Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

THE V. PEREZLABORATORYTECHNOLOGISTAndrew Hsia, B.S.COLLABORATORSRobert Fairchild, Ph.D. 1Peter Heeger, M.D. 1Bruce Ksander, Ph.D. 2Ann Marshak-Rothstein, Ph.D. 3Eric Pearlman, Ph.D. 4Luk Van Parijs, Ph.D. 51Dept. of Immunology, CCF2Schepens Eye Res. Inst.,Harvard Med. School,Boston, MA3Boston Univ. Sch. of Med.,Boston, MA4Dept. of World HealthMedicine, Case WesternReserve Univ., Cleveland, OH5Ctr. for Cancer Res., Mass.Inst. of Technol., Cambridge,MAVictor L. Perez, M.D., Ph.D.Immunological Regulation ofInflammatory Responses in the CorneaThe cornea is the structure of the eyeresponsible for allowing the entrance ofexternal images into the eye. Cornealclarity and transparency are the unique qualitiesof this tissue critical for visual function.Moreover, the cornea also serves as a mechanicalbarrier against infections and trauma, and isconsidered part of the bio-defense system of theeye. For these reasons, the immune-regulatorymechanisms used by this immune privilege tissue,are key in maintaining a balance between thesetwo important functions of the cornea. Failureof these mechanisms to control inflammation willresult in uncontrolled corneal inflammation,vascularization, fibrosis and scarring, that lead tosevere functional compromise and even blindness.Common and clinically relevant examples ofwhere the dysregulation of immuno-inflammatoryresponses of the cornea are critical for a goodvisual outcome include; microbial keratitis,herpetic keratitis, contact lensrelated ulcers, autoimmuneulcerative keratitis and cornealtransplant allograft rejection.One major interest in mylaboratory is the understandingof immune-regulatory mechanismsused by the eye during thedevelopment of an immuneresponse in the cornea. To studythis, we have developed an invivo model where animal corneascan be transfected with adenovirusvector containing any gene ofinterested and achieve proteinexpression in the corneal tissue.This technique has allowed us totest the role of differentimmunological molecules in theregulation of corneal inflammation.We have focused ourattention in Fas ligand (FasL), animportant molecule known toregulate immune responses inimmune privilege sites, whichalso can differentially regulate immune responsesin the cornea. Like other members of the TNFfamily, matrix metalloproteinase enzymes cancleave FasL from the cell surface to produce a 26-kD soluble protein. Therefore, FasL can be existsas a membrane-only (non-cleaved) or soluble(cleaved) form. We have used vectors containingthese different forms of FasL to demonstrate thatmembrane and soluble FasL have opposing effectson neutrophils-mediated inflammation in thecornea. Our studies show that the two forms ofFasL have opposite regulatory roles in the cornea:(i) membrane-only FasL initiates innate immunityand causes keratitis, and (ii) soluble FasLterminates innate immunity, protecting the corneafrom an uncontrolled inflammatory response thatwould jeopardize the function and integrity ofthe cornea. We believe that these two moleculeswill allow us to understand better the mechanismsof immune-regulation used by the corneaand could also represent a new modality oftreatment for inflammatory diseases of the eye.The other area of interest studied in mylaboratory the use of the eye to visualize in vivoimmune responses. The eye provides a uniquewindow into the body. Its translucent natureallows the visualization of events, such as cellgrowth, cell death, migration, and transformationas they occur in vivo, in a non-invasive manner.We are establishing new experimental systems tomonitor gene expression and cellular migration inresponse to inflammation and other injuries in theeye. Using a retrovirus-based technique togenerate mice that express GFP in the majorityof hematopoietic cells, we can detect GFPpositive cells infiltrate the cornea of live micewithin 60 minutes after the induction ofinflammation. Using in vivo fluorescent biomicroscopywith digital imaging system, real timeimages of cellular interaction in the cornea willbe capture for analysis. This technique will allowus to characterize in vivo the migration patternand interaction of inflammatory cells with othercells in the cornea.Lu, M., Perez, V.L., Ma, N., Miyamoto, K., Peng, H.B., Liao, J.K., and A.P. Adamis (1999) VEGF increasesretinal vascular ICAM-1 expression in vivo. Invest. Ophthalmol. Vis. Sci. 40:1808-1812.Perez, V.L., Biuckians, A.J., and J.W. Streilein (2000) In-vivo impaired T helper 1 cell development insubmandibular lymph nodes due to IL-12 deficiency following antigen injection into the anterior chamberof the eye. Ocul. Immunol. Inflamm. 8:9-24.Perez, V.L., and C.S. Foster (2001) Uveitis with neurological manifestations. Int. Ophthalmol. Clin.41:41-59.Segall, A.I., Vitale, M.F., Perez, V.L., and M.T. Pizzorno (2003) HPLC analysis of 5H-benzo[a]carbazolewith antifungal activity. J. Pharm. Biomed. Anal. 31:1021-1026.Thakker, M.M., Perez, V.L., Moulin, A., Cremers, S.L., and C.S. Foster (2003) Multifocal nodular episcleritisand scleritis with undiagnosed Hodgkin’s lymphoma. Ophthalmology 110:1057-1060.166