T84 monolayers with apical or basolateral adenosine induces A2b-receptor- mediated increase in IL-6 secretion which is polarized to the apical (luminal) compartment. In addition, Samonella typhimurium, TNF-~ and forskolin, known inducers of IL-6 secretion in intestinal epithelial cells, also stimulate IL- 6 secretion into the apical media. The secretion of IL-6 begins at 3 hours and is maximal at 6 hours. Exposure to adenosine for 10 minutes is sufficient to stimulate maximal IL-6 secretion. Experiments using various mutants of regulatory elements in the IL-6 promoter show that the induction of IL-6 by adenosine is transcription-dependent and occurs through cAMP- mediated activation of nuclear CREB. Adenosine induces phospho-CREB expression 5 minutes after apical or basolateral stimulation and IL-6 mRNA is seen beginning at 30 minutes after adenosine stimulation. Neither phospho-CREB nor IL-6 mRNA is detected in unstimulated monolayers. We also show that IL-6 released in the luminal (apical) compartment may function to activate luminal neutrophils since IL-6 induced elevated [Ca+ +] in neutrophils.

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