1004

Interferon-~ Mediates Total Parenterel Nofrition-Associated Loss of Epithelial Barrier Function Hun Yang, Daniel H. Teitelbaum, Univ of Michigan, Ann Arbor, MI

BACKGROUND: Removal of enteral nutrition with the administration of total parenteral nutrition (TPN) is associated with a loss of intestinal epithelial barrier function. Previous work from our laboratory has shown that intraepithelial lymphocytes (IEL) increased interferon-~/mRNA expression in a TPN model, Because interferon-,y (IFN--/) can decrease epithelial barrier function, we assessed the role of IFN--/on epithelial barrier loss in a TPN model. METHODS: Adult C57BIJ6J or mice with a targeted mutation of IFN-~ (C57BL/6-1fng"l=; IFNKO) (N =6/ group) received TPN or enteral diet (Control) for 7 days. Segments of small intestine from mice were then mounted in Ussing chambers. Epithelia[ barrier function was assessed with transepithelial resistance (TER), and transmural passage of 5~[Cr]-EDTA during a 9O-min incubation in Ussing chambers. The passage of 51[Cr]-EDTA was calculated and expressed by apparent permeability coefficient (Papp, 10 e cm/s). IFN-~, protein expression by IEL was detected using intracelluiar staining and flow cytometer. RESULTS: IEL IFN--,/protein expres- sion significantly (p<O.O5) rose in the TPN group (32.6 _+4.2% cells positive for INF-.y) when compared with controls (15.5 _+ 1.8 %). Epithelial barrier permeability significantly rose (increased Papp) with TPN compared with controls (see Table). Permeability in IFNKO TPN mice was significantly lower than in the TPN group. There was no significant difference of intestinal permeability between control and IFNKO control CONCLUSION: TPN results in an increase in both IEL derived IFN--~ expression and epithelial barrier permeability. IFN-~/appears to have an important role in this loss of epithelial barrier associated with TPN. The fact that epithelial barrier function in IFNKO TPN mice did not completely return to baseline suggests that the loss of epithelial barrier function may also be mediated additional factors and will require further investigated.

Group Control TPN INF- 7 KOCont INF-TKO.TPN

TER (O.¢m 2 ) 50.4±5.9* 38.8±3.8 52.6±5.5* 44.2±2.9* Papp(lO-6cm/s) 6.87 ± 1.3" 14.7 ± 4.4 6.4 ± 1.3" 9.2 ± 2.7*

*p<O.05 vs. TPN group.

1005

Alteration Of intestinal Epithelial Functions By Inlraepithelial Lymphocyte Homing. Takeshi Shibahara, Akinori Yanaka, Hideo Suzuki, Tsukuba Univ, Tsukuba Japan; James L. Madara, Emory Univ, Atlanta, GA

BACKGROUND:While intimate cross talk between intestinal epithelial cells and intraepithelial lymphocytea (IEL) play a critical role in mucosal immunity, little is known about the influence of lymphocyte migration into the epithelium, i.e. IEL homing, on epithelial functions. METHOD- S:We analyzed the alteration of epithelial functionsdue to IEL homing utilizing an in vitro IEL homing model consisted of culturedlEL lines and human intestinal epithelial T84 cells / HT29- 19A cells (AJP 1998; 275: G584). The expression of surface molecules (MHC class-I, -II, ICAM-1, -2, -3, integrin/~1, and CD44) on epithetbt celts was anatyzed by flow eytometry. The barrier function of epithelial monolayer was assessed by trans-eplthelial electrical resis- tance. The cytokine production (TNF-,~, TGF-/~, IL-8, GRO-~ and IP-IO) was measured by ELISA and northern blotting. RESULTS: (1) IEL homing induced a significant phenotype switch on epithelial cells- the up-regulation of MHC class I, ICAM-1, integrin ~ and CD44. IEL- derived IFN-.y could partially account for this alteration, since a neutralizing Ab to IFN-'y inhibited the up-regulation of those molecules, except for CD44. (2) A marked fall of trans- epithelial electrical resistance was observed even 4 hours after IEL homing started. A neutraliz- ing Ab to IFN--/(hut not Ab to TNF-~) showed a small inhibition of the fall of resistance. (3) The production of IL-B, GRO-~ and [P-IO (but not TGF-/3 nor TNF-(x) in epithelial monoiayer was markedly enhanced after IEL homing in a basolaterally polarized fashion. While IP-lO production was completely inhibited by a neutralizing Ab to IFN~y, neither Ab to IFN--/nor TNF-~ failed to inhibit I L-8 production. Northern blotting showed that those cytokines induced were chiefly derived from epithelial cells rather than IEL. CONCLUSION: Homed IEL obviously interact with epithelial cells and up-regulate adhesion molecules, alter barrier function, and enhance chemokine production in epithelial cells. Since such alterations might increase the permeability of luminal antigen or accelerate the migration of other inflammatory cells, IEL homing is a critical aspect of mucosal immune responses.

1006

Effects of Proinflammatory Cytokines on Cell Cycle Regulatory Protein Expression in Intestinal Epithelial Cells. Gary E. Wild, Line Dufresne, Chantal Cossette, Kevin A. Waschke, McGill Univ, Montreal Canada; Alan B.R. Thomson, Univ of Alberta, Edmonton Canada

Background: The up-regulation of epithelial cell proliferation is a characteristic feature of intestinal mucosal inflammatory states. This increased proliferative activity may be due to enhanced transition from G1 to S phase of the cell cycle. This is evidenced by induction of cyclin-dependent kinase 2 (Cdk2) activity and decreased expression of the universal inhibitors of the cyclin/Cdk complex, p21 (Wafl/Cipl)and p27 (Kip/Pic2. Aim: In the present study we sought to characterize the effects of the proinflammatory cytokines TNFc~ and IFN.y on the expression of cell cycle regulatory proteins in the Caco-2 cell model of enterocyte differentiation. Methods: Caco-2 cells, prior to attaining confluence, were exposed to a range of TNFc~(IO ng/ml to 100 ng/ml) and IFN~, (1 ng/ml to 200 ng/ml)for 24 hours. RNA abundance of Cdk2, p21 and p27 were measured by rihonuclease protection assay (RPA). The expression of Cdk2 was measured by kinase assay of immunoprecipitates and by immunoblotting; the relative levels of p21 and p27 protein were measured by immunoblotting. Exposure of Coco-2 cells to proinflammatory cytokines resulted in an increased Cdk2 kinase activity and this was seen initially at 10 ng/ml of TNFa or 50 ng/ml of IFN~/; increased concentrations of either TNFc~ or IFN~/did not result in any further increases in Cdk2 activity. The changes in CDk2 activity

seen in the presence of TNF~ or IFN~' were paralleled by corresponding changes in the levels of immunodetectable Cdk2 protein. In addition, exposure of cells to TNFo~ or IFN.y resulted in significant increases in Cdk2 RNA levels as measured by RPA. By contrast the levels of expression of RNA and protein of the universal inhibitors of the cyclin/Cdk complex, p21 and p27 were significantly decreased over the same time period of cytokine exposure. A similar pattem of responses of Cdk,?., p21 and p27 expression to TNF(z and IFN,y were seen in confluent (day 7) and post confluent (day 14 and day 21). As exposure of precorrrluent Coco- 2 cells to sodium butyrate has been previously shown to decrease Cdk2 and increase p21 expression, the effects of butyrate on these parameters were examined here. Incubation of cytokine-treated cells with 4.7 mM sodium butyrate diminished the effects of TNFo~ and IFN~, on Cdk2, p21 and p27 expression. Conclusion: Our data suggest that intestinal epithelial responses to TNF,z and IFN~/are associated with augmented G~ to S transition which is explained, at least in part, by increased Cdk2 expression and decreased expression of the cyclin/Cdk complex.

1007

Butyrate Modulates Cytokine induced ~-1 Preteinase Inhibitor Release in Intestinal Epithelial Ceils Dominik Faust, Sven Hormann, Michael Friedrich-Sander, Born Akoglu, Wolfgang W. Caspary, J W Goethe Univ, Frankfurt Germany; Juergen Stein, 2rid Oept of internal Medicine, Frankfurt Germany

Background: a-1 Proteinase inhibitor (~1 PI) is the main serine proteinase inhibitor in human plasma and is mainly liver-derived. Recent studies show that or1 PI is also synthesized st extrahepatic sites, such as epithelial cells as well as certain cell lines (e.g. Caco-2). Proinflam- matory cytokines seem to regulate local ~1 P] release in epithelial cells. In turn, butyrate regulates colonic epithelial cell differentiation and has antHnfiammatory effects. The aim of this study was to further characterize the mechanisms involved in ~-1 PI release in epithelial cells; we therefore investigated the effects of interleukins (IL)-1/3, IL-6, IL-8, and tumor necrosis factor-(x (TNF-~)in Coco-2 cells treated with butyrate. Methods: Differentiated Caco- 2 cells (day 14) were incubated with IL-1/3, IL-6, IL-8 or TNF-~z for 24h, with and without 2mM butyrate. After treatment the cells were lyzed and a-1 PI expression was detected by Western blot and cell culture supematants were assayed for a-1 PI concentrations by ELISA. Results: Treatment of Caco-2 cells with butyrate alone resulted in a 25% decrease of ~1 expression. In the absence of butyrate IL-1/3 and TNF-(x had no effect on a-1 PI secretion. Only IL-8 enhanced ~1 PI release significantly (2403 _+ 216/.q]/I vs. 1252 _+ 105 in controls; p<O.O01), which was accompanied by a 7O%-increase in lane density when ~-1 PI was detected in the corresponding cells by Western blot. When butyrate was added, IL-8 had no effect on ~1 PI release. In turn, butyrate plus IL-1/3 markedly decreased o,-1 PI expression in Caco-2 cells (75%-decrease in lane density vs. control). Treatment with TNF-<x plus hutyrate resulted in a 35% increase of ~1 PI expression in Caco-2 cells. Incubation with IL-6 had no effect, either in the presence or in the absence of butyrete. Conclusions: Our data imply that butyrate might amplify a-1 PI release after exposure to pro-inflammatory cytokines (IL-1/3, IL-8 and TNF-<x) in Caco-2 cells. Since butyrata is normally present in the colonic lumen, and since the colonic mucosa is exposed to cytokines during intestinal inflammation, it seems likely that imbalance in cytokine expression and decreased ~1 PI release may playa contributory role in ongoing inflammation in the gut.

1008

Differential Regulation of IFN~/and TNFa Production by Lamina Propda Lymphecytes (LPLs) as Evidenced by IL-12-Responsivenoss and Utilization of the C040/CD154 Ioforoction Ellen C. Ebert, Arthur I. Roberts, UMDNJ-Robert Wood Johnson Medical Sch, New Brunswick, NJ

LPLs produce large amounts of IFN,y, particularly in Cmhn's disease. IFN-y levels generally increase with IL-12 and IL-18. This study examines whether IL-12 and IL-18 up-regulate IFN-/ secretion by LPLs. LPLs, derived from normal human jejunal mucosa, were cultured with IL- 2 in the presence or absence of IL-12 or IL-18, stimuli that are likely to be present in vivo. The amount of IFN.y available to neighboring cells was measured by ELISA. LPLs produced more IFN-y than peripheral blood lymphocytea (PBLs) (930_+123 pg/ml for LPLs vs 49_+33 pg/ml for PBLs p<O.01). This up-regulation by LPLs was unlikely to he due heightened production of 11-12 since this cytokine could not be detected in culture supernates and since antibody neutralization of IL-12 did not reduce IFN-/production. Furthermore, supplementing the cultures with exogenous IL-12 substantially increased IFN,y output, indicating that the cultures were not saturated with IL-12. The production of IFN~, may be mediated through one or several interactions: CD2/CD58, CD28/B7, or CD4O/CD154. When antibodies blocking these interactions were added to the cultures, the most striking effect was the 76_+4% reduction in IFN.y output by LPLs after disabling the CD40/CD154 interaction. Surprisingly, TNFtz synthesis by LPLs was unchanged by IL-12 and was independent of CD40/CD154, demonstrating that TNFa synthesis was regulated differently from IFN-y secretion. Also unex- pected was the lack of effect by IL-18 on any of these events. In sum, LPL production of IFN-/was strongly up-regulated by IL-12 and required the CD40/CD154 pathway. Synthesis of TNF~ had neither of these characteristics.

Production of cytokines by IL-2-acSvated LPLs and PBI_s

LPL PBL IFNy 930~123" 66~33 TGFp O* 12+2

tn pg/mL p<O.01

A-191