Investigations on the regulation of normal intestinal immunity have described a fundamental property of the intestinal immune system that distinguishes it from other peripheral and tissue responses. "Physiological inflammation" is a state of immune[ regulation in the intestine characterized by hypo-responsive T lymphocytes. This unique, but molecularly undefined _roperty indicates that active tolerance is an essential component of mucosal immunity. We propose that regulated taucosal T cell hypo-responsiveness mediates intestinal immune tolerance, and that unregulated T cell activation i fitiates inflammatory bowel disease (IBD). In striking contrast to peripherial blood T cells (PBT), which are activated vi a the CD3 pathway, intestinal lamina propria T cell (LPT) responses are dominated by an alternate pathway that uses the CD2 receptor. We have developed an in vitro model in which LPT can toggle between a hypo-responsive (tolerant) s:ate and a responsive (protective) state of activation. Using this model we demonstrated that membrane proximal signaling within 2 minutes of activation through the CD3 receptor complex is markedly reduced in tolerant, but not pro ective LPT. We have also described that cholesterol-rich membrane microdomains, called lipid rafts, are not only structtLral components of the plasma membrane, but also localize proteins for the initiation of intra-cellular signal transductio: in PBT. With our expertise in characterizing LPT, isolating lipid rafts, and delineating signal transduction pathways iJ T cells, we are immediately poised to study mucosal T cell tolerance in the normal mucosa and aberrant T cell activat on in the IBD mucosa at a molecular level. Our ability to identify and characterize heterogeneity among lipid raft ct,nstituents in LPT will be combined with molecular, biochemical, and immunological techniques to test the following _:entral hypothesis: Lipid raft heterogeneity on intestinal T cells modulates their signaling, activation, and functi)n, thereby contributing to both normal mucosal immune tolerance and chronic inflammation. Our aims are a, follows: (1) Evaluate the structural mechanisms by which lipid raft heterogeneity modulates positive and negative rq_gulation of the TCR signaling complex in tolerant LPT. (2) Investigate the topological coupling of the cytoskeleton to lipid rafts via interactions through CD2 in LPT. (3) Explore the biochemical regulation of the second messenger Ras-MAPK pathway in lipid rafts from LPT. (4) Examine changes in lipid raft modulation of T cell activation from IBD mucosa. We believe this unique approach will generate important new information on intestinal immune tolerance and IBD, by examining the regulation of membrane proximal events in LPT signaling and how these events chaqge in the IBD T cell. PERFORMANCESITE(S) (organization,city, state) I Case We_stern Reserve University School of Medicine Cleveland, Ohio KEYPERSONNEL.Seeinstructions. Usecontinuationpagesas neededto providethe requiredinformationin theformat shownbelow. Startwith PrincipalInvestigator.Listall otherkey personnelinalphabeticalorder,lastnamefirst. Name Organization RoleonProject Levine, Alan D., Ph.D. Case Western Reserve University P.I. Brady-Kalnay, Susan, Ph.D Case Western Reserve University Consultant Brown, Deborah, Ph.D. State University NY at Stony Brook Consultant Eppell, Steven, Ph.D. Case Western Reserve University Co-Investigator Fiocchi, Claudio, M.D. Case Western Reserve University Co-Investigator Jacobberger, James, Ph.D. Case Western Reserve University Co-Investigator Nieminen, Anna-Liisa, Ph.D. Case Western Reserve University Co-Investigator Pilar, Guillermo Ph.D. Case Western Reserve University Consultant Shaw, Andrey, M.D. Washington University, St. Louis, MO Consultant ! Disclosure Permission Statement. Applicable to SBIR/STTR Only. See instructions. [] Yes [] No / PHS398(Rev.05/01) Page 2 FormPage2 Use1/2-inchMARGINS.Numberpagesconsecutivelyat the bottomthroughoutheapplication.Dono_uttsesuffixessuchas 3a, 3b. PrincipalInvestigator/ProgramDirector(Last,first,middle): Levine, Alan D. Thenameof theprincipalinvestigator/programdirectormust beprovidedat thetop of eachprintedpageandeach continuationpage. Typedensityandsizemustconformto limits andspecificationsprovidedinthe PNS398 Instructions. RESEARCH GRANT TABLE OF CONTENTS PageNumbers Face Page ......................................................................................................................................................... 1 Description,