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In vitro modulation of classic II MHC antigen expression by human cerebral endothelium and endothelial.. Huynh, Hanh Kim 1994-04-08

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IN VITRO MODULATION OF CLASS II MHC ANTIGEN EXPRESSION BYHUMAN CEREBRAL ENDOTHELIUM AND ENDOTHELIAL CELL -LYMPHOCYTE INTERACTIONS BY INTERFERONS y ANDibyHANH KIM HUYNHM.Sc., Cell Biology, The University of British Columbia, 1989A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFINTERDISCIPLINARY DOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Departments of Neuroscience and Pathology)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAApril 1994© Hanh Kim Huynh, 1994In presenting this thesisin partial fulfilmentof the requirementsfor an advanceddegree at the University ofBritish Columbia, Iagree that theLibrary shall makeitfreely available for referenceand study. I furtheragree that permissionfor extensivecopying of this thesisfor scholarly purposes maybe granted bythe head of mydepartment or by hisor her representatives.It is understoodthat copying orpublication of this thesisfor financial gain shall notbe allowed withoutmy writtenpermission.Department5 of i6t S(:iFCEz/The University of British ColumbiaVancouver, CanadaDate/qDE-6 (2/88)11ABSTRACTIn autoimmune demyelinating disorders of the central nervoussystem (CNS), theblood-brain-barrier (BBB) becomes permeable to plasma proteinsand circulating leukocytes.Studies on Multiple Sclerosis (MS) suggest the involvement ofT4+lymphocytes andIa+macrophages in lesion extension and demyelination. Circulatinglymphocytes recognizeantigen only when it is complexed with Ia molecules on the surfaceof antigen presenting cells.Recent studies aiming at defining cell populations in the CNS capableof expressing Ia antigen(Ia Ag) indicate Ia Ag expression by EC, astrocytes, microglia and macrophagesin MSlesions, while in experimental allergic encephalomyelitis, Ia Ag expressionby brainendothelium has been reported by some, but not other investigators. Therole of cerebralendothelium in CNS inflammation, therefore, remains ill defined and rather controversial.The objective of this thesis was to investigate the:1) Induction of Ia Ag on humanbrain microvessel endothelial cells (HBMEC) treated with interferons (IFN)y and , and 2)modulation of T-lymphocyte adhesion and migration across HBMECmonolayers by IFN-yand . To address these issues, an in vitro model of the humanBBB was utilized. The resultsindicate that HBMEC do not constitutively express Ta Ag.Treatment with IFN-y, not 3, resultsin de novo, polarized expression of Ia Ag. Co-incubation withIFN-13downregulates the IFNy-induced Ia Ag expression. Treatment with IFN-y, not1,induces morphological andfunctional changes of HBMEC associated with increased permeabilityto macromolecules.IFN-y induced changes do not appear in cultures incubated withboth cytokines.Treatment of HBMEC with IFN-y upregulates the adhesionand migration of restingand activated T lymphocytes across the monolayers. T-lymphocyteactivation alone greatlyaugments both processes. Treatment with IFN-f3has no effect on lymphocyteadhesion/migration, however, the IFN-y-mediatedincrease in adhesion/migration is111suppressed by IFN-3. MAb blocking studiessuggest a direct role of Ta moleculesinadhesion/migration.These studies indicate a potentially importantrole of HBMEC in CNS inflammationand increase our understanding of someof the factors involved in the recruitment oflymphocytes into chronic inflammatory sitesin the CNS. Therapeutic interventions utilizingmAbs against HLA-DR molecules as well asthe use of cytokines, such as TFN-3, that cansuppress TFN-y-mediated responses,may have considerable therapeutic potential.ivTABLE OF CONTENTSAbstract iiTable of Contents ivList of Table viiiList of Figures ixList of Abbreviations .xiiiAcknowledgements xviDedication xviiCHAPTER ONE INTRODUCTION1.1 Inflammation 11.1.1 Definition 11.1.2 General aspects 21.1.3 Role of EC 41.2 Interferons 71.2.1 IFN-y 81.2.2 IFN-3 91.3 Major histocompatibility molecules 91.3.1 Definition 91.3.2 Class IMHC 101.3.3 Class II MHC 10a) Role in autoimmune disorder 11b) Expression by EC 121.4 Central nervous system inflammation and autoimmunity. . . .151.4.1 Permeability of the Blood-Brain-Barrier 151.4.2 Lymphocyte infiltration 17a) Ia Ag association 17b) Lymphocyte-EC adhesion 18c) Transendothelial migration of lymphocytes 201.4.3 FVIIIR:Ag in primary cultures of HBMEC 211.5 Summary and objectives 24CHAPTER TWO MATERIALS AND METHODS2.1 Isolation and culture of HBMEC 272.2 Antibodies 282.3 Induction of Ia Ag expression on HBMEC by IFN-y and 3. . .292.3.1 Treatment of primary HBMEC cultures 29V2.3.2 Light microscopic immunocytochemicallocalization of Ia Ag 302.3.3 Immunoelectron microscopy 312.3.4 Enzyme linked immunosorbent assay (ELISA) 322.3.5 Quantitation of Ta Ag expression by HBMEC 322.4 Scanning electron microscopy 332.5 Permeability studies 332.6 Growth studies 342.7 Preparation of T lymphocytes 352.8 Activation of T cells 362.9 HBMEC-lymphocyte adhesion assay 372.10 HBMEC-lymphocyte migration assay 382.11 Monoclonal antibody-blocking studies 392.12 Quantitation of lymphocyte adhesion and migration 402.13 Transmission electron microscopy 402.14 Localization of FVIIIR:Ag in HBMEC 412.14.1 In vitro drug treatment of EC 412.14.2 Immunoelectron microscopy for FVIIIR:Ag 412.15 Statistical analysis 42CHAPTER THREE RESULTS3.1 Human brain microvessel endothelial cell 433.2 Immunocytochemical localization of FVIIIR:Ag 443.3 Induction of Ia Ag expression on primary cultures of HBMEC 453.3.1 Effects of recombinant human TFN-y 453.3.2 Effects of recombinant human IFN- 463.4 Kinetics of the downregulation of Ta Ag expression by IFN- .473.5 Effects of IFN-y andIFN-13on cell morphology,organization and growth 493.5.1 IFN-y 493.5.2IFN-13 503.6 Permeability of HBMEC monolayers 50vi3.7 Lymphocyte characterization 513.8 Human T lymphocyte adhesion to untreatedand cytokine-stimulated HBMEC .523.9 Adhesion of activated T-lymphocytes to untreatedand cytokine-stimulated HBMEC .533.10 Effects of blocking antibodies on lymphocyte adhesion 553.11 Transendothelial migration of resting T lymphocytes 553.12 Migration of activated T-lymphocytes acrossuntreated and cytokine treated HBMEC monolayers 573.13 Effects of blocking antibodies on lymphocyte migration. . . . 583.14 Effects of calcium ionophore A23 187, EGTA and IFN-y onthe constitutive pathway of factor VIIIR:Ag release 58CHAPTER FOUR DISCUSSION4.1 Influence of cytokines on Ia Ag expression on HBMEC. . . . 604.1.1 Human brain microvessel EC 604.1.2 Induction of Ia Ag expressionon primary cultures of HBMEC 604.1.3 Surface localization of Ia Ag on HBMEC 624.1.4 Effects ofIFN-13on Ia Ag expression by HBMEC. . .634.1.5 Regulatory mechanism of Ia Ag expression 644.1.6 Kinetic studies on the modulation of Ia Agexpression by interferons y and fE 644.2 Effects of interferons y and 3 on the morphologicalphenotype and growth of HBMEC, organization of themonolayers and permeability to macromolecules 664.2.1 Effects of IFN-y and IFN-f3 on HBMEC growth 674.2.2 Effects of interferons y and1on HBMECmorphology and organization of the EC monolayers. 674.2.3 Permeability of IFN-y treated HBMEC monolayersto macromolecules 694.3 Significance of Ia Ag expression on HBMEC 704.4 Adhesion of resting and anti-CD3 stimulated lymphocytes tountreated, IFN-y and/orIFN-13treated HBMEC 724.4.1 Activation of lymphocytes 724.4.2 Adhesion of resting lymphocytes to untreated,IFN-y and/or IFN-f3 treated HBMEC 734.4.3 Adhesion of activated lymphocytes to untreated,IFN-y and/orIFN-13treated HBMEC 78vii4.5 Migration of restingand anti-CD3 stimulatedlymphocytesacross untreated and cytokinetreated HBMEC844.5.1 Migration of resting lymphocytesacross untreated,IFN-y and/or IFN-3 treated HBMECmonolayers. . . . 844.5.2 Migration of anti-CD3stimulated lymphocytes acrossuntreated, IFN-yand/or IFN-3 treated HBMECmonolayers864.6 Effects of IFN-’y on thestorage and releaseof FVIIIR:Ag from HBMECin primary culture 914.6.1 Immunocytochemical localizationof FVIIIR:Agin HBMEC914.6.2 Effects of IFN-yon the storage and/or releaseof FVIIIR:Ag from HBMEC93CHAPTER FIVE CONCLUSIONS5.1 Summary and conclusions955.2 Future prospects985.3 Significance of this thesis101TABLE103FIGURES104REFERENCES214vii’LIST OF TABLETable 1. Permeabilityof HBMEC Monolayers to HRP: Quantitationof endocytosisand tight junction permeabilityof untreated and IFN-y-treated HBMEC103ixLIST OF FIGURESFigure 1. Diagram of the longitudinalsection of the double chemotacticculturing chamber104Figure 2. Primary cultures of HBMEC grownon plastic wells or cellagen discsform confluent monolayers106Figure 3. Immunoperoxidase stainingfor FVIIIR:Ag and UEA I confirming theendothelial origin of primary culturesof HBMEC108Figure 4. Ultrastructural studies demonstratingthe elongated morphology of ECwith their overlapping processes110Figure 5. Ultrastructural demonstrationof the pentalaminar configurationcharacteristic of the tight junctionsthat are present in areas ofcell to cell contact (a-d)112Figure 6. Cytoplasmic morphologyof an untreated HBMEC (a, b)117Figure 7. Immunocytochemicallocalization of FVIIIR:Ag in untreated EC(a-d) 120Figure 8. Time course of IaAg induction on HBMEC by IFN-y122Figure 9. Dose - responseof Ia Ag induction by IFN-y on HBMEC124Figure 10. Ia antigen expressionby HBMEC detected by immunogold silverstainingin untreated/IFN-y treated ECat various times and also in culturescoincubated with anti-IFN-yantibody126Figure 11. Immunogoldstaining of HBMEC for the demonstrationof Ia antigen inuntreated/IFN-y treated EC128Figure 12. Ia antigen expressionby HBMEC detected by immunogold silverstainingin cells treated with IFN-f3 alone ora combination of IFN-(3 andy 130Figure 13. Dose-responseof Ia Ag expression by HBMEC treated withIFN-y and/or IFN-3132xFigure 14. Effects of different treatments ofIFN-y and 3 on Ia Agexpression by HBMEC134Figure 15. Quantitation by ELISA of Ia Ag expressionby HBMEC treated withIFN-y and/or IFN-f136Figure 16. Phase contrast microscopicdemonstration of morphological alterationinduced by IFN-y treatment of confluentcultures of HBMEC 138Figure 17. SEM demonstration of morphologicalalteration induced byIFN-y treatment of confluent cultures of HBMEC140Figure 18. Effects of IFN-y andupon the growth of primary cultures of HBMEC. . ..142Figure 19. SEM of HBMEC grownin the presence ofIFN-13alone (a) or a combinationof IFN-(3 and y (b)144Figure 20. HRP localization in untreated (A) and(B) and IFN-y-treated (C-F)confluent HBMEC monolayers147Figure 21. Expression of IL-2R on resting and anti-CD3stimulated T lymphocytes . . . 151Figure 22. Immunoperoxidase stainingfor the demonstration of resting T lymphocyteand untreated HBMEC adhesion153Figure 23. Adhesion of resting lymphocytes toIFN-y treated HBMEC 155Figure 24. Adhesion of resting lymphocytes toIFN-f3 treated HBMEC 157Figure 25. Adhesion of resting lymphocytesto untreated andcytokine-treated HBMEC159Figure 26. SEM of adhesion ofresting T lymphocytes to untreated HBMEC161Figure 27. SEM of adhesion of restingT lymphocytes to IFN-y treated HBMEC166Figure 28. Adhesion of anti-CD3 stimulatedT lymphocytes to untreated HBMEC. . .. 169xiFigure 29. Adhesion of anti-CD3 stimulated T lymphocytes to IFN-y treated HBMEC. .171Figure 30. Adhesion of anti-CD3 stimulated T lymphocytes toIFN-y and 3 treated HBMEC 173Figure 31. Summary bar graph of adhesion of anti-CD3 activated lymphocytesto untreated and cytokine-treated HBMEC 175Figure 32. SEM demonstration of adhesion of anti-CD3 activated lymphocytesto untreated and cytokine-treated HBMEC 177Figure 33. Adhesion of stimulated lymphocytes to untreated HBMEC 181Figure 34. Adhesion of stimulated T cells to untreated HBMEC 183Figure 35. Light micrograph of adhesion of resting lymphocytes toIFN-y and anti-human HLA-DR treated HBMEC 185Figure 36. Light micrograph of adhesion of anti-CD3 stimulated lymphocytes toIFN-y and anti-human HLA-DR treated HBMEC 187Figure 37. Migration of resting lymphocytes across untreated and cytokinetreated HBMEC 189Figure 38. Summary bar graph of migration of resting and anti-CD3 activatedT cells across untreated and IFN-y and/orIFN-13treated HBMEC 191Figure 39. TEM examination of migration of resting T lymphocytes acrossuntreated HBMEC 193Figure 40. TEM examination of transendothelial migration of restingT lymphocytes across untreated HBMEC 198Figure 41. TEM examination of the integrity of the EC monolayers at the endof migration of resting lymphocytes across IFN-y treated HBMEC 200Figure 42. Light microscopic examination of migration of activated lymphocytesacross untreated HBMEC 203xiiFigure 43. TEM examination of migration of anti-CD3 stimulated lymphocytesacross untreated and IFN-y treated HBMEC205Figure 44. TEM examination of migration of anti-CD3 stimulated lymphocytesacross IFN-y treated HBMEC208Figure 45. Immunocytochemical localization of FVIIIR:Ag in chemical,cytokine-treated HBMEC210Figure 46. Summary bar graph of the immunocytochemical localizationof FVIIIR:Agin HBMEC212xliiLIST OF ABBREVIATIONSAD Autoimmune DisorderBBB Blood-brain-barrierBSA Bovine Serum AlbumincAMP Cyclic Adenosine MonophosphateCD3 Cluster of Differentiation 3CNS Central Nervous Systemcon A Concanavalin ACSF Cerebrospinal FluidEAE Experimental Allergic/Autoimmune EncephalomyelitisEAN Experimental AutoimmuneNeuritisEAU Experimental Autoimmune UveoretinitisEC Endothelial CellEGTA Ethyleneglycol-tetraacetic acidELISA Enzyme Linked ImmunosorbentAssayFACS Fluorescence Activated Cell SorterFCS Fetal Calf SerumFVIIIR:Ag Factor VIII related antigenGAMIgG Goat anti-mouse immunoglobulinGHBMEC Human Brain MicrovesselEndothelial CellxivHBSS Hanks’ Balanced Salt SolutionHDMEC Human Dermal Microvessel Endothelial CellHLA Human Leucocyte AntigenHRP Horseradish peroxidaseHS Horse SerumHUVEC Human Umbilical Vein Endothelial CellIa Ag Immune associated antigenICAM-1 Intercellular adhesion molecule-iIFN- Human recombinant interferon-betaIFN-y Human recombinant interferon-gammaIFNs InterferonsIgG Immunoglobulin G isotypeIL-i Interleukin-iIL-2 Interleukin-2IL-2R Interleukin-2 receptorLCA Leucocyte common antigenLFA- 1 Lymphocyte Function-associatedAntigen-imAb monoclonal antibodyMHC Major Histocompatibility complexmRNA messenger ribonucleic acidMS Multiple SclerosisxvNGS Normal Goat SerumPBS Phosphate buffered salinePECAM-1 Platelet/endothelial cell adhesion molecule-iRNA Ribonucleic acidSEM Scanning Electron MicroscopyTCR T cell receptorTEM Transmission Electron MicroscopyTNF-a Tumor necrosis factor-alphaUEA-I Ulex Europaeus type IVCAM-i Vascular cell adhesionmolecule-iVLA-4 Very late antigen-4xviACKNOWLEDGEMENTSI would like to thank my supervisor Dr. K. Dorovini-Zis for her guidance, supportand interestin my thesis, Mrs. R. Prameya for plating the EC, Donald Wong for his computer advice,Ms.Vivian Wu and Yolanda Bouwman for helping with the lymphocyte characterization.Thankyou all very much.xviiDEDICATIONI would like to dedicate this thesis to my supervisor, my mentor, DR. K. DOROVINI-ZIS,for her guidance, supervision, encouragement and patienceduring my Ph.D. training inNeuropathology Research, and to my very dear Mom (Huynh Thi Ngoc Suong),Dad (HuynhVan Tu), Brother (Huynh Kim Huu and family) and Sisters (HuynhThi Kim Lien, HuynhThi Tuyet Mai, Huynh Thi My Dung, Huynh Thi Hoang Yen,Huynh Thi Hoang Anh and theirfamily members) for their sacrifice, their support and their belief in me, andmost importantly,to my loving Mom and Dad who always believe that educationis the best gift that any parentscan give to their children, and last but not least to God for allof the blessings that I have beengiven. Thank you all for helping me through this.1INTRODUCTION1.1 INFLAMMATION1.1.1 DefinitionInflammation is a localizedprotective response whichoccurs as a defensivemechanism against the invasionof the host by foreign material, frequentlymicrobial in nature.Responses to toxins, neoplasms,and mechanical traumamay also result in inflammatoryreactions. Inflammation serves todestroy, dilute, or wall-off boththe injurious agent and theinjured tissue. The symptoms ofinflammation include redness(rubor), swelling (tumor),pain(dolor), heat (calor), and loss of function(functio laesa).According to Julius Friedrich Cohnheim,a pathologist in the late 1800’s, thefirst foursymptoms (redness, swelling,pain and heat) represent thecardinal signs of acuteinflammation, while the functionalloss (functio laesa) isin reality a resulting condition.Hedescribed “redness” as theoverloading of all blood vessels,“swelling” due to theincreasedvascular flilness, especiallythe great increase of transudation,“pain” due to the pressureon,and dragging of, the nervesof sensation by the overfilled vesselsand abundant transudation,and finally “heat” resulting froma more than normal amount of heatsupplied to the site fromwithin by the increasedblood supply (1).Inflammatory processesplay a central role in mediatingimmune host defense andwound healing, but unfortunately,they also participate in thepathogenesis of many diseases,e.g. allograft rejection. Informationconcerning the mechanismswhereby inflammatory cells2accumulate in tissues,as well as the mechanismswhereby such cellsare stimulated to damagetissues, should providebetter insightinto the pathogenesisof human diseasesand should alsoprovide clues for developingmore rational formsof therapy (2,3).1.1.2 General aspectsAs first witnessedby Cohnheim (1), and laterby Clark in 1935(4), using intravitalmicroscopy, the initialevent in leukocytelocalization to sitesof inflammation iscalled“margination”, wherebyleukocytes leave thecentral streamof blood flow in post-capillaryvenules. These leukocytesthen interact withthe endothelium liningthe vessel wallby“rolling” along theluminal surface, aprocess which occurswithin minutesof theinflammatory stimulus.As inflammation progresses,the number ofrolling leukocytesincreases along witha decrease in their velocity,and the process finallycomes to a halt.Consequently, “diapedesis”will take place,a process whereby the leukocytesmigrate throughthe endothelial cell(EC) junctions along thevessel wall and intothe tissues. The accumulationand subsequent activationof leukocytes are centralevents in the pathogenesisof virtually allforms of inflammation.The recruitment ofhumoral and cellularcomponents of theimmunesystem leads to theamplification and propagationof most forms of inflammation.Immunologically-mediatedelimination offoreign material proceedsthrough a seriesof steps. Firstly, thematerial to beeliminated (i.e. antigen)is recognized as being“foreign”by either specificor non-specific means:a) Specific recognitionis mediated byimmunoglobulins (i.e.antibodies) or byT cell receptors whichbind to specific determinants3(i.e. epitopes) on the antigen. b) Non-specificforms of recognition, suchas recognition ofdenatured proteins or endotoxins,can be mediated directly bythe alternative complementpathway or by phagocytes. Secondly,the binding of a recognition componentof the immunesystem to an antigen generally leadsto activation of an amplification system,initiatingproduction of proinflammatory substances.These mediators then, in turn,will alter the bloodflow, increase vascular permeability, augmentadherence of circulating leukocytesto vascularendothelium, promote migrationof leukocytes into the tissues, and stimulateleukocytes todestroy the inciting agent. The productionof inflammatory mediators which leadstoalterations of the normal functionof the blood vessels had been suggestedby Cohnheim morethan one hundred years ago based onhis research in inflammation. He speculatedthat ininflammation, a chemical changemust occur in the vessels to inducethe characteristiccirculatory disturbances that he observed(1).The actual destruction of antigensby immune mechanisms is mediatedby phagocyticcells. These cells may migrate freelyor may exist at the fixed tissue sitesas components of themononuclear phagocytic system. Macrophagesand related cells (e.g. Kupffer cells,type-Asynovial lining cells) are the central componentsof this system. Destruction ofantigensoutside of the mononuclear phagocyticsystem generally takes place intissue spaces and ismediated by polymorphonuclearleukocytes (neutrophils) or monocytes, whichare recruitedfrom blood.41.1.3 Role of endothelial cellsEndothelial cells (EC) are strategically locatedbetween the intravascular elements andthe parenchyma of every organ. Therefore,it appears reasonable to assume that,in addition toforming this crucial boundary, EC may participatein a number of important physiologic roles.However, in the past, the role of EC in inflammatoryprocesses had primarily been consideredto be passive. It was not untila series of breakthroughs occurred which allowedfor theisolation and subsequent culture of EC in vitro thatsubstantive questions could be addressedin a stepwise manner. Most of the information aboutthe structure and function of humanECcomes from studies of human umbilical veinEC (HUVEC) because these cells are relativelyeasy to obtain, isolate and culture. These studies haveprovided convincing evidence that ECnot only provide a nonthrombogenic surface tothe intravascular compartment but alsoperform a host of other functions. These include woundhealing, angiogenesis, production ofclotting factors, tumor metastasis, cytokine production, leukocytetrafficking, vascular tone,and many others. Consequently, far frombeing either passive elements in physiologicprocesses or non-participants in pathologic processes,it is now realized that EC are intimatelyinvolved in inflammation and help to create, modulate,and terminate inflammation. In vitrostudies of cytokine effects on EC have provided muchof the information regarding the role ofEC in inflammation (5).It is interesting to note that the observationmade by Cohnheim (1) more than onehundred years ago led to his remark,ttwehave here to deal with a molecular changeof thevessel walls”, and it took almost a century to establishthe molecular basis for leukocyte5interaction with endothelium.An early event in inflammation involvesthe adhesion ofpolymorphonuclear leukocytes(i.e. neutrophils) to the blood vesselwall (i.e. EC) which isacrucial step leading to leukocyteaccumulation within the inflammatorysite. EC exposed toagents such as histamine or thrombincan express signals (e.g. granulemembrane protein-140and platelet activating factor) which attractneutrophils and other blood leukocytesto leave themain vascular stream and marginatealong the vessel wall (6). Withinminutes of thistriggering, a phenomenon called11rolling” which involves leucocyticattachment to, anddetachment from endothelium isinitiated. During the initial stages ofadhesion, the neutrophilscome in close contact with theendothelium and extend pseudopodiathat attach to theendothelial surface or are directedtoward interendothelial junctions.The dynamic interactionbetween different adhesion moleculesexpressed by endothelium and bloodleukocytes playsan important role in leucocytic entry intotissues. The migration processstarts by insertion ofa portion of the cytoplasm betweentwo adjacent EC, followed bythe movement of the entirecell across the monolayer (7).It is still unclear how exactlythe neutrophils force theinterendothelial junctions apartand what molecular mechanisms areresponsible for thedisassembly and resealingof the tight junctions. It has beenshown that various cytokinesparticipate in this process,and interleukin-1 (IL-i), tumornecrosis factor-a (TNF-ct),andinterferon-y (IFN-y) areamong the most potent inflammatoryagents (8).Interleukin-1 and tumornecrosis factor appear to play pivotalroles in leukocyteendothelial adhesion. Theyact on neutrophils, renderingthem more??sticky??than normal.Through separate sets ofmechanisms, interleukin-1 and tumornecrosis factor also act onEC,6rendering these cells more adhesivefor neutrophils, monocytesand lymphocytes (9 - 11). Theeffects of IL-i and TNF-ct onEC can be blocked by RNAand protein synthesis inhibitors (10,11).Interleukin-1 is synthesizedby macrophages, microglia and astrocytes.Its effects includechemotaxis, induction of increased adherence,enhanced vascular permeability,stimulation ofthe release of platelet-activatingfactor and prostacyclin byEC (12, 13). Tumor necrosisfactor-a is synthesized predominantlyby macrophages, but alsoby T cells, astrocytes andmicroglia cells. This cytokine hasbeen linked to the inflammatory demyelinatingprocess ofexperimental autoimmune neuritis(EAN), experimental autoimmuneencephalomyelitis(EAE), and multiple sclerosis (MS)(8). MS is a chronic inflammatorydisease involvingdemyelinization of the central nervoussystem, while EAE is ananimal model for MS.Interferon-y, predominantly producedby CD4 T lymphocytes of the T helperinflammatoryphenotype, also exerts a multitudeof inflammatory effects. This cytokineis the most potentinducer or upmodulator of themajor histocompatibility classII molecules (MHC Class II), andit also enhances vascular permeability.The most convincing evidencethat IFN-y plays animportant role in inflammatorydemyelination arises from a clinicaltrial in which the systemicadministration of IFN-y to MS patientsresulted in clinical exacerbations.These exacerbationswere accompanied by increased numbersof monocytes expressing Human LeucocyteAntigen(HLA), especially HLA-DR subtype,enhanced proliferative responsesof peripheral blood Tcells and natural killer cell activity(15).Although many valuable observationshave been made duringin vitro studies of7human large-vessel EC, most physiologicand pathophysiologic events takeplace at the levelof the microvasculature. Furthermore,emerging evidence suggests that selecteddifferencesexist between BC of the microvasculatureand those that line the large blood vesselsas well asbetween BC from different vascularbeds. These include differences inmorphology, insecreted products, in expression of cell adhesionmolecules, in cytokine-induced regulationofcommonly expressed cell adhesion molecules,and in response to BC injury(5, 16 - 19).Subsequently, it is uncertain whetherthe observations on large vessel ECof other organs alsoapply to the human brain microvascularBC (HBMEC), so that the results shouldnot beextrapolated from one system to theother.1.2 INTERFERONSThe interferons (IFNs) representa group of glycoproteins discovered in1957 asbiological agents interfering withthe replication of viruses, hence the name“Interferon” (20).The IFNs can be producedby all nucleated cells and can be classifiedas cytokines. They aremultifunctional and are componentsof the host defenses against viral and parasiticinfections(e.g. chronic infection with hepatitisB and C viruses), and also certain tumors(e.g. hairy cellleukemia, Kaposi’s sarcoma, non-Hodgkin’slymphoma). They influence thefunctioning of theimmune system in various waysand also affect cell proliferation anddifferentiation. The IFNsexert their multiple activitiesprimarily by inducing the synthesisof many proteins (21). TheIFNs were originally classifiedby their sources as leucocyte, fibroblastand immune IFNs.Leucocyte and fibroblast IFNs,together, were also categorized astype 1 IFNs and immune8IFN as type 2 IFN. For the time being, the nomenclatureis based on sequencing data.Leucocyte IFNs as IFN-a and o, fibroblast IFN as IFN-f3, and immuneIFN as IFN-y.IFN-y will be examined in this study because of its role in inducing Ia Ag expression(22), activating BC to bind T lymphocytes (23) and in enhancing the migrationof lymphocytesacross EC monolayers (24). The effects ofIFN-13will also be determined because of its role indownregulating the effects seen with IFN-y (25 - 27) and more importantly, becauseof itspotential therapeutic application in treating autoimmune disorders of the CNS such as MS(28,282).1.2.1 IFN-yThe gene coding for IFN-y has been located on chromosome 12, and the matureIFN-y is made of 166 amino acids (29). IFN-y was initially consideredto be a secreted productexclusively of T lymphocytes, especially of the T helper subset.However, this interpretationwas later recognized to be too restrictive. Many cytolyticT cells also release IFN-y uponcontact with the specific target, such as virally infectedsyngeneic cells (30, 31). Natural killercells have also been shown to be another source of IFN-y whenthey are exposed to targetcells, interleukin-2 (IL-2) (32), or hydrogen peroxide (33).It is now recognized that the most important immunomodulatoryIFN is IFN-y. The proteinsinduced by IFN-y comprise those encoded by the major histocompatibilitycomplex class I andespecially class II regions. These proteins are involvedmainly in the processing, transport, andcell surface presentation of antigens, and also in cell to cell recognition(21).91.2.2 IFN-The gene coding for this cytokine has been located on the short arm of chromosome 9,and matureIFN-13is comprised of approximately 165 - 172 amino acids long (29). IFN- isexpressed exclusively by fibroblasts and it is induced essentially by viruses or double-strandedRNAs (34). As mentioned previously, the IFNs (i.e. IFN-a, j3, and y) were originally identifiedas antiviral proteins. Accumulating evidence, however, indicates thatIFN-13also plays animportant role in the control of cell growth and differentiation (35, 36). Together with IFN-y,IFN-f3 produces synergistic and antiproliferative activities (37). In contrast, theimmunomodulatory effects ofIFN-13include its capability to inhibit or down-regulate IFN-yproduction in MS (38, 39), to antagonize the Ia-inducing effect of IFN-y in vitro (40, 41) andto augment suppressor cell function in MS patients (42).1.3 MAJOR HISTOCOMPATIBILITY MOLECULES1.3.1 DefinitionThe major histocompatibility complex (MHC) molecules are proteins discovered bythe British geneticist Peter Gorer and by George D. Snell of Jackson Laboratory in BarHarbor, ME, as the cause of graft rejection. Their long-winded names are derived from theGreek word for tissues (histo) and the ability to get along (compatibility). These MHCmolecules can be categorized into two classes: class I and class II MHC molecules(43).101.3.2 Class IMHCThese molecules can be found in almost all types of body cells. They aretransportingproteins which are synthesized in the endoplasmic reticulum,and each MHC molecule has adeep groove into which a short peptide, or protein fragment,can bind. Class I MHC moleculesbind to peptides that orginate from proteins in the cytosolic compartmentof the cell, however,they can only hold short peptides because their bindingsite is closed off. This class 1-peptidecomplex is then transported to the cell surface, and if thepeptide is foreign to the cell, it willbe recognized by the passing T cells. These immune cells willrelease lymphotoxins or otherstructurally related molecules that destroy the cell presentingthe peptide. These T cells arereferred to as killer T cells (43).1.3.3 Class IIMHCClass II MHC or human leucocyte antigens (e.g. HLA-DR, HLA-DQ,HLA-DP) aredistributed on the cell surface as c43 heterodimers which can befound primarily on specializedantigen presenting cells such as macrophages, dendritic cellsand B lymphocytes (44). Theyplay an important role in the initiation of immune responses becausethe activation of T helperlymphocytes by an antigen is restricted to the presentation of the processedantigen togetherwith the homologous class II MHC molecule on macrophagesand other accessory cells (45).It is now realized that class II MHC molecules,HLA-DR antigen in particular (commonlyreferred to as Ia Ag), are actually peptide transport proteinswhich bind, transport to, anddisplay at the cell membrane peptides that are derived fromextracellular proteins. In contrast11to class I MHC molecules, class II MHC can bind to peptidesof different lengths, because thebinding site is open at both ends. They present these peptidesto helper T cells as part of themechanism for identifying foreign antigens and producing an immuneresponse (46 - 48).a) Role in autoimmune disorderAutoimmune disorders (AD) affect 5% to 7% of the population. TheAD are producedby autoimmunization secondary to a disruption of the normal self-tolerance whichErlichreferred to as “Horror autotoxicus” (49). The autoimmune processmay be initiated by anenoneous antigen presentation by the MHC molecules. It is important torealize that AD is notthe result of a unique mutation of the HLA alleles exclusivelyfound among the patients.Instead, the same sequence found among these patientscan also be found in the healthycontrols, although with a different frequency.With the recent development of biochemicaland molecular biological methods, it appears thatHLA class II molecules are important for at least some diseasesbecause: i) the HLA class IIspecific associations are maintained in different ethnicgroups (50), and ii) in some animalmodels, the induction of AD may be blockedby monoclonal antibodies directed against thealleged antigen, and the comparison of specificsequences between diseased and controlgroups has shown extremely strong associationswith specific class II alleles (51).The immune recognition of normal self componentswhich leads to tissue destructionand pathological abnormalities underlies a groupof disparate diseases such as insulindependent-diabetes mellitus, rheumatoid arthritis,myasthenia gravis, and multiple sclerosis,12collectively known as autoimmune disorders.It has been shown that the susceptibilityto thesediseases is strongly associated with particularclass II alleles, suggesting thatthe class IIMHC-restricted binding and presentationof specific autoantigens may be involvedin thedisease process (51 - 59). Class II MHC are alsoassociated with a heterogeneous groupofleukoencephalopathy including cerebrovascular disease,Alzheimer disease and mixeddementia of Alzheimer type (60), IgAnephropathy (61), anterior uveitis (62),and lupuserythematosus disseminatus (63).b) Expression by ECDue to the strategic location ofthe endothelium of blood vessels, i.e. asthe first celllayer that interacts with the blood constituents,the role of EC as active participants inimmuneregulation has been extensively investigatedin recent years. Thus, it has been shownthat ECof extracerebral blood vessels can beinduced to express class II moleculesand that theimmunogenic capacity of the EC is directlyproportional to the extent of class II MHCantigenexpression on the cell surface(64). There are conflicting reports as to whetherthe capillaryendothelium in brain expresses theHLA-DR antigen in normal or diseasedstates, e.g. theantigen could not be detected in ratbrain microvessels in both control or EAErats (65). Incontrast, the antigen is foundin approximately 10% of guinea pigbrain microvessels in thecontrol state and in 35% of guineapig brain microvessels in EAE(66). In humans, themicrovascular DR-antigenhas been reported to be rarely detectablein normal brain (67), butdiscontinuous vascular immunoreactivityfor the antigen can be found in multiplesclerosis13(68). However, Pardridge eta!. have demonstrated thatthe DR-antigen canbe detected in thebrain microvasculature ofboth normal subjectsand subjects with neurologicdisease (69). Invitro studies have shownthat untreated primarycultures of rat brainendothelium areabsolutely negative forclass II staining, but theseEC can be induced toexpress Ia Ag in vitrowhen they are treated withJFN-y (70).The ability of IFN-y to induceTa Ag expression has beenwell documentedin manydifferent cell types includinghuman and murine epithelialcells (71, 72), rat glomerularmesangial cells (73), murinemacrophages, mast cellsand myelomonocytic lines(74 - 77),human melanoma cells(78), monocytes (78, 79),dermal fibroblasts (80),human myoblasts(81) and myotubes (82)in vitro. Moreover, IFN-ycan induce class II MHCexpression onextracerebralEC such as HUVEC (22,83), and human dermal microvascularEC (HDMEC)(84) in cultures. These cellsdo not express class II MHCconstitutively understandard cultureconditions. The effectsof IFN-y on the aberrant expressionof Ta Ag on cell typesthat do notnormally functionas antigen presenting cellsmay lead to the presentationof cellular proteinsto the immune system whichcan then contribute tothe induction of autoimmunediseases (85,86) or graft rejection (87). Ithas been reported that IFN-y upregulatesclass II MHC expressionon heart EC which playsa role in the organ allograftrejection (64). Subsequently,manystudies have focussedon determining the mechanismsby which Ta Ag expressioncan bedownregulated, especiallyin situations involvingIFN-y upregulation.The IFNs have beensuggested to have therapeuticpotential in MSbecause of thehypothesis that a viralinfection, an immunoregulatorydefect, or both, may beimplicated in14the disease process (88 - 90). Reportsof deficient IFN-y productionin MS patients (91, 92)further suggested the possibility of usingIFN-y for therapeutic trial. In contrast,later studiesusing a sensitive solid phase radioimmunoassayto test for IFN-y levels reportedthat thelymphocytes isolated from untreatedMS patients actually producedmore IFN-y than thenormal controls (93, 94). Moreover,a small increase in cerebrospinal fluidIFN-y was alsoobserved in active MS patients (95).In the early 1980’s, when highly purified IFN preparationsbecame available, it waspossible to demonstrate for the firsttime the specific binding of IFNto cellular binding sites.The binding studies established that IFN-ybinds to a receptor different from that forIFN-3 (96- 100). However, several different groupshave also reported that thereis some cross-reactivitywith IFN-3 for the bindingsite for IFN-y (101 - 104). IFN-3 has beenshown to be one of thesubstances that can antagonize the inductionof Ia Ag expression by IFN-y. The antagonisticeffects ofIFN-13have been demonstrated incultured adult human astrocytes(25), humanglioma cells (26), murine macrophages (27,36), blood monocytes isolatedfrom MS patients(28), astrocytoma cell lines (37)and also in other EC systems (105,106).The ability of IFN-y and IFN-to induce and suppress Ta Ag expressionon HBMEC,respectively, was examined in thisstudy in an effort to provide betterunderstanding of thepotential role of HBMECas antigen presenting cells and to providean in vitro system forscreening drug therapies whichmay show promising value in MS.151.4 CENTRAL NERVOUSSYSTEM INFLAMMATIONAND AUTOIMMUNITY1.4.1 Permeability of the Blood-Brain-BarrierThe presence of tight junctions betweenadjacent EC, the absence ofvesiculartransport system and the paucityof cytoplasmic vesicles are uniquecharacteristics of brainmicrovascular EC, forming the basisof the blood-brain barrier (BBB)(107, 108). Undernormal physiological conditions, cerebralEC restrict the paracellular movementof proteins,ions, large lipid-insoluble nonelectrolytes,and the access of antibodies, complementmoleculesand white blood cells from the bloodto the brain parenchyma. Subsequently,the BBB controlsthe lymphocyte traffic into the CNSwhich is normally very limited.A variety of clinical and experimentalconditions may modify ordamage the BBB,leading to enhanced permeabilityto plasma proteins, ions, water andcirculating white bloodcells (108, 109). The cerebrospinalfluid (CSF): serum albumin ratioindicates the level ofvascular permeability inthe CNS. As long as the BBBis intact, the ratio between the tworemains relatively constant(110). Several studies havefocussed on the mechanismsofopening of the BBB under differentexperimental and clinical conditions.Transmissionelectron microscopic examinationusing horseradish peroxidase andlanthanum as electrondense tracers have demonstratedthat these molecules may traversethe endothelial barrier viaopened tight junctions (111- 114) or by means of acceleratedvesicular transport (115 -118).Moreover, the tight junctionsbetween the EC also becomepermeable to the electrondensetracers if a hyperosmotic solutionis administered into the rat via intracarotidinjection (113).It has been reported that in EAE,the experimental autoimmunedemyelination of the CNS16mediated by T cells, increase in the BBB permeability to small molecules occurs early in thedisease process (119 - 124). Alterations in BBB permeability in MS have also been detectedusing gadolinium contrast enhancement (125). The mechanisms responsible for the increasedBBB permeability in disease are still unknown. MS is a demyelinating disorder of the CNSwhere some inflammation exists. It is thought to be immune mediated. The presence oflymphocytes on brain slides suggests that they have egressed from the vessels. The reason whythey accumulate in the cerebral parenchyma is probably due to an increased exit from thecirculation (i.e. by an increase in the permeability of the BBB). Interestingly, IFN-y has beenshown to induce alterations in the morphology and permeability properties of cultured EC(84, 126 - 128). Studies on the effects of different combinations of cytokines, e.g. IFN-y,tumor necrosis factor-a (TNF-a), and interleukin-1a/13(IL-i a/f3), on HUVEC monolayerpermeability have indicated that IFN-y plays a central role in increasing the permeability ofEC monolayer (128). These results support previous in vitro observations on IFN-y effectson HBMEC permeability (127). Moreover, an increase in vascular permeability was detectedwhen Wistar rats were injected intradermally with IFN-y (129). The effects of IFN-y on ECpermeability are further confirmed by studies demonstrating augmentation of lymphocytemigration across HUVEC monolayers by IFN-y, and the effect has been shown to be due to aselective action on EC (24). Furthermore, direct interaction between memory T cells andvascular EC in a noncytolytic manner augments the EC permeability to macromolecules. It issuggested that modulation of endothelial permeability may be a critical factor contributing tothe preferential migration and accumulation of lymphocytes at chronic inflammatory sites17(130).1.4.2 Lymphocyte infiltrationAutoimmune demyelinating disorders of the CNS such as MS are characterizedbydemyelination and migration of acute and chronic inflammatory cells fromthe blood into thebrain parenchyma through the cerebral vasculature that normally excludes circulatingleukocytes from entering the brain. MS is a recurrent and progressive inflammation of theCNS, and it has been reported that the number of lymphocytes circulating through thecerebralmicrovasculature is always many folds higher than the disease-free control(110). Themechanism(s) leading to the migration of these cells from blood tobrain through the highlyspecialized BBB are largely unknown.a) Ia Ag associationEpidemiological studies suggest that common viral-likeinfections, primarilyrespiratory infections, are temporally associated with exacerbationsin MS (131). More recentstudies also show a close relationship betweencommon febrile events and new MS diseaseactivity (132). Poser has suggested that a systemicviral infection may play a role in thedisease process of MS whereby activated T cells can secreteIFN-y in response to a viralinfection (133). As mentioned previously, IFN-y is themost potent inducer of class II MHCmolecules (Ia Ag), and its induction has beenreported in extracerebral endothelium (22, 84,134) as well as cerebral endothelium (70, 127, 135,136). The expression of Ia Ag maysubsequently allow EC to present antigen to T cells (137)with the consequent release of18cytokines leading to the amplificationof the immune response. Immunohistochemicalstudiesin EAE and brain sections of MS lesionshave demonstrated Ia Ag expression onEC,astrocytes and macrophages, while theantigen is not detected in the normalbrain tissue (66,68, 138 - 141). Class II MHC (IaAg) expression appears 12 to 48 hoursafter exposure of ECto IFN-y in vitro, reaches a plateauby 4 to 6 days and is associated with the expressionof allknown class II antigens (80, 142).The mechanism of action of IFN-y is by increasingmRNAlevels for class II molecules (80,143). Moreover, untreated human EC have no detectablemRNA for class II antigen (80, 144).b) Lymphocyte - EC adhesionAdhesion of T lymphocytesto vascular endothelium is a necessary prerequisitetomigration of lymphocytes from the bloodinto chronic inflammatory sites. Studiesoflymphocyte-EC adhesive mechanismshave shown that IFN-y plays an importantrole inaugmenting lymphocyte-EC adhesion(23, 24, 134, 135, 145). IFN-y-mediatedincrease inadhesion occurred when EC were preincubatedwith this cytokine. In contrast,preincubationof lymphocytes with IFN-y did not affectthe adhesion, indicating that the action ofcytokinewas mainly on the EC (23).Moreover, lymphocyte adhesion to EC andexpression of HLADR antigens on EC are well correlatedin terms of both kinetics and the dose-responsepatternof IFN-y (134). The extent oflymphocyte adhesion to EC appears to dependon the densityof HLA-DR antigens expressed on theEC. Blocking studies with monoclonalantibodiesdirected against HLA-DR or CD4 moleculessignificantly inhibit lymphocyte-ECadhesion inboth autologous and non-autologoussyngeneic combinations, suggesting thatin IFN-y19enhanced binding, Ia Agplays a central role in the increasedadhesion of T cells toHUVEC(134, 145). CD4 molecules areexpressed in a subpopulationof lymphocytes, mainlyT-helpercells, and are known to havean affinity for classII molecules (146, 147), providingstrongsuggestion for the involvementof IFN-y-induced classII MHC as the correspondingendothelial adhesion molecule.To further support the abovestatements, in vivo studiesonEAE mice injected with mAb againstIa molecules showa decrease in lymphocyteaccumulation in the CNS(148). Subsequently, it is speculatedthat the anti-Ia antibodymusthave blocked the adhesion of Ia-reactiveT cells to Ia-expressing EC, leadingto inhibition of Tcell migration from blood intothe CNS. Masuyama etal. (134) have suggested thatT cellrecognition of HLA-DR antigenmay represent thesignal for the initiation of a subsequentadhesive processes whereby complementaryadhesion surface moleculesbecome engaged.These observations indicate thatthe release of IFN-y byactivated lymphocytes inchronicinflammatory sites can upregulatethe adhesion of circulating lymphocytesto the local EC.Since lymphocyte-EC adhesionrepresents the initial step of lymphocytemigration through themicrovessels (149), theIFN-y-mediated increase in lymphocyteadhesion on the surfaceofmicrovessels may lead tofurther influx of lymphocytesinto the site of chronic inflammation.In vitro studies have demonstratedthat lymphocyte-EC adhesioncan be significantlyupregulated by stimulatingthe T cells with phorbol esters(150 - 153), con A(154 - 156), orIL-2 (157, 158). Significantincrease in T-cell adhesionto purified ICAM-1 substrateshasalso been reported whenT cells were pretreated withanti-CD3 IgG and anti-IgG(159).Preincubation studies withphorbol esters have shownthat enhancement of the lymphocyte-EC20binding is entirely attributableto an effect on T cells, withno action on BC (150). Moreover,additive enhancement of adhesioncan be detected whenboth lymphocyte andBC are activated(150, 154). More recent studieshave demonstrated thatIFN-y treatment of passagedculturesof human cerebral BC furtherenhances the lymphocyte-BCadhesion. The activatedlymphocytes were isolatedfrom peripheral bloodof acute relapsing MSpatients duringexacerbations (160).c) Transendothelial migrationof lymphocytesIn vivo observations on T-lymphocyteentry into the CNShave determinedthatlymphocyte activationenhances their ability tocross the BBB and rapidlyappear in the CNStissue, irrespective of theirantigen specificity, MHCcompatibility, T-cell phenotype,and Tcell receptor gene usage (161).Moreover, the level oflymphocyte infiltrationinto the tissue issignificantly upregulatedin chronic inflammatory disorders,a process that may bemodulatedby the enhancement ofinteractions between differentadhesion molecules expressedon bothlymphocytes and vascularEC (150, 162). Studiesof lymphocyte traffic throughvariousorgans (e.g. lungs, liver,lymph nodes) have reporteddifferences in the migratorypatterns ofresting and activatedlymphocytes. Generally,resting lymphocytes moverapidly whileperipherally activatedlymphocytes movemuch more slowlythrough the organs.T-lymphoblasts canreadily leave the circulationand migrate into an inflammatorysite (163).Migration of activatedT cells from the peripheralblood to the CNS hasbeen demonstratedinEAE (164) along witha significant decreasein their activated T-cell levelsin the peripheralblood during exacerbations(165). A decreasedproportion of activatedT cells in the21peripheral blood of MS patients has also been reported (166, 167). Exacerbationsin MS wereusually accompanied by further decreases in activated T cells in the peripheralblood (167).Moreover, lower percentages of activated T cells were consistently foundin the CSF of MSpatients (168), suggesting that these cells may accumulatein the MS plaques. In vivotreatment of EAE rats with a monoclonal antibody specificfor activated rat T cells hasdemonstrated significant reduction in inflammation (169).Subsequently, these observations suggest that activationof both lymphocytes and ECplays a central role in upregulating the adhesive and migratory interactionsbetweenlymphocytes and EC. It is notable that studies of lymphocyte migrationacross cultured ratretinal microvessel EC have shown that con A activationof T-cells did not augment themigratory process. Furthermore, IFN-y treatmentof EC resulted in a slight, but notsignificant, increase in migration (170). The insignificant increasein migration oflymphocytes across rat retinal EC when both systemsare activated supports the concept ofimmune privileged site of the eye due to the presenceof the blood-retinal barrier. Theseresults further confirm the heterogeneity that existsamong EC isolated from vascular beds ofdifferent organs and species with regard to immunologicalresponses to cytokine treatment(19, 155).1.4.3 FVIIIR:Ag in primary cultures of HBMECFactor Vill-related antigen (FVIIIR:Ag) is a largemultimeric glycoprotein synthesizedand released by EC of large and small blood vessels,and it is widely considered as the most22specific marker for cells of endothelial origin (171,172). The protein has also been shown tobe synthesized in primary cultures of HBMEC asdemonstrated by immunofluorescence andimmunoperoxidase techniques at the light microscopiclevel (107). When vascular injuryoccurs, FVIIIR:Ag facilitates the adhesion of plateletsto the subendothelial matrix (173).Biochemical and immunohistochemical studies indicatethat FVIIIR:Ag is generally storedinrod-shaped, membrane-bound cytoplasmic organellescalled Weibel-Palade bodies (namedafter the investigators who first described them) whichare found exclusively in EC (174 -177). However, these organelles are rarely found inEC lining the cerebral capillaries, andthey are absent in primary cultures of HBMECwhen examined ultrastructurally (107).Weibel-Palade bodies are also absent inprimary cultures of microvessel EC derived frommouse (178) and rat (179 - 181) brain. Ithas been recently reported that IFN-y treatmentofHUVEC causes a decrease in the releaseof FVIIIR:Ag from these EC (182). The mechanismof IFN-y-suppressed release of FVIIIR:Ag is still unknownat the present time. Tannenbaumand Gralnick speculated that IFN-y-mediateddepression of FVIIIR:Ag release fromthe ECmay assist in maintaining blood fluidity during immuneactivation (182). Subsequently, thisstudy examined the distribution andfine structural localization of FVIIIR:Ag in primarycultures of HBMEC and also investigatedthe influence of IFN-y on the storageand release ofthis glycoprotein by HBMEC, consideringthe important role of IFN-y in autoimmunedisorders of the CNS.Consequently, the effects of IFN-yand IFN-f3 as potential factorsregulatinglymphocyte entry into the CNS will beexamined in this study. Their influenceson Ia Ag23expression, cell proliferation, alteration ofthe morphology and permeability characteristicsofHBMEC in cultures will be investigated. Furthermore,using primary cultures of HBMEC asamodel of the BBB, these cytokines will also be testedfor their effects on the adhesion andmigration of peripheral blood lymphocytes across HBMECmonolayers which may be relevantin understanding the in vivo immune reactions including increasedvascular permeability andcell shape changes. Blocking experiments withmAbs directed against human HLA-DRmolecules will further determine the role ofIa Ag in the adhesive and migratory processes.The results of this study will emphasize the potentiallycritical role of HBMEC in the initiationof the immune reaction in autoimmune disordersof the CNS. Elucidation of the factorsmodulating the adhesion and migration of lymphocytesacross microvascular EC can havegreat therapeutic potential. The informationmay assist in designing protocols that can preventfurther amplification of the unwanted inflammationor immune responses such as that inMS.In conclusion, understanding of the pathobiology ofHBMEC, such as their responsesto IFN-y andIFN-13treatments and their ability to mediate lymphocytetraffic into the CNS, iscentral to the understanding of CNSinflammation.241.5 SUMMARY ANDOBJECTIVESIn summary, a large body of evidence indicatesthat Ia Ag expression can be inducedby IFN-y in many different cell typesincluding EC. It has also been reportedthat IFN-3 iscapable of suppressing the IFN-y-inducedIa Ag expression. Furthermore, ithas beendemonstrated that lymphocyte-EC adhesionand migration can be augmented by IFN-y.Monoclonal antibody blocking studiesindicate that Ia Ag might play a criticalrole in theadhesive and migratory processes. Finally,IFN-y has been shown to alter the morphologyand permeability characteristics of theEC monolayers which may facilitate the influxoflymphocytes into chronic inflammatory sites. It is uncertainat the present time whether theobservations on EC of other organs andspecies also apply to the HBMEC. It is now wellaccepted that great heterogeneity existsamong different types of EC with regardto antigenicdeterminants, cell surface molecules,metabolic properties, permeability functionsandimmunological responses to cytokines.Cerebral EC are characterized by uniquefeaturesresponsible for the formation of the BBB.How this barrier is breached in inflammationandautoimmune disorders of the CNS is stillpoorly understood.The main objective of this thesis is todetermine the effects of IFN-y and IFN-f3on IaAg expression, proliferation, and alterationof the morphology and permeabilitycharacteristicsof HBMEC monolayers. Furthermore,the effects of these cytokineson the adhesion andmigration of resting/anti-CD3 stimulatedT lymphocytes across the EC monolayerswill beinvestigated. Finally, theeffects of IFN-y on the storage and releaseof FVIIIR:Ag byHBMEC will be examined.25My working hypothesis is that,in a chronic inflammatorysite, some activatedTlymphocytes, mainlyT helper/inducer phenotypes, releaseIFN-y locally. This cytokinecanthen activate the localcapillary EC by inducingIa Ag expression and morphologicalalterations on the EC,and by increasing the vascularpermeability. These changeswillaugment the adhesionand migration of the lymphocytesacross the EC, leading to aninflux oflymphocytes into the chronicinflammatory sites. The administrationof IFN- will play anantagonistic role on theIFN-y-mediated immunologicalresponses.IFN-13 will suppress theIFN-y-induced IaAg expression and morphological alterationon EC and the IFN-y-mediatedincrease in adhesion and migration,resulting in the reductionof lymphocyte numbersin thechronic inflammatory sites.Finally, the ability of IFN-yto affect the storage andrelease ofFVIIIR:Ag in HBMEC may havesome important implicationsin maintaining blood fluidityduring immune activation, consideringthe important role of IFN-yin autoimmune disordersof the CNS.The method of approach usedin this study involved thesuccessful application ofimmunogold labeling with silverenhancement techniquefor the detection of IaAg expressionat the light microscopic level.ELISA was further applied toconfirm the IFN-3 effectsinsuppressing the IFN-y-inducedTa Ag expression. Kinetic studiesof the cytokine effectson IaAg expression on HBMECwere also carried out inthis study. Ultrastructurallocalization ofTa molecules on the surfaceof EC was determined usingmAbs conjugated withcolloidal goldmarkers. Electrondense tracer studies wereapplied in order to assess thepermeability ofconfluent untreatedor IFN-y treated HBMECmonolayers. Immunoperoxidasetechniques26were used to study the IFN-y andIFN- effects on lymphocyte-ECadhesion and migration.Scanning and transmissionEM studies further showed theeffects of the cytokineson themorphological phenotypeof HBMEC, and on lymphocyte-ECadhesion and migration.Finally, immunoelectron microscopywas performed for theultrastructural localizationofFVIIIR:Ag in HBMEC, and theeffect of IFN-y on its storage andrelease.The Specific Aims of this thesisare:1) To determine,in vitro, whether primary culturesof HBMEC constitutivelyexpress Ia antigen, and whetherthe expression can bemodulated byinflammatory cytokines suchas IFN-y and IFN-f3.2) To determine whether IFN-yand IFN- exert antiproliferativeeffects on primarycultures of HBMEC, whetherthese cytokines can inducealterations in themorphology and organizationof the EC monolayers, andwhether IFN-y caninfluence the permeabilityand endocytotic characteristicsof HBMEC that wouldbe relevant to the in situ immune reaction.3) To determine whetherIFN-y and IFN-j3 treatmentof HBMEC can affect theadhesion of resting/activatedlymphocytes to these untreated/cytokine-treatedEC.4) To determine whetherIFN-y and IFN-El treatmentof HBMEC can influencethemigration of resting/activatedlymphocytes across untreated/cytokine-treatedEC.5) To determinethe influence of IFN-y on thestorage and release ofFVIIIR:Ag inprimary cultures of HBMECwhich may reflect the maintainanceof blood fluidityduring immune reactionsin vivo.27MATERIALS and METHODS2.1 Isolation and culture of HBMECHBMEC were isolated from normal brains obtained at autopsy andfrom temporal lobectomyspecimens removed for intractable seizure disorders and culturedaccording to methodspreviously described (107). The time interval between death andremoval of brain rangedfrom 3.5 hours to 15 hours. Pathologic abnormalities were not detectedin any of the brainsused for isolation of cerebral microvessels. Large vessels and theleptomeninges wereremoved from the cerebral cortex with fine forceps,and the tissue was cut into 1 to 2 mmcubes. The tissue was then incubated for 3 hours at 37°C in medium M199 (Gibco,Burlington, Ontario) containing 0.5% dispase (Boehringer Mannheim,Indianapolis, Indiana).Following centrifugation at 1000 xg for 10 minutes, the pellets were resuspended in mediacontaining 15% Dextran (Sigma, St. Louis, Missouri; averagemolecular weight, 70,000daltons) and centrifuged at 5,800 xg for 10 minutes in order to seperate the microvessels fromother brain components. The isolated microvesselswere then incubated in M199 containing 1mg/mi of collagenase/dispase (Boehringer Mannheim)for 12 to 16 hours at 37 °C. Followingincubation, the microvessels were resuspended inM199 containing 5% horse serum (HS)(Hycione Laboratories, Logan, Utah), layered overPercoll (Sigma) gradients prepared asdescribed by Bowman et al. (183) and centrifugedat 1,000 xg for 10 minutes in order toseparate the EC from red blood cells, pericytes andcellular debris. The layer containing theEC was washed in 10% HS in M199, and collected bycentrifugation for 10 minutes at 1,00028x g. Cell numbers were counted with a hemocytometer. Cell viability, as determined by thetrypan blue exclusion test, ranged between 85 and 90%.The isolated clumps of EC were seeded onto plastic wells (Coming Plastics, Coming, NY)previously coated with fibronectin (Sigma). The cultures were maintained inM199supplemented with 10% HS, 25 mM HEPES, 10 mM sodium bicarbonate, EC growthsupplement (20 ig/ml), heparin (100 tg/ml) (all from Sigma), and penicillin (100 tg/ml),streptomycin (100 tg/ml), and amphotericin B (2.5 tg/ml) (Gibco) at 37°C in a humidified2.5% C02/97.5% air atmosphere. The culture media were changed every second or third day.The endothelial origin of the cells was confirmed by their intense, granularperinuclearstaining for Factor Vill-related antigen (FVIIIR:Ag) and their binding of Ulex Europaeus-Ilectin as previously reported (107). Confluent, contact-inhibiting monolayers wereobtained 7to 9 days after plating. The monolayers were used once they reached confluency.2.2 AntibodiesMouse monoclonal antibody (mAb) against human recombinant interferon-y(KM48, IgG1)and mouse anti-human HLA-DR IgG (DK22, IgG,kappa) were obtained from DimensionLaboratories Inc., Missisauga, ONT. Goat anti-mouse IgG (GAMIgG)coupled to Snm goldparticles (Auroprobe LMGAM IgG) was obtained fromJanssen/Cedarlane Labs Ltd., (Hornby,ONT), GAMIgG coupled to peroxidase from JacksonImmunoresearch Lab Inc., PA, andmouse anti-human pituitary follicle-stimulatinghormone IgG from Biogenex Laboratories,CA. Mouse anti-human Leu-4 (CD3) mAb was purchasedfrom Becton-Dickinson, and mouse29mAb to human Leukocyte common Ag (CD45) from DimensionLab, Inc.,Mississauga, ONT.For the ultrastructural localization of FVIIIR:Ag, mousemAb raised against human factor VIIIantigen (Cedarlane Laboratories, Hornby,ONT) was used as the primary antibody and goatanti-mouse IgG coupled to Snm gold particles (Janssen/Cedarlane) as the secondary antibody.For the light microscopy immunoperoxidase staining,a rabbit antiserum to factor VIII antigen(Dakopatts, Santa Barbara, CA) was usedas the primary antibody and goat anti-rabbit IgGconjugated with HRP (Jackson ImmunoresearchLaboratories, West Grove, PA) asthesecondary antibody.2.3 Induction of Ia Ag expression on HBMECby human recombinant Interferon-gammaand beta2.3.1 Treatment of primary HBMEC culturesHuman recombinant interferon-y (IFN-y; CollaborativeResearch Inc., Bedford, MA) wasdiluted in complete media toa final concentration of 10, 20, 50, 100, 150 and200 U/ml.Confluent monolayers of HBMEC, grownin replicate wells were incubated with differentconcentrations of IFN-y for4 days and with 200 U/ml for 12 hoursto 4 days at 37 °C.Cultures used for these experimentswere derived from EC isolatedfrom several differentautopsy brains. The specificityof Ia Ag induction by IFN-y was tested in culturesco-incubatedwith IFN-y (200 U/mI) andanti-IFN-y mAb (optimal concentration,10 tg/ml) for 4 days. Inorder to study the reversibility of IaAg expression, monolayers previously treated withIFN-y30(200 U/mi) for 4 days were thoroughlywashed with M199 to removethe cytokine, then placedin complete media and returned tothe incubator for another 4 daysprior to Ia Ag detection.For the IFN-f3 studies, recombinant humaninterferon-f3ser(Triton BiosciencesInc., Alameda,CA) was used at 100, 250, 500, 1,000,2,000 and 6,000U/mi. These units were determined bythe National Institutes of Healthreference standard. ThisIFN- is the same cytokine thatisused for MS therapeutical trial,and it is now available for patientuse. Confluent cultures ofHBMEC grown in replicatewells were incubated with100 U/ml IFN-y with or withoutvarious concentrations of IFN-j3for 4 days. In separate experiments,replicate wells werepreincubated with IFN- (6,000U/mi) or IFN-y (100 U/ml) for2 days prior to coincubationwith IFN- andy for another 4 days.All experiments were performedin duplicate or triplicate wells.2.3.2 Light microscopic immunocytocLiemicallocalization of Ia AgFollowing cytokine treatment,the monolayers were washed3 times with buffer containingphosphate buffered saline (PBS- 10 mM, pH 7.2), 1% bovine serum albumin(BSA) and 1%normal goat serum (PBS/BSA/NGS)and incubated for 40 minutesat room temperature withmouse anti-human HLA-DRmAb at 1:30 dilution in carrier buffercontaining PBS, 5% BSAand 4% NGS. Followingbrief washing withPBS/BSA/NGS, the monolayers were incubatedwith the secondary antibody(Auroprobe LMGAM IgG coupledto 5 nm gold particles) diluted1:40 in carrier buffer for60 minutes at room temperature.At the end of the incubation period,the cells were washed withPBS/BSA/NGS, fixed in fresly preparedbuffered formaldehyde-31acetone fixative (20 mg Na2HPO4,100 mg K112P04,30 ml distilled H20, 25 ml 37%formaldehyde and 45 ml acetone) for 30 seconds, washed with distilled H20, andincubatedin silver enhancing solution(IntenseM, Janssen/ Cedarlane) for 25 - 35 minutes. Afterwashing with distilled H20, the monolayers were counterstained with Giemsaandcoverslipped using JB-4 plus (Polysciences/Analychem, Markham, ONT)as mountingmedium.Controls included untreated monolayers grown in the absenceof IFN-y andIFN-13and IFN-ytreated cultures incubated with 1) normal mouse IgG at the same concentration astheprimary antibody (5.9 tg/ml IgG), or 2) carrier buffer or 3) an irrelevant antibody(anti-human pituitary follicle-stimulating hormone IgG) instead of the primaryantibody.2.3.3 Immunoelectron microscopyHBMBC monolayers treated with 200U/ml IFN-y for 4 days were washed with buffercontaining PBS, 1% BSA and 0.2% NaN3 (PBS/BSA)and incubated with mouse anti-humanHLA-DR mAb at 1:30 dilution in carrier buffer containingPBS, 5% BSA and 4% NGS for 30minutes at room temperature. After washing withPBS/BSA, the cells were incubated with 5nm gold particle-conjugated secondary antibody (Auroprobe LMGAMIgG)at 1:40 dilution incarrier buffer for 45 minutes, washed, and fixed in periodate-lysine-paraformaldehydefixative(184) overnight at 4 °C. Following fixation, the cellswere washed in PBS, post fixed in 1%buffered 0s04,stained en bloc with uranyl magnesiumacetate overnight at 4 °C, dehydratedin graded series of methanol, and embedded in Epon-Araldite.Blocks cut out from the32embedded cultures were re-embedded for cross-sectioning. Thin sections were examined in aPhilips EM400 without heavy metal staining. Controls consisted of cells maintained in IFN-yfree growth media and monolayers incubated with normal mouse IgG or carrier buffer insteadof the primary antibody.2.3.4 Enzyme linked immunosorbent assay (ELISA)Following incubation with IFN-y and/orIFN-13,the cells were washed 3 times with PBS andfixed with 0.025% glutaraldehyde in PBS for 10 minutes at room temperature. Themonolayers were thoroughly rinsed with PBS, washed 3 times with PBS/BSA/NGS andincubated with mouse anti-human HLA-DR mAb at 1:30 dilution in carrier buffer containingPBS, 5% BSA and 4% NGS for 60 minutes at room temperature. After brief washing withPBS/BSA/NGS, the cells were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG diluted 1:5,000 in carrier buffer for 60 minutes at room temperature. Followingwashing in PBS, the cultures were incubated with o-Phenylenediamine (OPD) dihydrochloride(Sigma) (2 mg/ml) diluted in 0.1 M PBS containing 0.015% H20 for 45 minutes at roomtemperature. The reaction was stopped by the addition of 2 M H2S04.Absorbance wasmeasured at 490 nm on a Elisa Microtiter Plate Reader (Molecular Devices, CA). Allexperiments were performed in triplicate.2.3.5 Quantitation of Ia Ag expression by HBMECHBMEC monolayers stained for the light microscopic localization of Ta Ag were examined33under a Nikon Labophot light microscope. Quantitation of Ta Ag expression was performed bycounting one central and four peripheral randomly selected fields of each culture well with anocular grid under X200 magnification. All counts were performed blindly. Data are expressedas the mean±standard error of the mean.2.4 Scanning electron microscopy (SEM)Confluent HBMEC monolayers treated with 200 U/ml IFN-y, 6,000U/mi IFN-f3 or acombination of IFN-y (200 U/mi) and1E(6,000 U/mi) for 3 to 4 days, monolayerscoincubated with 200 U/mi IFN-y and 10tg/ml anti-IFN-y monoclonal antibody for the sameperiod of time, as well as untreated control cultures were processed for SEM as described bySchroeter et al. (185). Briefly, the cultures were washed in Hank’s balanced salt solutionandfixed in 2.5% glutaraldehyde in 0.05 M sodium cacodyiate buffer (pH 7.2) for 1 hour at 4°C. Following washing in cacodylate buffer, the cells were post fixed in buffered 1% 0s04for 1 hour, washed in buffer and treated with 1% Tannic acid for 1 hour. After further washingin cacodylate buffer, the monolayers were dehydrated in graded series of methanol up to 70%,and block stained with 4% uranyl acetate overnight at 4 °C. The cells were thendehydratedwith methanol up to 100%, critical point dried, gold coatedand viewed with a CambridgeStereoscan 250T scanning electron microscope.2.5 Permeability studiesConfluent HBMEC monolayers treated with IFN-y (200 U/mi) for 4 days were washedwith34serum-free aMEM and incubated in aMEM containing 1 mg/mi horseradish peroxidase (HRP;Sigma Type VI) for 5 - 10 minutes at 37 °C as previously described (186). At the end of theincubation period, the cells were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in0.1 M sodium cacodylate buffer (pH 7.4) for 1 hour at 4 °C. Following washing with buffer,the monolayers were incubated with 3,Y - diaminobenzidine (Sigma) for 1 hour at 4 °C,washed with cacodylate buffer, post-fixed in 1% buffered 0s04,stained en bloc with uranylmagnesium acetate, dehydrated with graded series of methanol, and embedded in EponAraldite. Thin plastic sections were examined in a Philips EM400 without heavy metalstaining. Controls consisted of identical age-matched primary cultures grown to confluence inIFN-y free media.Quantitation of the junctional permeability and pinocytotic activity of EC was performed bycounting the number of permeable and impermeable intercellular contacts and the number ofHRP labeled and unlabeled cytoplasmic vesicles in 100 IFN-y treated and 100 untreated cellsphotographed at standard EM magnification.2.6 Growth StudiesFreshly isolated BC were plated in replicate wells of Corning 24 - well plates at a density of 1X 10 cells/cm2on day 0. On day 1, all experimental wells were refed with complete mediumcontaining IFN-y (150 U/mi), IFN-3 (1,000 U/mi) or a combination of IFN-y and f3 (150 U/miand 1,000 U/mi, respectively). All experiments were carried out in duplicate and media werechanged every other day. Control cultures were maintained in growth media in the absence of35the interferons. The cells were viewed with a Nikon Diaphot TMD inverted microscope andphotographs of one central and four peripheral fields of each well were taken daily at loxmagnification. The number of cells in each photograph were counted and the data areexpressed as the mean±standard error of the mean.2.7 Preparation of T lymphocytesHuman peripheral blood lymphocytes (PBL) were prepared from heparinized venousblood ofnormal adult volunteers by Ficoll-Hypaque density gradient centrifugation (Histopaque -1077, Sigma, St. Louis, MO, USA) at 500 xg for 30 minutes. The PBL were washed threetimes in PBS and resuspended in RPMI 1640 (Gibco) containing 10% fetal calf serum (FCS)(Gibco), 2 mM Glutamine, 100 U/mI penicillin, 100 pg/ml streptomycin, 0.25ig/mlAmphotericin B, and 125 tg/ml gentamicin. By this procedure cell fractions containing85 -89% lymphocytes were obtained. Viability was 95 - 98% by the Trypan Blue exclusiontest. Tlymphocytes were prepared by the nylon-wool separation technique (187). Sterilenylon-wool(Robbins Scientific Corporation, Sunnyvale, CA) was packed into 6 ml syringesat 500mg/syringe. The columns were washed 10 times alternately with distilledH20 and 0.02 N HC1and then with 15 ml of 1X Hanktsbalanced salt solution (with Ca2and Mg2j,followed by15 ml of 10% Fetal Calf Serum in RPMI 1640 media (10% FCS/RPMI)per column. Thecolumns were used immediately or kept frozen at -20 °C for up to2 months. Frozen columnswere thawed for 30 minutes at room temperature, followedby another 30 minutes at 37 °C.They were then washed with 10 ml of 10%FCS/RPMI, and compressed to lml volume prior36to cell incubation. One ml suspension of isolated PBL (1 x i0 cells/ml) was placed over eachcolumn dropwise and the columns were incubated for 45 minutes at 37 °C. Non-adherent Tlymphocytes were eluted with 12 ml 10% FCS/RPMI. By this method, we routinely obtained5 to 6 Xio6cells/ml. Phenotypic characterization of the lymphocytes in the eluate wascarried out according to the following protocol: aliquots of lymphocyte suspension (200 [Licontaining 2 xio6celis/ml) were incubated for 30 mm at 4 °C with the following mousemAbs: anti CD2 (for T and Natural killer (NK) cells), anti CD4 (for T helper phenotype), antiCD8 (for T cytotoxic phenotype), anti CD16 and anti CD 56 (for resting NK cells), anti CD2O(for B cells), and anti-CD56 (for resting and activated NK cells) (Phycoerythrin-conjugatedanti CD2, CD8 and CD56 from Coulter Corporation, Hialeah, Fl., Fluorescein-conjugated antiCD4 and CD2O from Becton Dickenson, Mississauga, ONT). The tubes were then filled with2% FCS in TC 199 (Gibco) and centrifuged at 500 x g for 10 minutes. The supernatant wasdiscarded and 0.5 ml of 1% paraformaldehyde solution was slowly added while vortexing thetubes. Fluorescence was read on the Epic Profile I (Coulter Corporation, Hialeah, Fl.). Thequadrant was set by using isotypic control, and dead cells were gated out.CD4+cellscomprised 57% of the eluate whileCD8+cells constituted 22% of the cell suspension. Therewas a small number of NK cells (<10%), and B cells (4%).2.8 Activation of T cellsFreshly isolated lymphocytes were cultured for 72 hours at 37 °C with mouse mAb to humanLeu 4 (CD3) (10 ng/ml; Becton Dickinson, Mississauga, ONY). Before their use, lymphocytes37were washed extensively and resuspended in 10%FCS/RPMI. Lymphocyte activation wasdetermined by the following protocol: aliquots of lymphocyte suspension (200 ti containing2xio6cells/mi) were incubated for 30 mm at 4 °C with fluorescein-conjugatedmousemonoclonai anti CD25 antibody (for IL-2R, p55) (Becton Dickenson,Mississauga, ONT.).The cells were washed in PBS and centrifuged at 300 xg for 5 minutes. The pellet wasresuspended and fixed in 1% paraformaidehyde dilutedin PBS for 30 mm at roomtemperature. Fluorescence was read at 580nm. Approximately 2 fold increase in IL-2Rexpression was detected in anti CD3 stimulated lymphocytesin comparison to restinglymphocytes.2.9 HBMEC-lymphocyte adhesion assayConfluent monolayers of HBMEC were treated with recombinanthuman IFN-y (150 U/mi), orIFN- (2,000 U/mi) or a combination of IFN-f3 (2,000 U/mI) andy (150 U/mi) for 3 days.Prior to use, the cultures were washed extensively. 500tl of freshly isolated resting T cells oractivated lymphocytes containing 2 xio6cells/mi were incubated with the EC monolayers for1 hour at 37 °C. Following incubation, the monoiayers were washedthoroughly with warmedHankstbalanced salt solution (HBSS) and PBS(pH 7.2, 10mM) containing Ca2 and Mg2toremove non adherent lymphocytes, fixedin Acetone: Ethanol (1:1 ratio) for 7 minutes at 4°C,air dried and washed with Tris buffer. Blocking of theendogenous peroxidase was carried outby incubation with H20 and Methanol (1:4 ratio)for 30 minutes at room temperature. Afterseveral washes with Tris buffer, thecells were incubated for 90 minutes with mouse38monocional anti human leucocyte common antigen (LCA) (Dimension LaboratoriesInc.,Mississauga, ONT.) at 1:100 dilution, and then with horseradish peroxidase conjugatedgoatanti mouse IgG at 1:200 dilution (Jackson Immunoresearch LaboratoriesInc., West Grove,PA) for 120 minutes at room temperature. After washing with Trisbuffer, the cells weretreated with3,3t-diaminobenzidine (0.5 mg/mi; Sigma) and 0.01%H20 in Tris buffer (pH7.6) for 20 minutes at 4 °C, washed with H20, and counterstainedwith haematoxylin.Subsequently, the monolayers with adherent lymphocytes werecovered with JB4 mountingmedium and the walls of the wells cut out with a hot scalpel bladeprior to counting. Controlsconsisted of untreated HBMEC cultures incubated with restingor activated lymphocytes.For scanning EM studies of lymphocyte-EC adhesion,HBMEC were grown to confluency oncollagen discs (section 2.10) and then incubated withIFN-y (150 U/mi, 3 days) or leftuntreated (controls), followed by incubation witheither resting or anti CD3 stimulated T cellsfor 2 hours at 37 °C. The cells were then processedfor scanning EM as described above(section 2.4).2.10 HBMEC-iymphocyte migration assayFor the migration studies, HBMEC were grown onpermeable collagen membranes madeofhighly purified, pyrogen-free, pepsin-solubilizedcollagen forming the floor of 14mmdiameter wells (Cellagen Discs, ICNBiomedicals, Inc., Cleveland, OH) (Fig. 1). Immediatelybefore seeding the cells, collagen discs wereplaced inside 24-well culture platesandimmersed in M199. This double chemotactic chamberhas been previously demonstrated tobe39suitable for studying the adhesionand migration of humanpolymorphonuclear leukocytesacross cultured bovine brain microvesselEC (7). Plating of ECon collagen discs didnotrequire prior coating with fibronectin.Isolated clumps of HBMECwere plated at 50,000cells/cm2.Media were changed everyother day. Confluent monolayersof FVIIIR:Ag positiveEC were obtained by sixto eight days in culture. Confluentcultures of HBMEC weretreatedeither with IFN-y (150 U/mi),IFN-3 (2,000 U/mi) or acombination ofIFN-13(2,000 U/mi)and y (150 U/mi) for 3 days at37°C. Before their use,HBMEC were rinsed threetimes andthe last wash was replacedby 200 d of lymphocytesuspension containing 2Xio6cells/mi.Resting or activated lymphocyteswere incubated with ECfor 3 hours at 37 °C.Followingincubation, the monolayerswere washed with warmM199 and then PBS to removenon-adherent lymphocytes andprocessed for transmissionelectron microscopy (section2.16). Onetm thick and ultrathin sectionswere cut with an UltracutE ultramicrotome (Reichert-Jung,Austria). One tm thick sectionswere stained with toluidineblue, coverslippedand used forquantitation of lymphocytetransendothelial migrationby light microscopy (section2.12). Thinsections were stained withuranyl acetate and lead citrate.2.11 Monoclonalantibody-blocking studiesThe effects of mAbsto IFN-y and HLA-DR onthe adhesion and migrationof resting T cellsand activated lymphocytesacross HBMEC monolayerswere examined inseparateexperiments. Monolayerswere coincubated withIFN-y (150 U/mi) andmouse monoclonaianti human IFN-yIgG (10 tg/ml) for3 days at 37°C, or with IFN-y(150 U/mI) for 3 days at4037°C followed by incubation with mouse monoclonal anti human FILA-DR IgG (6 tg/m1) for2 hours prior to incubation with lymphocytes. Monolayers were thoroughly washed threetimes with M199 to remove the IFN-y and mAbs before the adhesion and migrationassays.Both antibodies were purchased from Dimension LaboratoriesInc., Mississauga, ONT.2.12 Quantitation of lymphocyte adhesion and migrationLymphocyte-EC adhesion was quantitated by counting the number of adherentlymphocytes toEC monolayers in one central and four peripheral randomly selected fields of eachculture wellusing a bright field microscope equipped with a 1 cm2 ocular grid underX 200magnification. Adhesion index is expressed as the number of adherent T cellsper mm2of themonolayer. Transendothelial migration was quantitated by countingthe number oflymphocytes that migrated across the monolayers by light microscopy in1 tm thick plasticsections stained with toluidine blue. For each treatment, 200 sections (40 tmapart), werecounted.2.13 Transmission electron microscopyEC monolayers were washed three times in serum-free media andfixed in 2.5%glutaraldehyde and 2% paraformaldehyde in 0.1M sodium cacodylate buffer(pH 7.35) for 1hour at 4°C. After washing with 0.2M cacodylate bufferfor 30 minutes, the cells were postfixed in 1% 0s04 in 0.1M sodium cacodylate buffer for1 hour at 4°C, block-stained withuranyl magnesium acetate overnight at 4°C, dehydrated and embedded inEpon-Araldite.41Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a PhilipsEM 400.2.14 Localization of FVIIIR:Ag in untreated, cytokine/chemical-treated HBMEC2.14.1 In vitro drug treatment of ECThe effects of calcium ionophore A23 187, ethyleneglycol-tetraacetic acid (EGTA) andinterferon-y (IFN-y) on the release of FVIIIR:Ag were studied in confluent 8 day old cultures.The cells were incubated with growth medium containing: a)lOitMCa2+ionophore A23187(Sigma), diluted from a 10mM stock dissolved in dimethylsulfoxide, for 10 minutes; b) 1mMEGTA (Sigma) for 10 minutes and c) 200U/ml IFN-y (Collaborative Research Incorporation,Bedford, MA) for 24 hours. At the end of the incubation period the cultures were washed withPBS and processed for immunoelectron microscopy.2.14.2 Immunoelectron microscopy for FVIIIR:AgEndothelial monolayers were washed with phosphate buffered saline (PBS) with 0.lg/l CaCl2and fixed in freshly prepared periodate-lysine-paraformaldehyde (PLP) fixative (184)overnight at 4°C. After fixation the cultures were washed with PBS for 30 minutes andincubated in cold 10% sucrose solution in PBS for 4 hours, 15% sucrose for 4 hrs and 20%sucrose overnight. The cells were permeabilized by incubation in 0.01% to 0.05% TritonX100 (Sigma) in freshly prepared PLP for 10 minutes at room temperature, washed with PLPfor 20 minutes and then with 0.1M glycine in PBS for 30 minutes. The monolayers were42incubated with 5% normal goat serum (NGS) in 0.1% BSA-Trisbuffer (BSA-Tris) for 20minutes and then with the primary antibody (mouseanti-human FVIII antigen) at 1:50 dilutionwith 1% NGS in BSA-Tris for 2.5 hours at room temperature.Following washing in BSATris, the cells were incubated with the secondary antibody(goat antimouse IgG) at 1:20dilution in BSA-Tris for 1.5 hours at room temperature.Cultures were further fixed in 1%glutaraldehyde in PBS containing 0.2% tannic acidfor 40 minutes at room temperature,washed in PBS and post-fixed in 0.5% osmium tetroxide(0s04)in PBS for 15 minutes.Following post-fixation, the cells were washed in acetate buffer, blockstained in uranylmagnesium acetate overnight at 4°C, dehydrated throughgraded series of methanol andembedded in Epon-Araldite. Blocks cut from the embedded cultures werere-embedded forcross-sectioning. Thin sections were examined ina Philips EM400 without heavy metalstaining.2.15 Statistical analysisStudent’s t-test, a procedure designed to test for differences in two groups,was used forstatistical evaluation of the data.Sigmastat program was used for this analysis.43RESULTS3.1 Human brain microvessel endothelial cellHBMEC grown on plastic wells or collagen membranesformed confluent monolayersby 7 to 10 days in culture. EC were elongated and grew in close associationwith each other.Upon reaching confluency, HBMEC exhibited density-dependent growthinhibition withsignificant decrease in mitotic activity and cellularproliferation. There was no difference inthe growth pattern between cells grown on plastic wellsand those cultivated on collagen discs(Fig. 2 a, b).Immunofluorescence and immunoperoxidase stainingfor Factor VIII related antigen(FVIIIR:Ag) revealed strongly positive, perinuclear, granular stainingof cells, thus confirmingtheir endothelial origin (Fig. 3a). Binding ofUlex europaeus type I (UEA I) lectin, a markerfor normal and neoplastic human endothelium (188,189), by HBMEC, was demonstrated bytheir strongly positive immunoperoxidase staining for UEA-1as previously described (Fig. 3b)(107). There were no other contaminatingcells in any of the cultures used for theseexperiments.Ultrastructurally, EC were elongated with overlapping processes(Fig. 4). Junctionalcomplexes with the characteristic pentalaminar configurationof tight junctions were present inareas of cell to cell contact (Fig. 5 a, b, c, d). Thecytoplasm was dense and containedprominent rough endoplasmic reticulum, frequentmitochondria and scattered 8 to lOnmintermediate filaments (Fig. 6a). Pinocytotic vesicleswere infrequently seen. Rod-shaped,44membrane bound cytoplasmic organelles with parallel arrays of tubular structures (WeibelPalade bodies) were not observed in the cytoplasm of HBMEC. A constant finding in all cellsexamined was the presence of dilated cytoplasmic vesicles or vacuoles bound by a singlesmooth limiting membrane. These vesicular structures were invariably located in the vicinityof the nucleus, in close association with the cisternae of the rough endoplasmic reticulum andwere absent from the most peripheral portions of the cytoplasm and the cell processes. Theyvaried in size from 0.15 to 1.1tm and they appeared empty or contained small amounts ofamorphous material (Fig. 6b).3.2 Immunocytochemical Localization of FVIIIR:AgIn cultures stained for FVIIIR:Ag with the immunogold technique, Snm gold particleswere distinctly localized within the vesicular profiles immediately adjacent to the roughendoplasmic reticulum (Fig. 7a) and close to Golgi cisternae in sections where the Golgiapparatus was present (Fig. 7b) as previously mentioned. The endoplasmic reticulum waslargely unstained with the rare exception of single isolated particles within its cisternae.Occasionally, labeled vesicles communicated directly with cisternae containing gold particles(Fig. 7c). The number of labeled vesicles and the degree of labeling (number of gold particlesper vesicle) varied among different cells. This is largely attributed to variable leakage ofintracellular proteins following permeabilization or to insufficient permeabilization andpenetration of individual EC by the antibodies. Low (0.01%) and high (0.05%) concentrationsof Triton X-100 resulted in poor labeling while a concentration of 0.03% was associated with45frequent and denser labeling. There was no staining of the cytoplasmicmembrane or thediscontinuous basement membrane-like material underlying the basal cell surface. The specificstaining was eliminated in control cultures incubated with normal mouse IgG or carrier buffer(Fig. 7d).3.3 Induction of Ia Ag expression on Primary cultures of HBMEC3.3.1 Effects of recombinant human IFN-yTreatment of cultures with IFN-y induced expressionof Ia Ag by EC, which wasdependent upon the concentration and length of exposure to IFN-y. Surfacelabeling wasobserved as early as 12 hours following incubation with 200U/mI in a small cell population(9.24±0.99%), increased up to 88.35±0.18% after 24 hours and reached 100% after 48 hours(Fig. 8). Ia Ag expression reached plateau levels after 2 days and persisted for4 days in thecontinuous presence of the cytokine. Expression was maximal with 100 - 200U/mI IFN-y(100% of cells) and minimal with 10U/ml (25.76 + 7%) (Fig. 9). Incubation with 20 U/ml ofIFN-y induced Ia Ag expression in 68.73±18.5% of cells, while 90.85±5.5% of cells werelabeled after treatment with 50U/ml. EC expressing Ia Ag showed diffuse surface staining inthe form of dark brown-black, granular deposits (Fig. bA). In marked contrast,untreated ECinvariably lacked Ia Ag expression as indicatedby their consistently negative staining withimmunogold (Fig. lOB). The staining intensity varied withthe concentration and length ofincubation with IFN-y. Thus, labeling was lessintense in cells incubated with 10 to 20U/mlfor 4 days or with 200 U/ml for 12 to 24 hours (Fig.bC), and most dense in cultures treated46with higher concentrations for 3 to 4 days (Fig. bA). Within the same culture, the larger cellswere usually stained most intensely. There were no differences in Ia Ag expression amongHBMEC monolayers originating from different individuals and subjected to identical cultureconditions and IFN-y treatment. Staining was not observed in control cultures incubated withnormal mouse IgG, carrier buffer or irrelevant antibody.In monolayers co-incubated with IFN-y and anti-IFN-y mAb for 4 days, induction of IaAg was completely abolished (Fig. 1OD) indicating that IFN-y specifically induces expressionof Class II MHC molecules on human brain EC. Treatment of cells with 200 U/mi IFN-yfollowed by withdrawal and culture in regular growth media resulted in complete reversal of IaAg expression and negative staining of the cultures.Ultrastructural examination following immunogold labeling showed that Ia Ag wasreadily detectable on the apical surface of EC. Gold particles were found at the cell membranewith a tendency to localize on or near thin cytoplasmic processes (Fig. ha). The basal cellsurface was not labeled. No labeling was seen in untreated control cultures (Fig. bib).3.3.2 Effects of recombinant human IFN-f3Treatment of primary HBMEC cultures withIFN-13for 4 days at concentrations of 100- 6,000 U/mi failed to induce surface expression of Ia Ag as indicated by the negative stainingof EC (Fig. 12a). Coincubation of EC with IFN-y and 3 resulted in downregulation of Ia Agexpression in a dose-dependent fashion. Thus, expression was decreased by approximately40% in monolayers treated with 100 U/miIFN-13and 100 U/ml IFN-y for 4 days. Increase of47the IFN- concentration to 250 U/mi and 500 U/mi resulted in 62% and 79% suppression,respectively (Fig. 12b). Downregulation of Ta Ag expression wasmaximal (89%) followingcoincubation with 100 U/ml IFN-y and 2,000U/mi IFN-; however, complete inhibition of IaAg expression was not observed even with IFN-3 concentrationsas high as 6,000 U/mi (Fig.13).3.4 Kinetics of the downregulation of Ia Ag expression by IFN-In order to obtain greater insight into the temporal effects of IFN-3on the induced IaAg expression by HBMEC, several treatment protocols wereapplied. Treatment of HBMECwith 100 U/ml IFN-y for 4 days induced Ia Ag expressionin 86% of the EC. Incubation of themonoiayers with a combination of IFN-f3 and IFN-y (6,000U/ml and 100 U/ml, respectively)for 4 days, significantly downregulated Ta Ag expression with positiveimmunogold staininglimited to <20% of EC (Fig. 14). Similar levels of downregulationwere also achieved whenBC were pretreated with IFN- (6,000 U/mi) for 2 days, followedby a combination ofIFN-13and IFN-y (6,000 U/mi and 100U/mi respectively) for another 4 days (Fig. 14).Interestingly, a much less significant decrease in TaAg expression was noted when cultureswere pretreated for 2 days with IFN-y(100 U/ml), followed by a combination ofIFN-13and‘(6,000 U/ml and 100U/ml respectively) for another 4 days. However, when cultures werepretreated for 2 days with a combinationof IFN- (6,000 U/mi) and y (100 U/ml), followed bya 4 day incubation with 100 U/mI IFN-y, no suppressionof Ia Ag expression was detected(Fig. 14).48These results indicate that downregulation of Ia Ag expression byIFN-13is mosteffective when HBMEC monolayers are coincubated with both cytokines with or withoutpretreatment withIFN-13alone. However, IFN-y-induced Ia Ag expression is not suppressedwhen treatment with IFN-3 and y is preceded or followed by incubation with IFN-y.The effects ofIFN-13on the IFN-y induced Ia Ag expression by HBMEC was furtherdetermined by Enzyme linked Immunosorbent Assay (ELISA) performed on primaryconfluent HBMEC cultures. Various concentrations of IFN- (100 to 6,000 U/ml) were usedin combination with an optimal concentration of IFN-y (100 U/ml) and different treatmentprotocols, similar to the ones used for immunohistochemistry were applied. The results weresimilar to those obtained by immunohistochemistry. Thus, treatment withIFN-13failed toinduce expression of Ta Ag. Coincubation with IFN-f3 andyresulted in significantdownregulation of Ia Ag expression that was dependent upon theTFN-13concentration (Fig.15). Significant reduction in Ia Ag expression was noted in cultures treated with 100 U/mIIFN-y in combination with 100 U/ml IFN-3 for 4 days (Fig. 15). Further suppression of Ta Agexpression was observed with higher concentrations of INF-3 (500U/mI to 6,000 U/ml),however, complete inhibition of Ia Ag expression byIFN-13was never achieved (Fig. 15).Downregulation of Ia Ag expression was maximal when EC were pretreated with IFN-f for 2days, followed by coincubation with IFN-3 and y for another 4 days. In monolayers incubatedwith IFN-y for 2 days followed by a combined treatment with IFN-y andifor 4 days, asimilarly significant suppression of Ta Ag expression was obtained.493.5 Effects of IFNj’ and 1FN43 on cell morphology, organizationand growth3.5.1 IFN-yPrimary cultures of HBMEC grownin regular medium in the absence of IFN-y formedhighly ordered confluent monolayers of elongated,closely associated, contact inhibiting cells(Fig. 16a). EC treated with 200U/mi IFN-y for 3 to 4 days acquired a spindle-like shapeandlong attenuated processes. These markedly elongatedcells frequently arranged themselvesinill-defined whorls and exhibited prominentoverlapping, thus contributing to auniqueappearance of the monolayers (Fig. 16b).These changes were most conspicuousunder SEM examination. Undernormal cultureconditions, elongated HBMEC growin close contact to each other and displaydistinctmarginal folds in areas of cell-to-cell contact(Fig. 17a). In contrast, EC treatedwith IFN-ybecame attenuated and their long,thin processes often extended over and coveredadjacentcells (Fig. 17b). As a resultof this rearrangement, intercellular contactsand marginal foldsbecame less prominent andthe monolayers lost their highly organizedappearance. The abovemorphological changes were reversed4 days following withdrawal of the cytokinefrom theculture medium and were not observedin cultures co-incubated with IFN-y andanti-IFN-yantibody.The effect of IFN-y on the growthof HBMEC was less profound. Thus, thenumber ofcells in primary HBMEC culturestreated with 150U/ml IFN-y from day 1, was slightly lessthan that in control cultures (Fig.18). This slight inhibitory growth effectof IFN-y providesfurther support to the observationthat re-arrangement and overlappingof HBMEC is the direct50effect of the cytokine and not the result ofcell overgrowth.3.5.2 IFN-Primary cultures of HBMEC grownin media containing IFN- (6,000 U/ml) for 4days showed no changes in cellmorphology and organization and were morphologicallyidentical to untreated cultures (Fig. 19a). When monolayerswere coincubated with IFN-j3and IFN-y (6,000 U/mi and 200U/mi, respectively) for 4 days, changes in cell shape andorganization of the monoiayers induced by IFN-y, werenot detected (Fig. 19b). Similarly,scanning EM studies of EC treated with IFN-f3 alone,or a combination of IFN-3 and IFN-y,confirmed the normal morphology and growth patternof HBMEC and the absence of themorphological phenotype and overlapping inducedby IFN-y (Fig. 19, arrows).On the other hand, the effect of IFN- on thegrowth of HBMEC was profound:inhibition of growth was observed whenHBMEC were treated from day 1 with IFN-3alone(1,000 U/mi) or a combination ofIFN-13and IFN-y (1,000 U/mi and 100U/mi, respectively)when compared to untreated cultures(Fig. 18).3.6 Permeability of HBMECmonolayersIn order to examine if IFN-y, in parallel to morphologicalchanges, also inducedchanges in the permeability of themonolayers to macromolecules, confluent treatedanduntreated cultures were incubated withHRP and the labeling of intercellular contactsandcytopiasmic vesicles was assessedultrastructurally. Intercellular contactsthat impeded the51tracer entirely or were penetrated only fora short distance from either the apical orbasal cellsurface by HRP, were considered impermeable.In untreated cultures, 75.2% ofinterendothelial junctions prevented the passageof HRP, in contrast with 36.6% in culturesincubated with the cytokine for 4 days (Table1). In untreated monolayers, EC formed a singlecell layer and were bound togetherby junctions, most of which were notlabeled with thetracer (Fig. 20a). Focally, HRP penetratedan intercellular contact for a short distancefrom thebasal aspect of the monolayer before being arrestedat a junctional complex of an otherwiseintact cleft (Fig. 20b). In treated cultures,interendothelial clefts were often penetratedby thetracer throughout their entire length(Fig. 20c). Overlapping of EC resulted in the formationof2 or more layers. Horseradish peroxidaseoften penetrated the intercellular clefts betweenECat the top layer, and extensive deposits werefound between adjacent cells at the lowerlayers(Fig. 20d). The number of cytoplasmic vesicleslabeled with HRP was equally low in controland experimental cultures (Table1), indicating that, contrary to the prominentconformationaland organizational changes, the pinocytoticactivity of HBMEC is not affectedby IFN-ytreatment.3.7 Lymphocyte characterizationThe different subsetsof lymphocytes obtained by the nylon-wool separationtechniquewere characterizedby using mouse mAbs directed against humanT cell surface molecules ofinterest. The results were determinedby Coulter cytometry with the Epics ProfileAnalyzer.Greater than 90% of cells (91.3±2.8%) recovered after nylon-wool seperationwere T cells.52When peripheral blood lymphocyteswere activated by incubationwith mouse anti-humanLeu-4 (CD3) monoclonal antibodyfor 3 days at 37 °C, up to91.5±6% of cells were Tlymphocytes. Stimulation with anti-CD3resulted in more than 2 foldincrease in Interleukin2 receptor (IL-2R) expression as comparedto resting T lymphocytes (Fig.21). Scanning andtransmission EM studies showed thatthe surface membrane of activatedT cells appearedruffled with numerous folds on thecell surface. In contrast, resting lymphocytesexhibited asmooth surface membrane. Subsequently, anti-CD3treated T cells provided morphologicaland functional evidence of activation.3.8 Human T-iymphocyte adhesionto untreated and cytokine-stimulatedHBMECConfluent cultures of HBMEC grownon plastic wells for 7 days were treatedwithIFN-y (150 U/mI) or IFN- (2,000 U/ml),or a combination of IFN-y (150 U/mi) and1(2,000U/mi) for 3 days. Controls consistedof monolayers grown to confluency in theabsence ofcytokines.By light microscopy, following immunoperoxidasestaining for leukocyte commonantigen, a small number of resting lymphocytesadhered to untreated HBMEC(50±10 Tcells per mm2of EC monolayers)(Fig. 22, 25). Treatment of HBMEC withIFN-y for 3 daysresulted in more than 3 fold increasein adhesion over control values (165 +13 T cells permm2 of EC monolayers) (Figs 23, 25). Treatmentof the monolayers withIFN-13had no effecton the basal adhesion of lymphocytes to endothelium(p = 0.953) (Figs. 24, 25). In culturesincubated with both cytokines,adhesion was not significantly different fromthat observed in53control cultures(p= 0.004) (Fig. 25).Examination by SEM revealed thatuntreated HBMEC monolayersconsisted of closelyassociated cells with marginal folds inareas of cell to cell contact.A similar morphology andgrowth pattern was observed in ECcultures treated with IFN-f3 or witha combination of IFN-yand Resting T lymphocytes first adheredto the endothelium by extendingpseudopodia thatcontacted the endothelial surface(Fig. 26a). Eventually they positionedthemselves betweenadjacent EC (Fig. 26b), andbegan migrating across the monolayer(Fig. 26c). Lessfrequently, lymphocytes were seen penetratingthe apical EC plasma membraneand movingthrough the endothelial cytoplasm(Fig. 26d). Monolayers treatedwith IFN-y exhibitedelongation and overlapping of EC.A large number of lymphocytes establishedcontact withthe endothelium via pseudopodia (Fig.27a), and singly or in smallaggregates alignedthemselves preferentially alongthe borders between adjacent ECin preparation for crossingthe monolayers (Fig. 27b). Adhesionand penetration of EC cytoplasmby lymphocytes wasencountered much less frequently.Ultrastructurally, lymphocytes firstestablished contact withintact or cytokine treated EC by extendingfinger-like cytoplasmic processesto the surface ofthe endothelium. The two cell membranesbecame closely apposed.3.9 Adhesion of activatedT-lymphocytes to untreatedand cytokine stimulatedHBMECActivation of T-lymphocytes withanti-CD3 resulted in a threefold increase inadhesion to untreated ECover control values (202+ 36 activated T cells permm2 of EC54monolayers) (p = 0.0 10) (Figs. 28, 31).Following pretreatment of EC with IFN-y, adhesion ofactivated T cells to endothelium was 2 fold greater than adhesionto untreated EC (403±29activated T cells per mm2 of EC monolayers) (Figs. 29, 31)(p= 0.012). Pretreatment ofHBMEC with IFN-j3 had no effect on the adhesion of activated Tcells to the endothelium.Preincubation of EC with a combination of IFN-y andi,however, resulted in levels ofadhesion comparable to those obtained when activated T cells were incubatedwith untreatedEC (Fig. 30, 31), indicating that IFN-3 downregulates the IFN-y-mediatedincrease in adhesion(218 activated T cells per mm2of EC monolayers)(p0.788) (Fig. 31). Examination bySEM revealed prominent changes in the morphology of activated Tlymphocytes whichappeared larger and exhibited a ruffled cell membrane with numerousfolds. Activatedlymphocytes adhered to the endothelium in large numbers (Fig. 32a-c). They appearedconsiderably larger than resting T cells and their surface was decorated with numerous foldsand cytoplasmic projections. As observed with resting T cells, activated lymphocytesestablished close contacts with EC by means of cytoplasmic projections and usually positionedthemselves along the borders between adjacent EC in both untreated and cytokine-treatedcultures in preparation for migration. Direct penetration of the endothelial cytoplasm byadherent lymphocytes was rarely observed. In such instances, a protuberance on the apicalsurface of the endothelium, having the size and shape of an activated T cell, indicatedmovement through the EC cytoplasm (Fig. 32, arrows). Ultrastructurally, activatedlymphocytes displayed abundant cytoplasm, increased numbers of mitochondria and variablenumbers of cytoplasmic vacuoles containing amorphous, flocculent material (Fig. 33). The55cell surface was extremely irregular due to the presenceof numerous thin, finger-like, variablyundulating, cytoplasmic processes. Several pointsof close cell-to-cell contact betweenendothelium and processes of adherent lymphocyteswere present (Figs. 33, 34).3.10 Effects of blocking antibodies on lymphocyteadhesionThe ability of mAbs to IFN-y and human HLA-DR to blockthe adhesion of restingand anti-CD3 activated T cells to HBMECwas examined. In monolayers coincubated withIFN-y and mAb to IFN-y for 3 days, adhesion ofresting and activated T-lymphocytes wassignificantly decreased(p = 0.002 and p = 0.001, respectively) (Figs. 25, 31). When ECcultures were treated with IFN-y for 3 days, followedby incubation with mAb to humanHLA-DR for 2 hours prior to incubation of T cells withEC, marked suppression of IFN-yinduced adhesion of resting and activated T-cells was observed(p = 0.012 and p = 0.014,respectively) (Figs. 25, 31, 35, 36).3.11 Transendothelial migrationof resting T lymphocytesTransendothelial migration of restingT lymphocytes across untreated HBMECmonolayers was minimal (Figs. 37a, 38). Significantincrease in migration, up to 3 fold, wasobserved when cerebral EC were pretreatedwith an optimal concentration of IFN-y (150U/ml), known to induce maximalTa Ag expression, for 3 days prior to incubation with thelymphocytes(p < 0.001) (Figs. 37b, 38). In contrast, IFN- treatment had no effect onmigration as the numbers ofT cells detected underneath the EC monolayers werecomparable56to those that migrated across untreated HBMEC(p = 0.304) (Fig. 38). Moreover, the INF-ymediated increase in migration was markedly suppressed when EC were preincubatedfor 3days with a combination of IFN-y and IFN- and then allowed to interact with therestinglymphocytes for 3 hours (Figs. 37c, 38). Adhesion of lymphocytes directly to collagenmembranes in the absence of EC was not observed. The results indicate thatIFN-13significantly downregulated the IFN-y-induced increase in transendothelial migration(p <0.001).One tm thick, toluidine blue stained cross sections of the monolayers revealed that Tcells initially attached and subsequently moved across the endothelium. At the end of theirmigration, lymphocytes positioned themselves underneath the monolayer between EC and thecollagen membrane and assumed a flattened, elongated shape. The endothelial monolayersoverlying the migrated lymphocytes appeared to retain their continuity (Fig. 37a to c).Examination by TEM revealed that resting T-lymphocytes initiated their migrationacross the EC monolayers by directing one or more cytoplasmic processes betweentwoadjacent EC (Fig. 39a). Eventually, a small segment of the cytoplasm, without thenucleus,was inserted between the two EC and was followed by the remaining cytoplasm and nucleus(39 b, c). After passing between the EC, the lymphocytes became elongated and flattenedandremained between the overlying EC and the underlying collagenmembrane (39d).Throughout the migratory process, lymphocytes remainedin close contact with the EC, theadjacent plasma membranes of the two cell types beingtightly apposed (Figs. 39a to d).Infrequently, lymphocytes migrated by moving through the cytoplasmof EC. A lymphocyte57was considered moving through rather than between adjacent EC only when the cytoplasm ofthe EC completely surrounded the lymphocyte (Fig. 40). At the end of the migration period,EC monolayers rapidly assumed their continuity and appeared structurally intact.Culturestreated with IFN-y showed variable overlapping of EC. Lymphocytes that had completed theirmigration across adjacent EC of the top layer, would then proceed to migrate across thesecondlayer of EC. The integrity of the monolayers was reestablished once resting T cells completelymigrated across the untreated/cytokine treated EC (Fig. 41 a, b).3.12 Migration of activated T lymphocytes across untreated and cytokinetreatedHBMEC monolayersTo determine whether nonspecific activation of T lymphocytes had any effectonmigration, peripheral blood lymphocytes treated with anti-CD3 antibody for 3 days wereincubated with HBMEC for 3 hrs.Activation of T cells resulted in a four fold increase in the number of cellsthatmigrated across untreated monolayers of HBMEC as compared with the migrationof restinglymphocytes across untreated brain endothelium(p < 0.001) (Figs. 38, 42). Pretreatment ofEC with IFN-y further increased the migratory response by approximately 30%(p< 0.00 1)(Fig. 38). In contrast,IFN-13had no effect on the basal level of migration of activated T cells(p = 0.341). Moreover, when HBMEC were preincubated with a combination of IFN-y andIFN-13,the level of migration was not different from that obtained whenactivated Tlymphocytes migrated across untreated EC monolayers(p = 0.268), indicating thatIFN-1358downregulated the IFN-y-mediated increase in migration (Fig. 38).Ultrastructurally, large numbers of activated lymphocytes migrated across theendothelial monolayers. Although migration proceeded in a fashion similar to the oneobserved during migration of resting T cells (Figs. 43 a, b), crossing of the monolayers bymeans of moving through the cytoplasm of EC, was not observed in any of the materialexamined. Migration of activated lymphocytes was not associated with any apparentdisruption of the monolayers (Fig. 43b). Lymphocytes that had completed their migrationacross adjacent EC of the top layer, would then proceed to migrate across the next layer of EC(Fig. 44).3.13 Effects of blocking antibodies on lymphocyte migrationT lymphocyte migration across IFN-y-treated HBMEC monolayers was significantlyblocked by preincubation of EC with a mAb to human HLA-DR (p < 0.00 1) regardless of theactivation status of lymphocytes. The level of suppression approximated that obtained bytreating EC with a combination of IFN-y and IFN-f (Fig. 38). These results suggest that classII MHC molecules (Ia Ag) play a central role in the IFN-y-induced upregulation of Tlymphocyte migration across HBMEC, irrespective of the activation status of the lymphocytes.3.14 Effects of calcium ionophore A23187, EGTA and IFN-y on the constitutivepathway of factor VIIIR:Ag releaseTreatment of the monolayers with 10.tMCa2+ionophore A23187 for 10 minutes59resulted in almost complete loss of staining (Fig. 45a), while incubation with 1mMEGTA for10 minutes was associated with slightly increased numbers of labeled vesicles (Fig.45b).When the monolayers were preincubated with200U/ml IFN-y for 24 hours, there was asignificant increase in the number of immunostained vesicles over the untreated cultures(Fig.45c). In order to quantitate these findings, the number of labeledand unlabeled vesicles wascounted in 100 cells in each group of treated and in untreated cultures.Fig. 46 summarizesthese results. The differences reflect variations in the percentageof immunostained vesicles.There was no appreciable difference in the number of gold particlesper vesicle betweentreated and untreated cells. The difference in the number of labeled vesicles between controlsand IFN-y treated EC was statistically significant(p = 0.000), while no significant differencewas found between controls and EGTA treated cultures(p = 0.21).60DISCUSSION4.1 INFLUENCE OF CYTOKINES ON IaAg EXPRESSION ON HBMECThe first specific aim of this thesis was to determine whetherIa Ag is constitutivelyexpressed in primary cultures of HBMEC and whether its expressioncan be induced andmodulated in vitro by the cytokines IFN-y and IFN-.4.1.1 Human brain microvessel ECHuman brain microvessel EC in primary cultureform confluent contact-inhibitingmonolayers composed of elongated, closely associatedcells. EC are uniformly positive forFactor VIIIR:Antigen, the most specific marker for cellsof endothelial origin, and bind thelectin Ulex europaeus, a marker for human EC. CulturedHBMEC contain few pinocytoticvesicles and are bound together by tight junctionalcomplexes that restrict the paracellularmovement of macromolecules. Primary culturesof HBMEC retain their human EC properties,exhibit morphological and permeability characteristicssimilar to cerebral endothelium in vivoand, therefore, provide a useful in vitromodel for studying the biology and immunopathologyof these cells.4.1.2 Induction of Ia Ag expression on primary culturesof HBMECThe present studies demonstrate that humanrecombinant IFN-y induces de novo61expression of class II MHC antigen(Ta Ag) by HBMEC in primary culturein a time andconcentration - dependent manner.Unstimulated HBMEC grown under standardcultureconditions do not constitutively express TaAg as indicated by lack of immunogold stainingonlight and electron microscopy.Previous in vivo immunohistochemical studieshavedemonstrated absence of IaAg expression by EC within the normal humanCNS with lowlevels of reactivity detected in blood vesselsof patients with brain neoplasms, abscesses,autoimmune connective tissue disease,cerebral infarcts and in older patientswithoutidentifiable CNS lesions (68, 190- 192). Although the EC used in our studieswere isolatedfrom normal brains of several donors with a wide agedistribution, expression of TaAg was notobserved in any of the untreatedcultures. A similar lack of constitutiveexpression of class TImolecules has been observed in primary culturesof rat brain endothelium (70), in freshlyisolated human umbilical vein EC (HUVEC)(22, 193), in serially passaged cultures of humancerebral vascular EC (136) and HUVEC(194), as well as in human glioblastoma multiformecells (195), and cultured adult humanastrocytes (25) maintained under normalcultureconditions. Contrary to thesereports, EC of normal guinea pig CNS displaysurface MHC invivo and in vitro (196) and minimalbasal expression has been reported inprimary cultures ofrhesus monkey cerebral endothelium(197), while cultured rat heart vascular ECconstitutivelyexpress considerably higher levelsof Ia Ag (64). It is apparent, from the abovestudies, that thepresence of Ia Ag on normal, unstimulatedvascular endothelium may vary among differentspecies and vascular beds.Previous studies on Ta Ag inductionby IFN-y on HUVEC report a rapid increaseof62MHC class II mRNA that precedes surface expression by 1 - 2 days and rapidly declines toalmost undetectable levels following withdrawal of the cytokine, while surface expressiondeclines slowly after 4 days (80). In HBMEC, removal of IFN-y from the media results inuniform loss, rather than decrease to lower levels, of class II MHC surface expression after4days. Rat heart endothelium, however, behaves in a much different way, since withdrawalofIFN-y is not followed by return of the Ia Ag expression to basal levels after 3 days (64).4.1.3 Surface localization of Ia Ag on HBMECPrevious immunohistochemical studies on MS and EAE have demonstrated thatsurface expression of Ta Ag on EC is discontinuous along the microvessel lumen, so thatIa+cells are interposed between EC lacking Ia Ag expression (68, 139). A similarly variableexpression of Ta Ag was observed in vitro when HBMEC were treated with low concentrationsof IFN-y or with higher concentrations for less than 2 days. Taken together with the in vivostudies, these observations may indicate individual cell variationin the regulation of class IIMHC molecule expression.Induction of Ia Ag expression on HBMEC was restricted to the apical portionof thecell membrane. Immunogold particles were not identified on the lateral or basal cell surfaces.Our findings correlate with previous immunohistochemical studiesin acute EAEdemonstrating Ta expression on the luminal but not abluminal surface of cerebralmicrovesselEC (139) and with similar observations in a variety of epithelial cells inmice treated withIFN-y (72). Although the mechanisms responsible for the asymmetrical presentationof Ta Ag63on the cell membraneare not known, polarizationof expression is probablyof functionalsignificance since itwould enable circulatingT lymphocytes to recognizeantigen inassociation with classII MHC molecules on theluminal surface of thecerebral endotheliumand then migrate to sitesof inflammation.4.1.4 Effects of IFN-j3on Ia Ag expression by HBMECHuman recombinant IFN-3failed to induce expressionof Ia Ag on HBMEC atallconcentrations tested.A similar lack of IaAg expression has been previouslyreported incultured adult humanastrocytes (25), human dermalmicrovascular EC(84), and humanglioblastoma multiforme cells(26) treated with IFN-3.In our studies, the results obtainedfrom the immunocytochemicalstaining and ELISA indicatethat IFN- downregulatestheIFN-y-induced Ia Ag expressionin a concentration-dependentmanner. Immunocytochemicallabeling indicates thetotal number of Ia-positivecells in the cultures,but providesnoinformation on the membranedensity of HLA-DR moleculesper cell. ELISA providesrelative measurementof the total density of HLA-DRmolecules within the culturebut with noindication of thenumber of cells expressingIa Ag. The suppressive effectof IFN-f3 on Ia Agexpression has been previouslyobserved in other EC systems(105, 106). Downregulationofthe IFN-y inducedIa Ag expressionbyIFN-13 has also been reported in cultured adult humanastrocytes (25), humanglioma cells (26), murinemacrophages (27, 40),blood monocytesisolated from MS patients(28), and in an astrocytomacell line (41).644.1.5 Regulatory mechanism of TaAg expressionIt has been shown that the inductionof Ta Ag on macrophages byIFN-y operates at thelevel of transcription and requiresde novo synthesis of a new protein(s)(198). It has beenreported that the plateau values ofHLA-DR mRNA content in HUVECand human dermalfibroblasts treated with IFN-y precede maximalsurface expression by1 to 2 days (80). Thiscould explain the lag periodof 12 to 24 hours between additionof IFN-y to the media anddetection of Ta Ag surface expressionby immunohistochemistry on HBMEC(127).Interferons-13 and y bind to different receptors on the cell surface (21) and theinhibitory effectofTFN-13 onIFN-y induction of the Ia Ag genesis exerted at the transcriptional level(41, 75).It has been suggested that there isa complex interplay of trans-actingfactors involved inmodulating the expression of the Iagenes product and the subsequentexpression of theirpeptide products on the cell surface(41, 75). The fact that IFN- failedto completely inhibitthe induction of Ia Ag by IFN-y isnot fully understood at the presenttime; further studies arerequired in order to elucidatethe exact mechanism(s).4.1.6 Kinetic studieson the modulation of Ta Ag expression by interferonsy and13In our studies, the most significantdownregulation of the IFN-yinduced Ia Agexpression was found whenHBMEC were either coincubated withthe two cytokines for 4days or pretreated with IFN-13for 2 days and then treated witha combination of IFN-13 andyfor 4 days (approximately80% reduction). In contrast, significantdecrease in Ta Agexpression was not observed(0% to 15% suppression) when EC werepretreated with TFN-1365and y for 2 days, followed by another 4 day treatment with IFN-y, or pretreated for 2 dayswith IFN-y followed by coincubation with IFN-3 and y for another 4 days. Similarobservations have been reported in cultured adult human astrocytes. The addition of IFN-ct or3 24 hours after incubation of astrocytic cultures with IFN-y did not significantly alter HLADR expression, while IFN-a orIadded 24 hours before or at the initiation of incubation withsuboptimal concentrations of IFN-y reduced the extent of HLA-DR expression (25). Furtherwork using human astrocytoma cell lines demonstrated that the suppressive effect of IFN-3 onthe HLA-DR induction by IFN-y was relatively gene-specific since IFN-j3 could not impairthe induction of intercellular adhesion molecule-i (ICAM-i) expression by IFN-y in these celllines (4i). Similar results were observed in HDMEC, and the authors speculated that theeffect of IFN-y on HDMEC may be mediated through multiple distinct pathways which can beindependently regulated (i06). Consequently, the results could not be explained by IFN-f3downregulation of IFN-y receptors or defective receptor-linked signal transduction. Theinhibition was also suggested to be tissue-specific becauseIFN-.13did not antagonize IFN-yinduction of HLA-DR expression in human monocytes (4i). The results of the present studiesindicate that in order to effectively suppress the induction of Ta Ag by IFN-y in vitro,IFN-13must be present continuously in the culture media. It is also shown that once the cells havebeen activated by IFN-y, downregulation of Ia Ag expression does not occur in the continuedpresence of IFN-j3 in the culture media. Taking into consideration that increased levels of IaAg have been associated with induction of autoimmune disorders of the CNS such as MS (68,i99), and that antibody blocking directed against class II MHC determinants can prevent the66induction of experimental autoimmune disease(200), these findings may partly explain thesignificant therapeutic potential ofTFN-13in MS (281). Inaba et al. have shown that thereis acorrelation between downregulationof Ta Ag expression and reduced levels of antigenpresentation by macrophages in vitro(27). Together with the finding that IFN-y is unsuitablefor use as a therapeutic agent in MS(15), Joseph et al. suggested that administration of IFN-flin patients with MS could have beneficialeffects if reduced Ta Ag expression occurs,and thereis reduced antigen presentation inthe CNS (26). The expected results would be longerremission periods or fewer relapsesin MS patients. Therapeutic applicationof recombinantinterferon beta-lb for the treatment ofMS has recently reported that the cytokineis welltolerated and has a beneficial effecton the course of relapsing-remitting MS (281). Basedonthe results obtained from our studieson HBMEC and others (26 - 28, 40), a repetitive dosingwith IFN-3 may be essentialto effectively downregulate Ia Ag expression.4.2 EFFECTS OF INTERFERONSy AND f3 ON THE MORPHOLOGICALPHENOTYPE AND GROWTH OFHBMEC, ORGANIZATION OF THEMONOLAYERS AND PERMEABILITYTO MACROMOLECULESThe second specific aimof this thesis was to determine whetherIFN-y and IFN-3 exertantiproliferative effects on HBMECand whether treatment with these cytokinescanmodulate the morphological phenotypeand organization of the EC cultures,and alterthe permeability of the monolayersto macromolecules.674.2.1 Effects of IFN-y and IFN-3 on HBMEC growthThe anti-proliferative effect of IFN-y on primary cultures of HBMECcorrelates withprevious studies demonstrating inhibition of cell growth by IFN-y inducedon extracerebrallarge and small vessel endothelial cultures in a dose-dependent manner (84, 126, 201,202) andpossibly through modulation of the EC growth factor receptors (202). Lower concentrationsofIFN-y (10 - 100 U/mi), however, appear to have a stimulating effect on cultured HUVECbothin the absence and presence of EC growth factor (203). In addition, IFN-y significantlyinhibitsformation of endothelial tubular structures in in vitro models of angiogenesis(204, 205).When the cells were treated withIFN-13or a combination of IFN-3 andy, significantgrowth inhibition was detected. The antiproliferative effectsof IFN-y and especially of IFN-f3on primary cultures of HBMEC correlate with previous studiesdemonstrating inhibition ofcell growth by these cytokines induced on human dermal microvascularEC (HDMEC) (84),human glioblastoma multiforme cells (195), cultured human brain tumors(206), and humanvascular smooth muscle cells in vitro (207).4.2.2 Effects of interferons y and on HBMEC morphologyand organization of the ECmonolayersIFN-y-treated EC undergo unique changes in their morphologyand organization, whichare associated with a considerable increase in the permeabilityof confluent cultures tomacromolecules. Treatment of HBMEC with IFN-yinduces marked elongation of EC,prominent overlapping and frequent arrangement ina whorled pattern. A similar alteration of68the morphological phenotype and monolayer organization has been previously reported incultures of HUVEC (126, 202) and HDMEC (84) treated with IFN-y for 3 to 4 days and hasbeen shown to be associated with reorganization of the cytoskeletal filaments and considerableloss of the fibronectin matrix (126).It has been previously demonstrated that IFN-3 alters the morphology of culturedHDMEC.IFN-13treated cells become spindle - shaped, an alteration which was also observedin cultures treated with IFN-y in comparison to untreated cells that showed the typicalmorphology of human EC (84). In contrast, morphologic changes were not observed inHUVEC treated with IFN- (126). Furthermore, treatment of HBMEC with IFN-3 failedtoinduce structural or organizational alterations on the monolayers; in fact, it inhibitedthemorphological changes induced by IFN-y when the cells were incubated simultaneously with acombination of IFN- andy. These observations further emphasize the heterogeneity whichexists between EC derived from different organs or species (19).Our studies, therefore, demonstrate that IFN-3 downregulates the expressionof Ia Aginduced by IFN-y on HBMEC and alone or in combination withIFN-y has greaterantiproliferative effect on these cells than IFN-y. In addition,IFN-13downmodulates the IFNy-mediated changes in cell morphology and organization of theEC monolayer which may berelevant to the in vivo immune response. These findings and the workof other investigatorswould indicate that in situ vascular changes take place in responseto cytokines generated atthe inflammatory site. Thus, IFN-y alone or in combination withother locally generatedcytokines, induces changes that may mimick immuneregulatory events which signal69endothelial preparation for inflammatory cells to adhereand transmigrate, while IFN- mayplay a negative regulatory role in inflammation or disorders upmodulatedby enhanced IFN-ysecretion. In fact, it has been reported that systemic administrationof IFN-f3 to MS patientsinhibits endogenous IFN-y synthesis in their peripheral blood mononuclear cells(38). At thepresent time, it is not known whether cerebral ECin vivo undergo the same or a similarspectrum of changes in response to cytokines in inflammatory fociof the human CNS.4.2.3 Permeability of IFN-y treated HBMEC monolayers to macromoleculesHuman cerebral microvessel EC in primaryculture are bound together by tightjunctions and have a paucity of cytoplasmic vesicles, twoimportant morphologicalcharacteristics of their in vivo counterparts(107, 186). Under standard culture conditions, thegreat majority of interendothelial junctions restrict thepassage of HRP. In cultures incubatedwith IFN-y, an increase in the permeabilityof the monolayers was observed that coincidedtemporally with changes in morphology and rearrangementof the cells. The number of labeledcytoplasmic vesicles was not increased in IFN-y treated monolayersindicating that increasedjunctional permeability is primarily responsiblefor the permeability changes of themonolayers. The mechanism(s) responsible for the increased junctionalpermeability are notknown at present. Recent in vitro studies have demonstratedthat tumor necrosis factor (TNF)treated aortic EC cultures undergo prominent cytoskeletalchanges similar to those induced byIFN-y alone or in combination withTNF, which are temporally related to increase inthepermeability of the monolayers to macromoleculesand are regulated by G protein (208). The70fact that leakiness of intercellular contactsappears concomittantly with the morphologicalchanges of the endothelium following IFN-ytreatment may indicate that physiologically??tightfltight junctional complexes fail to formduring the extensive rearrangement ofthe cellsand their cytoskeleton. However, othermechanisms, such as modulation of regulatoryproteinsor cell surface molecules by IFN-y cannot be excluded.Disruption of the BBB has beenpreviously described as an early and critical eventin the evolution of EAE (119, 120, 209,210). Recent electron microscopic studiesindicate that increased junctional permeability aswell as increased interendothelial space and migrationof inflammatory cells are primarilyresponsible for the increased permeabilityof the BBB to macromolecules in this disease(211).The functional significance of the in vitro morphologicaland permeability changes ofHBMEC, observed in this study, is presently unknown.If, however, similar changes areinduced in situ on cerebral EC by cytokinesreleased locally by activated T-lymphocytes,theywould provide an additional mechanism forthe opening of the BBB and could facilitatethetransmigration of inflammatory cellsfrom blood into brain across the endothelial barrier.4.3 SIGNIFICANCE OF Ia Ag EXPRESSIONBY HBMECExpression of Ia Ag in situby cerebral vascular endothelium has been previouslydemonstrated in autoimmune demyelinatingCNS disorders. Thus, class II MHC moleculeshave been localized on the surfaceof EC lining microvessels at the edge of demyelinatingplaques as well as within the adjacentwhite matter in acute, active and silent chronicMSlesions (68, 138). The presenceof Ia positive EC has also been documentedin acute EAE71(139, 140), while expression of Ia Ag by cerebral endothelium in chronic relapsing EAEappears to coincide with the appearance of inflammatory cell infiltrates and diminishes wheninflammation subsides (141). In addition, murine cerebral EC isolated from SJL mice withEAE are able to present antigen to sensitized syngeneic lymph node cells following incubationwith IFN-y in vitro (137). Contrary to these observations, cultured rat brain EC are noteffective at stimulating T-cell division and therefore, have not been considered important asantigen presenting cells (212). Recent studies using murine endothelial and fibroblast celllines to determine their capabilities in presenting antigen to helper T cells have reported thenecessary requirements for costimulatory signals (213 - 216). The lack of signals such as B7,CI’LA4 molecules on the surface of the potential antigen presenting cells can result in theinability of T helper cells to proliferate in response to a specific antigen. Consequently, theconclusions drawn by Pryce et al. (212) that rat brain EC are not important antigen presentingcells, prior to the realization that costimulatory signals may be required for the efficientfunction of antigen presenting cells, deserve further investigation. The present workdemonstrates that class II MHC molecules are not detectable on intact HBMEC isolated andcultured from normal human brain microvessels by the methods employed in our study, butcan be specifically induced in vitro by human recombinant IFN-y in association withprominent alterations in the morphology, organization and permeability of the monolayers tomacromolecules. Although the ability to present antigen by HBMEC has yet to beunequivocally proven, our findings indicate a possibly important role of the human cerebralendothelium in lymphocyte-endothelial interactions, lymphocyte recruitment and alteration of72blood-brain barrier permeability in immune - mediated CNS inflammation.4.4 ADHESION OF RESTING AND ANTI-CD3 STIMULATED LYMPHOCYTESTO UNTREATED, IFN-y and/or IFN- TREATED HBMECThe third specific aim of this thesis was to examine the effects of cytokine treatmentofHBMEC on the adhesion of resting and anti-CD3 stimulated lymphocytesto theendothelium.4.4.1 Activation of lymphocytesThe CD3-molecular complex is comprised of a series of noncovalentlylinkedpolypeptides (217). Monoclonal antibodies directed against the CD3 moleculeshave beenwidely used to study T cell activation (218, 219). The binding of anti-CD3antibodies to Tcells leads to the rapid hydrolysis of phosphatidylinositols and results in anincrease in freeintracellular calcium concentration, in generation of diacylglycerol, and activationof proteinkinase C (220, 221). It is notable that binding of mAbs toCD3 molecules clearly results in thegeneration of activation signals (220), but this situation has been reported tobe insufficient toinitiate T cell proliferation (222). Moreover, Ledbetter etal. (223) have demonstrated thatbinding of anti-CD3 antibody to T cells results in a rapid rise(4 to 6 fold) in cyclic adenosinemonophosphate (cAMP), and high levels of cAMP are knownto inhibit T cell growth. It hasalso been shown that other cAMP-elevating agents suchas prostaglandin E2, cholera toxin,73and the cell-permeable analog 8-bromo-cAMPcan inhibit nuclear IL-2 transcription anddecrease the stability of IL-2 mRNA (224, 225).Interestingly, other studies have shown that Tcells proliferate in response to soluble anti-CD3mAb in the presence of IL-2, a lymphokinecentral to the mediation of antigen-activated Tcell proliferation. IL-2 is produced by activatedT cells, and binds to the IL-2 receptor of secretingT cells and other antigen-stimulated cells(226). Since the expression of IL-2 receptoris reported to be increased when T lymphocytesare treated with anti-CD3 antibody (227, 228), activationof peripheral blood lymphocyteswith anti-CD3 mAb was confirmed in thisstudy by the upregulation of IL-2 receptor ratherthan by the conventional tritiated thymidine uptakeassay. The results obtained byFluorocytometry demonstrated more than 2 fold increasein IL-2R expression. By SEM,lymphocytes treated with CD3 mAb appeared larger thanresting T cells and their surfacemembranes exhibited a ruffled appearance in contrast to the smoothmembrane of resting Tcells, confirming their activated state. Moreover, transmissionEM studies showed numerouscytoplasmic folds and finger-like projectionsat the surface of activated lymphocytes alongwith a significant number of vacuoles in thecell cytoplasm. Subsequently, anti-CD3 activatedT cells are functionally and morphologically differentfrom resting T lymphocytes.4.4.2 Adhesion of resting lymphocytes to untreated,IFN-y and/or IFN-3 treated HBMECThe present studies demonstrate that lymphocyte adhesionto cultured HBMEC can bemodulated by treatment of the EC withIFN-y and/orIFN-13.A low basal level of adhesionbetween resting T cells and untreatedEC was detected. Pretreatment of EC withan optimal74concentration of IFN-y (150 U/mi) known to induce Ia Ag expression (127),significantlyaugmented lymphocyte-EC adhesion. Scanning EM studies demonstrated thata small numberof resting lymphocytes, with relatively smooth surface membranes, adheredto confluentmonolayers of untreated HBMEC. The lymphocytes lined up on the borders betweenadjacentEC, and occasionally adhered to the apical surface of the endothelium. Significant increaseinadhesion of resting lymphocytes to IFN-y-treated EC was noted by light microscopy andSEM.By SEM, increased numbers of resting T cells were detected along the overlappingprocessesof IFN-y-treated EC. The results suggest that the changes in the organizationof themonolayers induced by IFN-y may further facilitate lymphocyte migrationacross these EC.Previous studies on HUVEC (23, 134), rat retinal endothelium(156), and rat (229) and mousebrain EC (135) have also demonstrated IFN-y-mediated increase in lymphocyte-endothelialadhesion. Moreover, it has been shown that treatment of rat brain endotheliumwithcycloheximide, an inhibitor of protein synthesis, inhibits the IFN-y-mediatedincrease inadhesion, implying the requirement for new protein synthesis (154). It is notablethat theoptimal concentration of IFN-y required for maximal adhesive response varieswith differentspecies of EC. Our results, therefore, provide additional evidence forthe existence ofheterogeneity among EC of different organs or species with regard tocytokine responses.The adhesion of lymphocytes to mouse and rat brainEC has also been reported to increasewith the length of IFN-y treatment (from 4 hours to 2 days)when compared with the controls(135, 154), suggesting that different adhesion molecules with different kineticsof inductionmay participate in the lymphocyte-EC adhesive mechanismsover different time periods.75HBMEC treated for 3 days with IFN-j3 (2,000U/mi) showed no increase inlymphocyte adhesion as compared with the baselineof adhesion in untreated control.Moreover, IFN- actually suppressed the increase in adhesioninduced by IFN-y when brainEC were treated with a combination of IFN-y and IFN-f3. Thisobservation indicates apotentially important role of IFN-f in downregulatingimmune responses mediated, at leastpartly, by IFN-y.The mAb blocking studies indicate that de novoexpression of class II MHC Ag byHBMEC is largely responsible for the increased T lymphocyte-EC adhesion.Similar resultshave been reported in mouse brain EC (135). These investigatorsfurther confirmed theirobservations by transfecting a murine lung EC linewith cDNA for the class II MHCmolecules in order to demonstrate the role of Ia Ag in lymphocyte-ECadhesion. The adhesiverole of EC is totally distinct from any antigen presenting function, as thelymphocytes are non-activated and there is no antigen present in eithersystem (135). It is notable that completesuppression of IFN-y-enhanced adhesionby anti-human HLA-DR antibody was not achieved,indicating that other mechanisms, not related toDR antigens, operate during lymphocyte-ECbinding. MAb blocking studies in HUVEC have alsoreported similar observations (134).Working with retinal capillary EC, Liversidge et al.(230) have suggested that, if severaladhesion pathways are available for cellular interactions,then mAbs blocking one pathwaymay be ineffective in completely reducing the numberof cells bound, since alternative ligandswould be utilized. MAb blocking studieswith anti-human IFN-y indicate that the increaseinadhesion observed with resting T cellsis specifically mediated by IFN-y.76The results of the present study provide evidence that IFN-3,used at a concentrationknown to significantly suppress the IFN-y-inducedIa Ag expression (2,000 U/mi), candownregulate the increase in adhesion mediated byIFN-y. Taken with theIFN-13effects onthe IFN-y induced Ia Ag expression, these results suggest thattheIFN-13suppression of theIFN-y-mediated increase in adhesion may be largelydue to the downregulation of Class IIMHC molecules by IFN-f3.The role of class II MHC molecules in antigen presentation has been well documented(137); however, the role of class II MHC molecules in lymphocyte-ECadhesion remainscontroversial. Curtis (231) was the first to suggestthat class II MHC molecules may functionin the adhesion of non-activated lymphocytes to endothelium and,together with Rooney (232),they state that these molecules may also participate in contactinhibition between epithelialcells, a process which partially involves cell adhesion.Doyle and Strominger report that Blymphocytes expressing class II MHC molecules couldbind to CD4 transfected fibroblasts invitro and speculate that the interaction betweenthese two molecules would cause cell-celladhesion independently of antigen presentation (147).They also suggest that at morephysiological levels of expression, it ispossible that CD4 molecules and class II antigens helpto mediate low affinity, transient interactions among lymphocytes,and together with otherspecific and accessory adhesion molecules, functional cell-cellinteractions can take place(147). Studies with HUVEC systems have alsoshown that HLA-DR molecules playanimportant role in IFN-y-mediated HUVEC-lymphocyteadhesion (134). Furthermore, mAbblocking experiments implicate CD4-classII MHC interaction in IFN-y-induced endothelial77lymphocyte adhesion (145, 233). Interestingly, there is no significantdifference in theadhesion between autologous and allogeneic assays which suggeststhat the interaction is notsimply alloreactive, but may be part of a physiologicalmechanism for the adhesion, migrationand accumulation of lymphocytes at sites of chronic inflammation(145). It has also beendemonstrated that experimental allergic encephalomyelitis (EAE),a model disease for MS invivo, can be prevented by administration of monoclonal antibodiesto class II molecules(234). Alteration in the homing of lymphocytes to the brain in EAE has been implicatedas thepossible mechanism for the prevention of disease development in anti-Ia antibodytreatment(235). Finally, Ia Ag expression on EC in EAE has been shown toprecede lymphocyteinfiltration (66). Taken together, these results suggest an importantrole for class II MHCmolecules in the interactions between lymphocytes and cerebralEC and in the development ofthe disease process.The adhesion of circulating lymphocytes to brain EC in vivo takes place ina dynamicrather than in a static system. Furthermore, the adhesive capacityof Ia Ag in situ would be ofa much lower affinity than observed in vitro, since the concentrationof IFN-y and thesubsequent level of Ia Ag expression on individual EC and its distribution wouldprobably belower. Nevertheless, if under the influence of focallyincreased concentrations of IFN-y in thecerebral microvessel microenvironment, a minimal levelof initial adhesion could take placevia class II MHC molecules, followed by antigenpresentation to specific T cells, leading tothe production of more cytokines including IFN-y,class II MHC expression can then beelevated and subsequently, enhance lymphocyte-ECadhesion. Working with HUVEC,78Masuyama and his coworkers (134) have suggested that T cell recognitionof HLA-DRmolecules may be the signal for the initiation of subsequent adhesiveprocesses in whichcomplementary adhesion surface molecules becomeengaged. McCarron et a!. (236) havefurther speculated that the CNS-immune cell interactions maybe responsible for localizedalterations in the BBB permeability, resulting in the subsequent influxof non-specificinflammatory cells. In addition, the IFN-y-induced changes inEC morphology and monolayerpermeability, observed in our studies, may further facilitatethe movement of lymphocytesacross the BBB.4.4.3 Adhesion of activated lymphocytes to untreated,IFN-y and/or IFN- treated HBMECLymphocyte activation results in great increase in adhesionof activated T cells tountreated HBMEC, indicating that the activation statusof the lymphocytes plays an importantrole in lymphocyte-EC adhesion. The adhesive interactionis further augmented by treatingthe EC with IFN-y. Studies with HUVEC (150),human (230) and rat retinal EC (156), rataortic and brain microvascular endothelia (154, 155)and activated lymphocytes have alsoreported significant increase in lymphocyte-EC adhesion.These studies have shown that theadhesion between lymphocytes and ECis dependent on the state of cell activation: maximallevel of adhesion occurs when activatedT cells interact with cytokine-stimulated EC.Scanning EM studies further confirmthe light microscopic observations. Activatedlymphocytes preferentially adhere at theborders between adjacent and overlapping EC.MAb blocking studies with anti-human IFN-y indicatethat the enhanced adhesion of activated79lymphocytes to IFN-y-treated EC is specifically mediated by IFN-y. It has been reported thatirrespective of the state of cell activation, the level of lymphocyte adhesion to CNS-derivedendothelium is generally lower than that reported for extracerebral large and small vessel EC(154 - 156, 237, 238). Subsequently, this low level of adhesion may account for the limitedlymphocyte traffic through the CNS of normal healthy individuals. Furthermore, it is notablethat irrespective of the mode of lymphocyte activation (150, 154 - 158, 230) (i.e. ConA,Phorbol ester, Phytohaemagglutinin, anti-CD3 antibody), adhesion of activated lymphocytes toEC is significantly upregulated in comparison to that of resting T cells.Significant increase in adhesion of anti-CD3 activated T cells to intercellular adhesionmolecule-i (ICAM-1) substrates has been previously reported (159). ICAM-i is a member ofthe immunoglobulin gene superfamily; it is expressed constitutively by HBMEC and canbeupregulated by various cytokines such as IFN-y, TNF-cx and IL-13 (239). Its counter receptoris the lymphocyte function-associated antigen-i (LFA-i) which belongs to the integrin family;LFA-1 is expressed on T lymphocytes, not EC, and comprised of heterodimeric, divalentcation-dependent adhesion molecules (162). MAb blocking experiments directed againstICAM-i and the a and f3 subunits of LFA-1 molecules completely block the anti-CD3stimulated adhesion to purified ICAM-1 (159). Since there is no significant change in LFA-iexpression by T cells treated with anti-CD3 antibody versus resting lymphocytes, theauthorsconclude that the stimulated increase in T-cell adhesion seems to be duemainly to an increasein LFA-i avidity (159). Fluorescence-activated cell sorter (FACS) studies with mAbs directedagainst both a and f3 subunits of LFA-1 molecules on the adhesionof resting or stimulated T80cells to HUVEC have also shown that the increasedadhesion of stimulated lymphocytes to ECis possibly due to the altered function, not increase in expression,of the LFA-1 molecules(240).The binding of anti-CD3 antibodies to T cells triggers phosphatidylinositolturnoverand elevates cytoplasmicCa2+(220, 221). It has been demonstrated in rat cerebraland aorticendothelia that the removal ofCa2+from the media can effectively inhibit lymphocyte-ECadhesion (155). Interestingly, earlier work indicates that aCa2+dependentepitope on LFA-1,termed L16, is a prerequisite for LFA-1 to mediate cell adhesion andmay distinguish restinglymphocytes from activated lymphocytes (241). Subsequently, it hasbeen shown that L16epitope is expressed when the lymphocytes are stimulated byphorbol ester or T cellreceptor/CD3 (TCR/CD3) complex. It has been suggested that thereare possibly 3 distinctforms of LFA-1: a) an inactive form, partially exposed epitope, is presenton resting T cells,b) an intermediate one can be found on mature or previously activatedcells, and finally, c) anactive epitope, capable of high affinity ligand binding, can be demonstratedafter TCR/CD3 orphorbol ester activation (241). Studies with humanretinal pigment epithelial cells havedemonstrated that these cells constitutively expresshigh levels of ICAM-1, and thesemolecules are functional in binding activated Tlymphocytes but not resting T cells (230).LFA-1-dependent pathway has been implicatedin the increased adhesion of activatedlymphocytes to vascular endothelium such as highEC (HEC) and HUVEC (151, 158, 240).Therefore, these results suggest thatLFA-1/ICAM-1 interactions may also play a significantrole in the increased binding between anti-CD3 activatedT cells and untreated HBMEC81observed in this study.Total inhibition of binding by monoclonal antibodies directed against LFA-imoleculeshas not been observed (151, 240). Approximately 20% to 40% of adhesion betweenseveralT cell leukemia cell lines and HUVEC can not be suppressed by blocking withanti-LFA-1and anti-VLA-4 antibodies (242). In fact, these observations are in accordancewith otherstudies indicating that there are at least three to four other mechanismsor pathwayscontrolling lymphocyte-EC adhesion (243, 244). Studies on adhesion of activatedT cellleukemia cell lines to HUVEC have suggested that the LFA-1 adhesive mechanism dominatesthe interaction; however, very late antigen-4 (VLA-4) is used by T lymphocytesto bind ECwhen LFA-1 is not expressed or not functional to mediate adhesion (242),pointing to aselective use of different adhesion receptors by the T cells. In contrast, restinglymphocytesuse both LFA-1 and VLA-4 adhesion pathways (242). Like LFA-1, VLA-4also belongs tothe integrin family of cell surface heterodimers; it is expressed by lymphocytesand caninteract with vascular cell adhesion molecule-i (VCAM-1) (245). VCAM-1is a member ofthe immunoglobulin superfamily like ICAM-1 (246); however, VCAM-1is not expressed byperipheral blood lymphocytes (247, 248). Primary cultures of HBMEC havebeen shown toexpress low levels of VCAM-1 constitutively (249).Blocking experiments with niAbsdirected against LFA-1, ICAM-1, VLA-4, and VCAM-imolecules on the adhesion of restingT cells to untreated/activated HUVEC have demonstrated thatthe VLA-4/VCAM-1 adhesivemechanism is largely responsible for the adhesion of T lymphocytes to cytokine-treatedEC,while LFA-i/ICAM-i pathway mediates much of the bindingof T cells to unstimulated EC.82Furthermore, the binding of activated lymphocytes is not blocked by antibodies to VLA-4or VCAM-1, irrespective of the activation status of the EC. However, antibodies to LFA-1 orICAM-1 can modestly inhibit the adhesion of activated T cells to HUVEC. It is notable thatcomplete inhibition of T ceil-EC interactions by these antibodies has never been detected(152). Other studies with high EC and rat cerebral EC have also indicated that VLA-4/VCAM-1 mechanisms do participate in lymphocyte-EC adhesion (158, 250). Finally, the participationof other pathways besides the LFA-1/ICAM-1 system in the T cell - EC binding has beenexamined using LFA-1-deficient T cell clones generated from a patient with leukocyteadhesion deficiency. The results of these experiments confirm previous observations by Dustinet a!. (243) and Shimizu et al. (244) stating that there are other pathways mediatinglymphocyte-EC adhesion in addition to LFA-1/ICAM-.1 mechanism. In fact, VLA-4/VCAM-1represent the alternate receptor/ligand pairs which mediate the binding of LFA-1-deficient Tcells to HUVEC (153).Treatment of HBMEC with IFN-y and IFN-3 further modulates the anti-CD3stimulated T lymphocyte-EC interactions. Activation of HBMEC with an optimalconcentration of IFN-y (150 U/mi), known to induce maximal Ia Ag expression and markedlyupregulate the binding of resting T cells to EC, results in a 2 fold increase in adhesion ofactivated lymphocytes to cytokine treated brain EC. Further enhancement of lymphocyte-ECinteractions when both systems are activated has been reported with rat retinal (156), aortic(155) and brain endothelia (154), and also HUVEC (150). Interestingly, the level of adhesionbetween activated T cells and IFN-f3 treated HBMEC is comparable to that of untreated EC,83indicating that IFN-alone has no influence on adhesion.Moreover, a combined treatmentofhuman brain EC withIFN-y and IFN-f3 in thepresent studies, demonstratedthat IFN-j3actually inhibits theIF’N-y-mediated binding. These observationsfurther support the negativeregulatory role of IFN-13on changes inducedby IFN-y. The IFN-y-mediatedincrease inadhesion of activatedlymphocytes to HBMEC can alsobe suppressed by mAbsdirectedagainst human HLA-DR. Inaddition, blocking studieswith anti-human IFN-y indicate thatIFN-y is responsible forthe increased adhesion betweenactivated T cells and IFN-y-treatedHBMEC most likely throughinduction of class II molecule expressionby HBMEC. Theseresults indicate that class IIMHC molecules play a centralrole in mediating the increasedadhesion of activated Tcells to IFN-y-treated HBMEC.The fact that IFN-3 suppressestheIFN-y-induced Ia Agexpression on HBMEC also suggeststhat IFN-3 downregulates the IFNy-mediated binding via the Ia Agmechanism, which is furthersupported by the resultsobtained from mAb blocking experiments.In this respect, IFN- downmodulatesthe IFN-yinduced increased binding between Tlymphocytes and HBMEC regardlessof the activationstatus of the lymphocytes. Studieson lymphocyte-EC binding withmAbs directed against aand1EJsubunits of LFA-1 molecules have demonstratedsignificant inhibition of bindingbetween resting or stimulated T cells to untreatedHUVEC; however, these antibodieshave noinfluence on the adhesion of lymphocytesto cytokine-treated EC (240). Theseauthors havesuggested that the mechanism of bindingof T cells to unstimulated endotheliadiffers fromthat to stimulated endothelia, and the latterappears to be independent of LFA-1. Maleet al.have also indicated in their work with ratcerebral EC that the control ofbasal binding and84binding to activatedendothelia are regulated by different mechanisms.This system wouldallow brain endothelium to havelow basal binding to minimizelymphocyte traffic into thebrain normally, while permittingrapid increase in traffic if the cerebral EC arestimulatedappropriately (155).4.5 MIGRATION OF RESTINGand ANTI-CD3 STIMULATED LYMPHOCYTESACROSS UNTREATED, and CYTOKINETREATED HBMECThe fourth specific aim of this thesiswas to determine whether migration ofresting andanti-CD3 stimulated lymphocytesacross cerebral endothelium can beinfluenced bytreatment of HBMEC with interferonsyand/or Migration of resting lymphocytesacross untreated, IFN-y and/or IFN- treatedHBMEC monolayersIf the low basal level of migration of resting Tlymphocytes across untreated HBMECmonolayers in vitro reflects the limitedlymphocyte traffic into the CNS in vivo, it wouldcontribute to the relative immunological isolation ofthe brain under normal physiologicalconditions. As observed with adhesion, migration ofresting lymphocytes was also regulatedby cytokine treatment of the cerebral endotheliumin this study. Treatment of HBMEC withIFN-y results in a 3 fold increase in migrationcompared to that of untreated EC, suggestingthat IFN-y enhances the migration of T cells acrossthe EC monolayers possibly by a direct85action on the endothelium. Lightmicroscopic and TEM studies demonstratelarge numbers ofmigrated lymphocytes underneath the monolayersof HBMEC previously treatedwith IFN-y,while fewer lymphocytes crossed untreatedEC. Migration of resting T cells is not associatedwith damage to the integrity of the monolayers ineither untreated or IFN-y treated monolayer.Lymphocyte migration usuallytakes place between adjacent EC. Migrationthrough thecytoplasm of EC is a less common route ofmigration across the monolayers. IFN-y has beenpreviously reported to significantly upregulatethe migration of lymphocytes across HUVEC(24) and rat retinal EC (170). Oppenheimer-Marksand Ziff (24) observed that the augmentingeffect of IFN-y on lymphocyte transendothelialmigration is not dependent on the presence ofan exogenously added chemotactic factor belowthe EC monolayer. Using passaged culturesof rat cerebral EC, Male et al. (251) demonstratedthat the activation status of the endotheliumhas no influence on the migration of activated Tcells, however, they observed that the brain-specific surface phenotype of the cultured cells deteriorated afterthe first passage (251). Ourstudies, therefore, indicate that IFN-y upmodulates both adhesionand migration of resting Tcells across the cerebral endothelial banier. Whether thelevel of lymphocyte migration in thebrain is a reflection of the level of lymphocyte-EC adhesion orthe two events arepathophysiologically different and under the control of distinct influences by IFNs and/orother cytokines remains to be further investigated.The level of migration of resting T cells across IFN-13 treated HBMEC is comparableto that of untreated endothelia, indicating that IFN-f3 has no directeffect on lymphocytemigration. Treatment of HBMEC with a combination of IFN-y and IFN-j3 results in significant86suppression of the IFN-y-mediated increased migration which further supports thedownregulatory role of IFN- Migration of anti-CD3 stimulated lymphocytes across untreated, IFN-y and/or IFN-treated HBMEC monolayersIn this study, nonspecific stimulation of T lymphocytes with anti-CD3 generates asignificant increase in migration across untreated monolayers of HBMEC as compared to themigration of resting lymphocytes across untreated EC. In accordance with the observations onresting T cells, the level of stimulated lymphocyte traffic in the CNS most likely reflects thelevel of adhesion of activated T cells to HBMEC: the rate of increase of lymphocyte migrationis comparable to that of adhesion. It has been previously demonstrated that lymphocyteactivation induces three to four fold increase in migration across HUVEC in vitro whencompared to resting T cells (151). MAb blocking studies directed against various adhesionmolecules and their ligands including LFA-1, ICAM-1, VLA-4 and VCAM-1 have reportedthat the LFA-1/ICAM-1 interaction plays an important role in transendothelial migration ofactivated lymphocytes through HUVEC (151, 152). In contrast, VCAM-1 has thus far notbeen found to be utilized during the migration process, regardless of the activation status of theT cells or EC (152). Migration of activated T cells is not entirely blocked by mAbs to LFA-1and ICAM-1, indicating that additional surface molecules are required for transendothelialmigration (151, 152). Furthermore, studies on patients with LFA-1 deficient leukocytes haveshown the presence of lymphocytes in inflammatory lesions of these patients, indicating that87lymphocyte migration into inflammatory foci isnot entirely dependent upon the expression ofLFA-1 molecules (252). Thefact that migration of activated lymphocytes across untreatedHBMEC is significantly greater than thatof resting T cells through IFN-y treated cultures,indicates that antigen-nonspecific stimulationof lymphocytes plays a critical role in theiremigration from the blood into the perivasculartissue. In vivo observations in the rat havereported that activated lymphocytes can rapidlyenter into the CNS tissue once they areintroduced into the circulation, irrespective ofantigen specificity, MHC compatibility, T-cellphenotype or T-cell receptor gene usage (161).Furthermore, it has been shown that activatedlymphocytes can increase thelevels of the enzyme heparan sulfate endoglycosidase (253), andsubstances that inhibit this enzymaticactivity can prevent the development of EAE which isdependent upon T-cell entry into the CNS (254).These features may play some role in themigration of activated T cells, however, othermechanisms may also participate in thismigratory process.Immunohistochemical studies on the migration of T cellsacross HUVEC cultureshave shown the presence of ICAM-1 along the intercellularcontacts between EC that are incontact with the migrating lymphocytes as well as on the basalmembrane of the EC. Thepresence of ICAM-1 molecules at these sites as well as at sites ofcontact between the ECmembrane and the leading edge of migrating T cells suggests a criticalrole of ICAM-1 intransendothelial migration of T cells. The authors conclude that, as theT cells migrate acrossthe EC layer, migration proceeds by the successive formation ofadhesive bonds betweenreceptors on T lymphocytes and their counter-receptors on EC, likea “zipper’tmechanism88(152). Recent studies havedemonstrated that primary cultures of HBMEC express relativelyhigh levels of ICAM-1 (up to 40%)constitutively (239). As discussed previously, activationof lymphocytes may lead to an increasein avidity of LFA-1 molecules present on the T cells(159) which can further enhancethe interaction of LFA-1 to its counter receptor, ICAM-1.MAb blocking studies directed against LFA-1molecules have found that migration of restingT cells acrossHUVEC is not inhibited, while migration of activated T lymphocytes can besuppressed to a comparable level with resting T cells (151).Subsequently, the LFA-1/ICAM-1 dependent pathway may play a central rolein the marked increase in migration of anti-CD3stimulated lymphocytes across untreated HBMEC.Studies on platelet/EC adhesion molecule 1 (PECAM-1)have recently indicated thatthis molecule may also play an importantrole in transendothelial migration of leukocytes(255). PECAM-1 is a member of the immunoglobulingene superfamily (256), appears to beconcentrated at the junctions between EC (257) and is expressed on the surfaceof monocytes,neutrophils, and a small subset of lymphocytes (258 - 260).Muller et al. have suggestedseveral possible roles of PECAM-1 in transendothelialmigration of leukocytes. The mostobvious role involves PECAM-1 as a direct adhesion molecule binding the leukocytetightly tothe HUVEC during its passage through the junctions (255), since PECAM-1 has beenshownto be concentrated at the intercellular junctions, with approximately 15% exposed tothe apicalsurface (257). These authors suggest that an apical-basal gradient of PECAM-1 mayexistthrough the HUVEC junction which can act similarly to a surface-bound chemotactic gradientto produce directed migration of leukocytes through the junction. Another possible role is that89PECAM-1 may be ligated on the surfaceof leukocytes, which can then activate CD11/CD18binding activity, and thismechanism could apply as well if an apical-basal gradientofPECAM-1 exists (255). Finally, inductionof PECAM-1 has been reported on activated T cells(258), thus suggesting a possible roleof this adhesion molecule in mediating migration ofactivated lymphocytes across untreated monolayersof HBMEC in addition to the LFA1/ICAM-1 mechanism.Migration of activated T lymphocytes acrossEC monolayers is further enhanced byIFN-y treatment of HBMEC. Similarobservations have also been reported for the migration ofresting or activated lymphocytes across untreated andcytokine treated HUVEC (24, 151). Incontrast, treatment of rat retinal EC with IFN-y is associatedwith a small, but not significant,increase in the level of activated T-cell line lymphocytemigration (170). In addition, Male eta!. have reported that migration of activatedlymphocytes across rat brain endothelia does notappear to depend on the activation state of the EC (251).A possible explanation for theseresults could be the relatively low concentrations of IFN-yused. Alternatively, inherentdifferences in the culture systems used could account for thesediscrepancies.Transmission EM studies show significant numbers of activated lymphocytesmigrating across untreated as well as IFN-y treatedHBMEC. Activated T cells displayincreased size and altered appearance. Finger-like projections decoratethe cytoplasmicmembrane and significant numbers of mitochondria and vacuoles occupythe cell cytoplasm.Migration of both resting and anti-CD3 stimulated lymphocytes acrossuntreated and IFN-ytreated HBMEC is not associated with disruption of themonolayers. The integrity of the90monolayers is reestablished once lymphocyte migration iscompleted. Similarly, migration ofbovine peripheral blood lymphocytes across the endothelium ofpulmonary artery intimalexplants has been shown to cause no damage to the continuityof the vascular endothelium(261).The optimal concentration of IFN-y used for the adhesion and migration assayshasbeen shown to induce maximal Ia Ag expression on HBMEC in primaryculture (127). Weobserved no significant change in the degree of activated T cellmigration acrossIFN-13treated EC as compared to controls, indicating that IFN-3 has no direct effecton the migrationprocess. However, the IFN-y-mediated increase in migration is downregulated by IFN-f3,sincemigration of activated lymphocytes across EC coincubatedwith IFN-y and IFN-3 iscomparable to that of controls. Blocking experiments with mAbs against humanHLA-DR inIFN-y treated HBMEC show comparable levels of decrease inmigration to those obtained bytreating HBMEC with both cytokines.Taken together, the results of our studies on the migration of resting andnonspecifically stimulated T lymphocytes across untreated and cytokine treated HBMECmonolayers, indicate that induction of class II molecules on the surface of HBMEC by IFN-yis, at least in part, responsible for the increased migration of T cells across the monolayers.IFN-f3 has no direct effect on lymphocyte-EC binding, but downregulates the IFN-y-mediatedincrease in transendothelial migration most likely through downregulation of the IFN-yinduced de novo expression of class II MHC molecules by HBMEC.914.6 EFFECTS OF IFN-y ON THESTORAGE AND RELEASE OF FVIIIR:AgFROM HBMEC IN PRIMARY CULTUREThe fifth and final specific aim of thisthesis was to investigate the effects of IFN-y on thestorage and release of FVIIIR:Ag followingits immunocytochemical localization inprimary cultures of HBMEC.4.6.1 Immunocytochemical localization ofFVIIIR:Ag in HBMECHuman brain microvessel EC in primaryculture synthesize FVIIIR:Ag as indicated bytheir positive, granular, perinuclear stainingfor FVIIIR:Ag with the immunoperoxidasetechnique. By immunoelectron microscopyFVIIIR:Ag is localized within cytoplasmic vesiclesclosely associated with the rough endoplasmic reticulumand Golgi apparatus in theperinuclear region. Treatment of EC withCa2+ionophore A23 187 results in marked reductionin labeled vesicles, while preincubation with IFN-yleads to increase of intracellular poois ofFVIIIR:Ag.EC lining large vessels and arterioles synthesizeand secrete FVIIIR:Ag and store thenewly synthesized glycoprotein within cytoplasmic organelles uniqueto these cells, known asWeibel-Palade bodies (174 - 176). These rod-shaped structuresare absent in primary culturesof microvessel EC derived from rat (179 - 180), mouse (178) andbovine cerebral cortex (262)and bovine retina (263), but have been reportedto be present in EC derived from rat andbovine brain white matter (264). Weibel-Palade bodies are extremely rareor absent in normal92human cerebral capillaries (265, 266). Theyhave been observed in the orbital cortex of normalaged humans (267) and with increased frequencyin certain brain tumors (266, 268). HBMECin primary culture are similarlydevoid of Weibel-Palade bodies. The perinuclear, granularstaining for FVIIIR:Ag with the immunoperoxidasetechnique corresponds to variably dilatedvesicular profiles within which deposits of colloidalgold were observed ultrastructurally. Thesingle limiting membrane of these vesicles is notdecorated with ribosomes and therefore, it isunlikely that they represent dilated cisternae of roughendoplasmic reticulum. Their constantpresence near the Golgi apparatus and the rough endoplasmicreticulum suggests that theimmunolabeled vesicles belong to the polymorphous vacuolesthat form part of the transmostGolgi section (269). These trans Golgi elements havebeen found to be part of the pathway ofnewly synthesized molecules (270). It is, therefore, likely that,following synthesis in theendoplasmic reticulum and extensive modification inthe Golgi apparatus (271), the newlysynthesized FVIIIR:Ag is transported to the trans Golgi polymorphousvesicles where it isconcentrated. In the absence of Weibel-Palade bodies in cerebral microvesselEC, thesevesicular bodies most likely represent sites of short-term storage ofFVIIIR:Ag prior to release.Previous in vivo studies on the localization of FVIIIR:Ag in vascular endotheliumof normalhuman extracerebral tissues and one capillary hemangioma by immunoelectron microscopydemonstrated immunolabeling of endoplasmic reticulum and cytoplasmic vesiclesandvacuoles in addition to Weibel-Palade bodies (176). These vesicular profiles strongly resemblethe ones observed in the present study. A similar localization of FVIIIR:Agwithin cytoplasmicvesicles has been reported in EC lining the saphenous vein (172).934.6.2 Effects of IFN-y on the storage and/orrelease of FVIIIR:Ag from HBMECLarge vessel EC secrete the newly formedFVIIIR:Ag via two pathways (272, 273).The regulated pathway involves release ofthe large multimeric forms of the glycoprotein fromthe specific storage organelles, the Weibel-Palade bodies.In vitro studies on the secretion ofvon Willebrand factor by umbilical vein EC indicatethat this pathway is highly polarized anddependent upon intact microtubular system, sincemicrotubule-depolymerizing agents inhibitthe regulated release (274). The majority of FVIIIR:Agsynthesized by EC is secretedconstitutively in the form of small multimers. In contrast to the regulated pathway,constitutiverelease is not affected by microtubule-depolymerizing agents (275) and is notpolarized. SinceHBMEC do not store FVIIIR:Ag in Weibel-Palade bodies, it isquite possible that they secretethe newly synthesized protein through the constitutive pathway only, with theGolgi-associatedcytoplasmic vesicles serving as temporary storage pools following multimerization and priorto release.A variety of stimuli can lead to increased release of FVIIIR:Ag from EC invitro. Mostof these factors stimulate the regulated pathway of secretion.Thus, treatment of ECmonolayers with calcium ionophore A23 187, thrombin or phorbol-myristate-acetate results inrelease of the large multimeric forms of FVIIIR:Ag and a simultaneousdisappearance ofWeibel Palade bodies from EC (272 - 274, 276) in association with a rise in the concentrationof intracellular calcium. The effect of calcium ionophore was inhibited by EGTA in a dosedependent manner (276). The basal secretion was apparently not affected by these treatments.In the present study short preincubation of HBMEC with calciumionophore led to rapid94reduction in the number of labeled cytoplasmic vesicles. In contrast, addition of the calciumchelating agent EGTA to the culture media resulted in slight increase in immunostainedvacuoles, which was not statistically significant when compared to untreated cells. Loss ofstaining following calcium ionophore treatment may represent rapid release of FVIIIR:Agfrom intracellular pools, although other mechanisms, such as antigen degradation followingionophore - mediated protease activation, cannot be ruled out. These findings indicate that atleast some of the factors that stimulate the regulated pathway in large vessel EC, similarlyinfluence the release of FVIIIR:Ag from microvessel endothelium in the absenceof WeibelPalade bodies. Whether the FVIIIR:Ag secreted by cerebral small vessel endothelium is in theform of small or large multimers, is presently unknown.Incubation of HBMEC with IFN-y for 24 hours resulted in significant increase in thenumber of immunostained vesicles suggesting that IFN-y interferes with the releaseand/orstorage of FVIIIR:Ag. Recent studies on the effect of cytokines on the release of vonWillebrand factor indicate that IFN-y decreases the constitutive and regulated release fromcultured HUVEC reversibly and in a time and dose-dependent manner (182). Althoughtheexact mechanism of action is not presently known, it is possible that IFN-y exerts its effectbymodifying the concentration of intracellular calcium. It has been recently demonstrated thatIFN-y can activate the calcium-dependent pathway through activation of phospholipaseC andinduce, in addition, a significant outflux of calciumions from EC (277). Inhibition ofFVIIIR:Ag release by IFN-y may be important considering its pivotal roleas mediator of thelocalized immune response in autoimmune diseases of the central nervous system.95CONCLUSIONS5.1 SUMMARY AND CONCLUSIONSThe main objective of this thesis was to examine the effects of IFN-y andIFN-13on IaAg expression, cell proliferation, and alteration of the morphology and permeability propertiesof HBMEC using an in vitro model of the human BBB. In addition, the adhesion andmigration of resting and anti-CD3 stimulated T cells across untreated and cytokine-treatedHBMEC was studied. Finally, the effects of IFN-y on the storage and release of FVIIIR:Ag byHBMEC was determined.The working hypothesis of this thesis was that in chronic inflammation, some activatedT lymphocytes will release inflammatory cytokines including IFN-y. This cytokine can theninduce the local brain endothelia to express Ta Ag on the cell surface, to alter their morphologyand increase their permeability to macromolecules. These changes will facilitate the adhesionand migration of resting lymphocytes across the cerebral endothelial barrier. Lymphocyteactivation will further augment adhesion and migration. Finally, IFN-y by inhibiting therelease of FVIIIR:Ag from HBMEC will contribute to the maintenance of blood fluidityduring the immune reaction.In this study, IFN-y induces de novo expression of Ia Ag on HBMEC in a time anddose-dependent fashion. Primary cultures of HBMEC do not express Ia Ag constitutively.The expression of Ia molecules on HBMEC can be detected as early as 12 hours followingincubation with IFN-y and reaches plateau levels by 48 hours. Surface labeling for Ta Ag is96maximal with 100 to 200 U/mi IFN-y and minimal with 10 U/mi. In contrast, treatment ofHBMEC withIFN-13has no influence on Ia Ag expression. Moreover, incubation of HBMECwith a combination of IFN-y and IFN-f3 results in downreguiation of IaAg expression. IFNsuppresses the IFN-y.-induced expression in a dose-dependent manner, however, completeinhibition was not detected. Kinetic studies on the effects of IFN-y and IFN-3 onIa Agexpression indicate that administration of IFN-j3 prior to or simultaneously withIFN-ytreatment generates the most significant downregulation of IFN-’-induced IaAg expression.These observations may partly explain the results of recent therapeutic trials withIFN-13inMS.Treatment of HBMEC with IFN-y results in changes in cell shape and organizationofthe EC monolayers. The IFN-y-treated endothelia acquire a spindle-like shape andlongattenuated processes. Prominent overlapping and ill-defined whorls areunique features ofIFN-y-treated EC monolayer. The IFN-y-induced phenotypic alterationson HBMEC areinhibited when EC are incubated simultaneously with IFN-y and IFN-3;the monolayersresume their highly organized growth pattern with prominent marginalfolds in the areas ofcell to cell contact. The morphological changes are associated withincreased permeability ofconfluent monolayers to horseradish peroxidase as compared withuntreated cultures. Thenumber of HRP labeled vesicles was not increased inIFN-y treated EC as compared tountreated EC.Lymphocyte-EC adhesion is significantly upregulated whenHBMEC are pretreatedwith IFN-y, while IFN-f3 inhibits the IFN-y-enhanced adhesion whenthe brain endothelia are97incubated with a combination of IFN-y and IFN-3. IFN-3 alone has no effect on lymphocyteEC interactions since the level of adhesion is comparable to that of untreated EC. Nonspecificactivation of T lymphocyte causes a significant increase in lymphocyte-EC adhesion; in fact,the level of adhesion of activated lymphocytes to untreated EC is greater than that of resting Tcells to IFN-y treated EC, suggesting that lymphocyte activation plays an important role in Tcell-EC adhesion. Similar to the responses obtained with resting lymphocytes, IFN- inhibitsthe IFN-y-enhanced adhesion of activated T cells when EC are treated simultaneously withIFN-y and IFN-3. However, IFN- treatment alone has no effect on the adhesion of activatedT lymphocytes to EC. MAb blocking studies against IFN-y and human HLA-DR moleculesindicate that the enhanced binding is specifically induced by IFN-y and HLA-DRmoleculesplay a central role in the IFN-y upregulated adhesion.Migration of resting lymphocytes is markedly augmented when HBMEC are treatedwith IFN-y, but not IFN-. Treatment of cerebral EC with a combinationof IFN-y andIFN-13significantly downmodulates the IFN-y-mediated migration. Activation oflymphocytes isassociated with a dramatic increase in migration across untreated EC, and the levelofmigration is greater than that of resting T cells through IFN-y treated EC. IFN-y,but not IFN, further augments the migration of activated T cells. The IFN-y-enhancedmigration issuppressed byIFN-13treatment. Blocking studies with mAbs against HLA-DRmoleculesindicate that Ta Ag plays a central role in the IFN-y mediated migration of bothresting andanti-CD3 stimulated lymphocytes.Finally, it has been determined that IFN-y suppresses the releaseof FVIIIR:Ag from98HBMEC which suggests that IFN-y may also play a role in maintainingblood fluidity duringthe immune reaction.In conclusion, the results of these studies demonstrate the critical functionof IFN-y inupregulating the immune response which playsan essential role in the host defensemechanism. Indeed, autoimmune disorders of the CNS such asMS may arise as the results ofunwanted inflammatory or immunological responses. Subsequently,the ability of cytokinessuch as IFN- to reduce or suppress the IFN-y-enhancedreactions may explain theirtherapeutic effect. The facts that IFN- is able to inhibitthe IFN-y-induced Ia Ag expressionand to suppress the IFN-y-mediated increase in lymphocyte-ECadhesion and migrationindicate that Ia Ag plays a central role in these immunologicalresponses. This statement isfurther supported by results obtained from mAb blocking studies directedagainst humanHLA-DR. Therefore, the results of this thesis demonstrate theimportant role of HBMEC inCNS inflammation and enhance our understandingof some of the factors involved in therecruitment of lymphocytes into chronic inflammatorysites in the CNS.5.2 FUTURE PROSPECTSThe results obtained from this study indicate that class IIMHC participates in the IFNy-mediated increase in lymphocyte-HBMEC adhesionand migration. An avenue forimmediate future research would be to determine therole of HBMEC as antigen presentingcells in CNS inflammation using this in vitro BBB model. HBMECcan be induced to expressIa Ag by IFN-y and allowed to endocytose and processmyelin basic protein (MBP), a protein99component of the myelin shealth. At the appropriate time, MBP-specificlymphocytes areincubated with these HBMEC, and lymphocyte proliferation can be determinedwith tritiatedthymidine assay.Since it has been demonstrated in this thesis that antigen-nonspecificstimulation ofperipheral blood lymphocytes plays a central role in markedlyupregulating the lymphocyteEC adhesion and migration, another avenue for future research wouldbe to examine themolecular mechanisms that are responsible for augmenting theadhesion and migration ofactivated lymphocytes to HBMEC. The application ofmAbs directed against specificadhesion molecules such as ICAM-1, LFA-1, VCAM-1, VLA-4,that are present on thesurface of both lymphocyte and EC will help to determinethe molecules responsible forupmodulating the immunological reactions.Finally, a concern that also needs to be addressed in futureresearch in CNSinflammation is the question: What is the significanceof peripherally activated Tlymphocytes in the development of immune reactionsin the brain?In one version of rat EAR, autoimmune demyelination can beinduced by immunization withmyelin basic protein, and systemic injectionof a specific monoclonal antibody directed againstmyelin/oligodendrocyte glycoprotein can amplify demyelination.Immunotherapy of thisantibody-induced demyelination is possible with anotherspecific mAb directed to an antigenon activated rat T cells, suggesting an important roleof T lymphocyte activation in the diseasedevelopment (169). Studies on the migration ofactivated lymphocytes across rat brainendothelium lead Male and his coworkers (154) to speculatethat lymphocyte activation in the100periphery may lead to increased traffic through the brain. This lymphocyte traffic can haveserious consequences if antigen specific interaction develops between circulating T cells andantigen presenting cells in the CNS. Amplification of the immune reaction can result fromcytokine release and local activation of the brain endothelium, causing further increases incellular migration. As suggested, this scenario is possible in Bordetella pertussis vaccinationin a small proportion of individuals. In accord with the above speculation, in vivo studies ofT-lymphocyte entry into the rat CNS by Hickey and his coworkers (161) have reported thatactivated T cells appear to enter the CNS in a random manner, irrespective of their antigenspecificity, MHC compatibility, T cell phenotype, and T-cell receptor gene usage. Theseauthors also showed that only T-lymphocytes that are able to recognize a specific antigen inthe CNS of the host remain beyond 72 hours in the target organ while non-specificallyactivated T-cells exit the CNS within 1 to 2 days.The above observations raise question(s) about the “immunological privilege” status ofthe CNS because any T-cell that is activated in the tissues of the immune system can possiblygain access to the brain in a random manner once it enters the circulation. Transplantation ofallogeneic tissue into the CNS is well accepted by the recipient; however, the graft is quicklyrejected when the same alloantigen is exposed to the host periphery (278). In concordancewith the results described by Hickey et al. (161), in vivo studies of mice EAE, Cross et al.(279) have shown that 14C-labeled CNS antigen-specific T cells home to the CNS endothelia24 hours prior to and during the initial clinical disease, but these cells always remain withinthe perivascular area. The antigen-specific lymphocytes only represent 1% to 4% of the101inflammatory cells that are present in the brain parenchyma during diseasedevelopment.These investigators conclude that the inflammatory cells are predominantly ofrecipientderivation. More recently, Caspi et al. (280) have demonstrated thatin experimentalautoimmune uveoretinitis (EAU), a T cell-mediated autoimmunedisease in rat serving as amodel for a number of human blinding ocular diseasesof a presumed autoimmune nature,only mild or essentially no disease can be inducedby the CNS antigen-specific T cell lines inunreconstituted athymic rats. However, the situation canbe significantly reversed by infusionof naive cell populations containing immunocompetentT cells. Subsequently, the recruitmentof naive T cells constitutes an amplification mechanism thatis central to the expression andpathogenesis of uveitis. The phenomenon of recruitmentcan magnify the effect of a tinynumber of antigen-specific “pathogenic” T lymphocytesinto a destructive inflammation.Consequently, the results that are available hitherto do supportthe significant role thatperipherally activated, antigen-nonspecific T cells can havein the development of immunereactions in the brain. The ability to suppress these activatedlymphocytes, for example, withanti-LFA-1 monoclonal antibodies to block the LFA-1 dependentpathway of adhesion duringan unfavorable inflammation of the CNS may havegreat therapeutic potential.5.3 SIGNIFICANCE OF THIS THESISPrimary cultures of HBMEC provide a usefulin vitro model for investigating theeffects of cytokines on the morphologyand function of the cerebral endothelium and on thecomplex processes of lymphocyte adhesion andmigration across the cerebral endothelium102barrier. These studies indicate that interferonsyand 3 are important mediators of the localizedimmune response within the human CNS and that classII MHC molecules, induced de novoon HBMEC, play a pivotal role in lymphocyte-EC interactionsin the CNS.As pointed out by Wekerle at al. (14),the detailed analyses of the cellular andmolecular mechanisms involved in the interaction betweenT cells and the BBB are oftremendous importance for various reasons:a) Such knowledge will give insight into thedevelopment of CNS disorders with putative autoimmunepathogenesis, e.g. multiple sclerosis.b) The information obtained will provide a better understandingof the mechanisms involve inphysiological immune surveillance, as they are relevantin the prevention and the control ofinfectious diseases within the CNS. c) On the basis of such knowledge,it may be possible todesign novel specific therapies of CNS (autoimmune)disease.103Table 1Permeability of HBMEC monolayers to HRPNo. of labeled No. of interendothelialcytoplasmic vesicles*tightjunctions**Permeable ImpermeableControl 2.0±1.7 vesicles/cell 24.8±2.7% 75.2±2.7%Experimental2.4±2.1 vesicles/cell 63.4±5.2% 36.6±5.2%*Numbers represent mean + SD of labeled vesicles in 100 controland 100 IFN-Y treated cellsfrom one experiment. P 5 0.05* *Numbers represent mean±SD of 400 junctions (200 treated and 200 untreated) from twoexperiments using two different isolates. P < 0.05104Figure 1: Diagram of the double chamber chemotaxis system used to study themigration of lymphocytes across confluent HBMEC monolayers. Thecellagen membrane is a firm membrane, made up of solubilized collagen,that forms the bottom of 14 mm diameter wells. These wells are placedinside larger wells of 24-well plates. Four support feet separated the innerfrom the outer chamber. HBMEC are seeded onto the cellagen membranesand grown inside the inner chamber. Initial attachment of EC to themembranes does not require precoating with fibronectin. This system canbe used to study the interactions between EC and inflammatory cells suchas lymphocytes or polymorphonuclear leukocytes.S(ThCD0.‘C)—CD—‘CDL05C’.)CHC.)CDCii106Figure 2: Primary cultures of HBMEC grown on plastic wells (a) or cellagen membranes(b) and maintained under standard culture conditions form highly organized,confluent, contact inhibiting monolayers composed of elongated endothelialcells. Bars = 20 tm.107F.z108Figure 3: Intense, predominantly perinuclear cytoplasmic staining of cultured HBMEC forFVIIIR:Ag with the immunoperoxidase reaction (a). Lectin binding by HBMECis indicated by their positive immunoperoxidase staining for UEA 1(b). Bars =20 tm.109it,14441.40,a’aa SF?.3110Figure 4: Confluent monolayersof HBMEC (EC) grown oncellagen membrane (C).Elongated cells with focally evidentfinger-like cytoplasmic projections form acontinuous cell layer firmly attachedto the substrate. Bars = 2 tm.Ill.112Figure 5: Primary cultures of HBMEC (EC) cultivated on cellagen membranes (C) (a - d).Intercellular contacts vary in length and complexity. Tight junctional complexes(arrows) with pentalaminar configuration are present in areas of cell to cellcontact. Bars = 0.2 tm.p4Cr’00cJ0’‘9[‘‘v’I*3 ‘V:%9*4*’’1wI117Figure 6: The cytoplasmof cerebral microvesselendothelial cells (EC) containsprominentrough endoplasmicreticulum (smallarrows), small to largemitochondria(arrowheads), and avariable number of smalland large vesicles (V) injuxtanuclear position.Small amounts of amorphousmaterial are present,otherwise, the vacuoles are clearand bound by a single limitingmembrane. Theendoplasmic reticulum isclosely associated with thevesicular profiles. EC weregrown on cellagen membranes(a) or plastic wells (b).Weibel-Palade bodies arenot present. C = cellagen membrane;N = nucleus. Bars =1 tm.£6ct-LI’1F. 61’120Figure 7: Immunogold staining of intact endothelial monolayers for FVIIIR:Ag. (A) Fivenm gold particles form dense aggregates within several cytoplasmic vesicles(large arrowheads). Other vesicles contain scant particles (small arrowheads),whereas still others are not stained. Labeled vesicles are located close to theGolgi apparatus (B) and the endoplasmic reticulum (A). (C) Occasionally, a fewgold particles localize within cisternae of endoplasmic reticulum (arrowhead)next to a labeled vesicle. (D) Staining is absent in control cultures incubated withcarrier buffer instead of primary antibody. N, nucleus; G, Golgi. Bars = 1 tm.I4122Figure 8: Time course of Ia antigen induction on human cerebral endothelium.ConfluentHBMEC cultures were incubated with 200 units/mi IFN-y for 0.5 to 4 days andthen stained with the immunogold technique for the immunohistochemicaldemonstration of Ia antigen. Results are expressed as percentage of labeled cellsin treated cultures. Untreated cells were not labeled. Bars represent the mean±SEM of duplicate wells of two separate experiments.123C/)C)0ci)ci)0N00-Fig. 8lime course of Ia Aginductionon HBMEC by IFN-y1008060402000.5123 4Time (days)124Figure 9: Dose response of Ia antigen inductionby IFN-y on HBMEC. Confluentmonolayers were incubated for 4 days with10 to 200 units/mi IFN-y and thenimmunostained for the demonstration of Ia antigen. Resultsare expressed aspercentage of labeled cells in treated and untreated cultures. Barsrepresent themean±SEM of duplicate wells of three separate experiments.125Fig. 9Dose-response of Ia Ag inductionby IFN-y On HBMECC,)ci)C)ci)ci)-Q001008060402000 10 20 50 100 150 200IFN-’Y Concentration (U/mi)126Figure 10: Ia antigen expression by HBMEC detected byimmunogold silver staining. A,endothelial cells incubated with 200 units/ml IFN-y for 4 days demonstratingintense granular surface staining for Ia antigen. B, control untreated monolayersnot expressing Ia antigen. C, endothelial cells treated with 200units/ml IFN-y for24 hours exhibiting less dense labeling. Individual cell variationin stainingintensity is apparent in A and C. In D, cultures coincubated with anti-IFN-yantibody failed to label with the immunogold reagent. Bars = 20 tm.I<qI0Figure 11: Immunogold stainingof HBMEC for the demonstration of Ta antigen. A,endothelial cells incubated with 200 units/mi IFN-y for 4 days. Fivenanometer gold particles focally decorate the apical surface of endothelialcells (arrowheads) with a tendency to localize close to fingerlike cytoplasmicfolds. The basal cell surface is not labeled. B, staining isabsent in untreatedcells. Bars = 0.5 tm.12841I,•%111)4’4q“‘‘111*1I’1434b‘1‘“4tee14‘I14130Figure 12: Ia antigen expression by HBMEC detectedby immunogold silver staining.a) endothelial cells incubated with6,000 units/mlIFN-13for 4 days failedto express of Ta Ag as indicated bythe negative staining of EC. b) Incultures coincubated with IFN-y (100 units/mI) and IFN-3 (500units/ml)expression of Ia Ag is limited to a small number ofendothelial cellsdisplaying positive surface labeling with immunogoldsilver staining(arrows). Bars = 20 tm.rH,IId0aa132Figure 13: Dose response of Ia Ag expression by HBMEC treated with IFN-y and/orIFN-j3. Confluent monolayers were examined untreated, or followingtreatment with IFN-3 (6,000 units/mi) or IFN-y (100 units/mi) or with acombination of IFN-y (100 units/mi) and 13(100 to 6,000 units/mi) for 4days. At the end of the incubation period, monolayers were stained withthe immunogoid siiver staining technique for the surface detection of IaAg. Resuits are expressed as percentage of iabeied ceiis in treated anduntreated cultures. Bars represent the mean±SEM of triplicate wells ofthree separate experiments.133Fig. 13 Dose-response of Ia Ag expressionby HBMEC treated with IFN-1and/orIFN-p% labeled cells0 20 40 60 80 100untreatedIFN-6cJoIFN- 100cIFN-yioo/_________IFN-13100EIFN-yioo/_________,__ IFN- 13250a,-FN-135Y3F—IFN-y ico/_______FN-13ux0IFN-’ lao!______FN- 132000IIFN- y 100/IFN-13aOcxJ134Figure 14: Effects of differenttreatments of IFN-y and 3 on Ta Ag expressionbyHBMEC. Confluent monolayerswere left untreated, or treated with TFN-13(6,000 units/mi) or TFN-y (100 units/mi)or a combination of IFN-y (100units/mi) and1(6,000 units/mi) for 4 days, or treatedwith TFN-y or IFN1alone for 2 days, followed by a combinationof TFN-y and for another 4days [TFN-y(2) or IFN-13(2)/IFN-+y(4)],or with a combination of IFN-j3(6,000 units/mi) and y (100units/mi) for 2 days, followed by IFN-y alonefor another 4 days [IFN-3+y (2)/IFN-y (4)]and then immunostained forthe demonstration of Ta Ag. Results areexpressed as percentage of labeledcells in treated and untreated cultures. Barsrepresent the mean±SEM oftriplicate wells of two separate experiments.135Fig. 14Effects of different treatmentsof IFN- andfon Ia Ag expressionby HBMEC% labeled cells0 2040 60 80 100untreatedIFN-y icCaIFN-13÷yIFN-y(2)/IFN-13+y(4)FN-13(2)/IFN-13+y(4)IFN-p+Y (2)/IFN-y (4)136Figure 15: Quantitation by ELISA of Ia Ag expression by HBMECtreated with IFN-yand/or IFN-. Confluent monolayers ofHBMEC were left untreated, ortreated with IFN-y (100 units/ml), or IFN- (6,000 units/mi), or withacombination of JFN-y (100 units/mI) and 3 (100 to 6,000 units/mI) for 4days, or with IFN-i or y alone for 2 days, followed by a combinedtreatment withIFN-13and y for another 4 days(132/y2+ f3y4). Valuesrepresent mean±SEM of triplicate wells.Ia Ag expression was measured in triplicate wells of confluentHBMECcultures.137Fig. 15Quantitation by ELISA of Ia Agexpression by HBMEC treated withIFN-yand/orIFN-pAbsothace at 490 nm0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8I I I I I I IUntreatedR1-y 100P14 6,000R1-y+ 100R1-y+P1-y+ 1,000IR1-y+ 6,000 I2+Y4 Iy2+y4138Figure 16: A, HBMEC maintained understandard culture conditions formed highlyorganized, confluent, contact inhibiting monolayers composedof elongatedcells. B, endothelial cells incubated with IFN-y (200 units/ml) for4 dayshave become spindle shaped, overlap, and focally arrange themselves intowhorls. Bars = 10 tim.13917.140Figure 17: Scanning electron micrograph of HBMEC grown in the absence(A) orpresence (B) of IFN-y in the culture media. A, endothelialcells closelypacked without apparent intercellular spaces. Marginal folds (arrows)arepresent in areas of cell-cell contact. In B, incubationwith IFN-y (200units/ml) for 3 days induces marked attenuation of cellcytoplasm anddisorganization of the monolayer due to the tendency ofendothelial cellprocesses to extend over and under adjacent cells. Bars = 20tm.141. . .. : . •• . .• •. • . .. . : .;;•wç.•• . . .—b- • —.-- -::::;E______--——:—— —;i:__c—- .- -—‘-—N- --N142Figure 18: Effects of IFN-y and f3 upon the growth of primarycultures of HBMEC.Cells were left untreated or treated from day 1 with IFN-y (150 units/mi) orIFN- (1,000 units/mi) alone, or with a combination of IFN-yand (100units/ml and 1,000 units/mi, respectively). Barsrepresent the mean±SEMof triplicate wells.Cells/Well- 00oc,)(D:T1 —aCl)r\)01001001000000C)) 0 00 :3Untreated IFN-y IFN-FN-y+3144Figure 19: Scanning electron micrograph of HBMEC grown in the presence of IFN-falone (a) or IFN-3 and y (b) in the culture media. a) endothelial cells aremorphologically identical to untreated cultures, they are closely packedwithout apparent intercellular spaces. Marginal folds (arrows) are presentin areas of cell-cell contact. In b) incubation with a combination of IFN-13and‘y(6,000 units/ml and 200 units/mi, respectively) prevents theoccurrence of the IFN-y induced changes in cell morphology andorganization of the monolayers. Arrows point to marginal folds in areas ofcell-cell contact. Bars = 50 tm.45&liiIIi6IL13147Figure 20: Permeability of untreated (A and B) and IFN-y-treated (C - F) confluentHBMEC monolayers to HRP. A, under standard culture conditions, tightjunctions at intercellular contacts (between arrowheads) impede thepassage of HRP. B, HRP penetrates a short segment of an intercellular cleftfrom the basal cell surface, forming small deposits at the basal aspect ofthe cleft (arrowheads) and stopping at a junctional complex (arrow). Theremaining interendothelial cleft is free of HRP. Untreated Cultures. In C,following 4 days’ incubation with IFN-y (200 units/ml), heavy deposits ofHRP are seen under the basal cell surface, and the tracer permeates theentire length of a long intercellular cleft. The proximal portion of the cleftis focally dilated(*).There is no increase in the pinocytotic activity of theendothelium. D, in monolayers treated with IFN-y, HRP penetrates theintercellular clefts and forms extensive deposits between the layers ofoverlapping EC. Bars = 0.5 tm. E, F, Permeable interendothelial clefts ofIFN-y treated monolayers exhibit HRP deposits throughout their length.Bars = 0.5!m.-c0c0151Figure 21: Expression of IL-2R on resting T cells and anti-CD3stimulated (a-CD3)lymphocytes. Approximately 2 fold increasein IL-2R expression isobserved when lymphocytes are stimulated with a-CD3mAb for 3 days at37 CC.152Fig. 21Expressionof0C,)Anti-CD3stimulated25lymphocytesIL-2Ron Restingand2015 -1050a-CD3UntreatedTreatment153Figure 22: Adhesion of resting T lymphocytes to untreated confluentHBMECmonolayers. At the end of the incubation period, EC cultures with adherentlymphocytes were fixed and stained with the immunoperoxidase techniquefor leukocyte common antigen (LCA). Small number ofLCA positivelymphocytes (L, arrowhead) adhere to the untreated endothelial cells (ec).Bar= 1Oim.4’1%S4.1trD155Figure 23: Adhesion of resting T lymphocytesto IFN-y (150 units/mi) treatedHBMEC as demonstrated by immunoperoxidasestaining for leukocytecommon antigen (LCA). Large numbers of LCA positive lymphocytes(L,anowhead) adhere to the IFN-y treated endothelial cells(ec). Bar = 10 tm.$S157Figure 24: Adhesion ofresting T lymphocytes toIFN-13(2,000 units/ml) treatedHBMEC as demonstrated by immunoperoxidase stainingfor leukocytecommon antigen (LCA). A small number of LCApositive lymphocytes (L,arrowhead) adhere to IFN- treated endothelialcells (ec). Bar = 10 tm.159Figure 25: Adhesion of resting lymphocytes to untreated and cytokine-treatedHBMEC. Confluent monolayers of EC were left untreated or treated withIFN-y (150 units/ml) or IFN-3 (2,000 units/mi), or with a combination ofIFN-y (150 units/mi) and1E(2,000 units/mi) or IFN-y (150 units/mi) andanti-IFN-y antibody (ay - 10 fig/mi) for 3 days prior to incubation withresting T cells. For the mAb blocking studies, cuitures were treated withIFN-y for 3 days, followed by 2 hr incubation with anti-human HLA-DRmAb (ala) prior to incubation with resting T lymphocytes (T). Barsrepresent the mean±SEM of triplicate weils of two separate experiments.160Fig. 25 Adhesionof resting Lymphocytesto untreatedand cytokine-treatedHBMECCells/mm2EC+TEC+R4-y +TEC+FN-p +TEC+IRI-y+p +TEC+1R4-y+ay+TEC+1B4-y+ aIQ+T50 100 1500 200I-H161Figure 26: Scanningelectron micrograph demonstratingthe adhesion of restingTlymphocytes to untreatedHBMEC. a) Lymphocytes(L) first adhere to theendothelium (BC)by extending pseudopodiathat contact the endothelialsurface (arrowhead). Marginalfolds (arrow) are presentin areas of cell-cellcontact. Bar = 20 tm.b) Lymphocytes eventuallyposition themselvesbetween adjacent EC(bar = 4.5 tm), and c) beginmigrating across themonolayer (bar =1.8 tm). d) Lymphocyteswere infrequently seenpenetrating the apicalEC plasma membrane (arrowheads)and movingthrough the endothelial cytoplasm(bar = 4.36 tm).oQ6 aCc3p4166Figure 27: Scanning electron micrograph of the adhesion of restingT lymphocytes toIFN-y (150 units/ml) treated HBMEC. IFN-y treatment of the endothelialcells (EC) induces reorganization of the monolayer anda tendency of ECprocesses to overlap (arrow). a) Large numbers of lymphocytes(L)establish contact with the endothelium via pseudopodia(arrowheads). Bar= 50 m. b) Lymphocytes (L), singly or in small aggregates(arrowheads),align themselves along the borders between adjacent EC inpreparation forcrossing the monolayers. Bar = 20 tm.Fig.ca169Figure 28: Adhesion of anti-CD3 stimulated T lymphocytes to untreated HBMEC asdemonstrated by immunoperoxidase staining for leukocyte commonantigen (LCA). Significant numbers of LCA positive activatedlymphocytes (L) adhere to untreated endothelial cells (EC). Activated Tcells are larger than resting lymphocytes and display irregular, folded, cellmembranes. Bar = 10 rim.j7OEC171Figure 29: Adhesion of anti-CD3 stimulated T lymphocytes to IFN-y (150 units/ml)treated HBMEC as demonstrated by immunoperoxidase staining forleukocyte common antigen (LCA). Large numbers of LCA positiveactivated lymphocytes (L) adhere to endothelial cells (EC). Focally,lymphocytes begin to migrate across the monolayer by extendingpseudopodia between EC (arrowheads). Bar 10urn.‘USfr173Figure 30: Adhesion of anti-CD3 stimulated T lymphocytes (L) toIFN-y (150units/mi) and1E(2,000 units/mi) treated HBMEC as demonstrated byimmunoperoxidase staining for ieukocyte common antigen (LCA).Leucocyte-EC adhesion is comparable to that observed between anti-CD3stimulated T cells and untreated EC. Bar = 10 tm.41:IE.S.4lbIIEC‘>4175Figure 31: Adhesion ofanti-CD3 activated lymphocytesto untreated and cytokinetreated HBMEC. Confluentmonolayers of EC were leftuntreated ortreated with IFN-y (150 units/mi) orIFN-3 (2,000 units/mi), or withacombination of IFN-y (150 units/mi)and f3 (2,000 units/mI) or IFN-y(150units/mi) and anti-IFN-y antibody(cry - 10 tg/m1) for 3 days priortoincubation with activated T ceiis. Forthe mAb blocking studies, cultureswere treated with IFN-y for 3days, followed by 2 hr incubationwith antihuman HLA-DR mAb (ala) priorto incubation with activated Tlymphocytes (Tcd3). Bars represent themean±SEM of triplicate wells oftwo separate experiments.176Fig. 31Adhesion of anti-CD3 activatedlymphocytes to untreated andcytokine-treated HBMECCells/mm20 90 180 270EC+Tcd3EC+R1-y +Tcd3EC+FN-p +TcdSEC+IFN-y + p+Tcd3EC+FN-y+ a7 +Tcd3360 450HEC+R1-y+ ala+1177Figure 32: Scanning electron micrographof the adhesion of activated T lymphocytesto untreated (a) and IFN-y (150units/mI) treated HBMEC (b, c). IFN-ytreatment of endothelial cells (EC) inducesoverlapping of EC processes(*in b). Activated lymphocytes (L) adhereto the endothelium (EC) in largenumbers, and they appear enlarged and exhibita ruffled cell membranewith numerous folds in comparison tothe resting lymphocytes (r)(arrowheads). Activated lymphocytes establishclose contact with EC bymeans of cytoplasmic projections (small arrows)and usually positionthemselves along the borders between adjacent EC inboth untreated andIFN-y treated EC. Protuberances on the apical surfaceof the endothelium,having the size and shape of an activated T cell,indicate movementthrough the EC cytoplasm (large arrows) (a- c). Bars = 50 tm (a ,b) and19 tm (c).F. 30to—3eZCLgo181Figure 33: Adhesion anti-CD3 activated lymphocytes to untreated HBMEC. Activatedlymphocytes (L) display abundant cytoplasm, increased numbers ofmitochondria (m) and variable numbers of cytoplasmic vacuoles (V)containing amorphous, flocculent material. Several points of close cell tocell contact between endothelium (EC) and processes of adherentlymphocytes are present (arrows). C, cellagen membrane. Bar =’S----4t.J183Figure 34: Adhesion ofanti-CD3 stimulated T cells to untreated HBMEC.The cellsurface of activated T lymphocytes (L)is extremely irregular due to thepresence of numerous thin, finger-like cytoplasmicprocesses (arrows).Variable numbers of cytoplasmic vacuoles(V) containing amorphousflocculent material are present inthe lymphocyte cytoplasm. EC,endothelial cell; N, nucleus; C, cellagen membrane. Bar= 1tim.:0::.-ijA185Figure 35: Adhesion of resting lymphocytes(L) to IFN-y (150 units/mi) and antihuman HLA-DR mAb treated HBMEC asdemonstrated byimmunoperoxidase staining for leukocyte common antigen (LCA).FewLCA positive lymphocytes adhere to endothelial cells (EC). Bar = 10 tm.‘18I4a—ECa187Figure 36: Adhesion of anti-CD3 stimulatedlymphocytes (L) to IFN-y (150 units/ml)and anti-human HLA-DR mAb treated HBMEC asdemonstrated byimmunoperoxidase staining for leukocyte common antigen (LCA).Thenumber of lymphocytes adhering to endothelial cells (EC) is comparabletothat observed in the absence of pretreatment with mAb. Bar = 10im.188189Figure 37: a) Transendothelialmigration of resting T lymphocytes (arrow) acrossuntreated HBMEC (EC) monolayers is minimal. C, cellagen membrane. b)Significant increase in migration of lymphocytes (arrows) was observed inHBMEC pretreated with IFN-y (150 units/mi) for 3 days. c) Coincubationof EC with IFN-y (150 units/mi) and 13(2,000 units/mi) for 3 days,significantly suppresses the IFN-y-enhanced migration (arrow). Bars = 10tm (a-c).190‘3-f-191Figure 38: Migration ofresting and anti-CD3 stimulated T lymphocytesacrossuntreated and IFN-y and/or IFN-3 treated HBMEC.Confluent monolayersof EC were left untreated or treated with IFN-y(150 units/mi) or IFN-(2,000 units/mI), or with a combination of IFN-y(150 units/mI) and 3(2,000 units/mi) for 3 days prior to incubationwith resting (T) or anti-CD3stimulated lymphocytes (Tcd3) for 3 hours. For theblocking studies, ECwere treated for 3 days with IFN-y (150 units/mi),followed by 2 hours ofincubation with anti-human HLA-DR (ala) mAbbefore incubating with Tor Tcd3 for 3 hours. Bars represent the mean±SEM of 200 differentlevels.192Fig. 38Migration of resting and anti-CD3activated T cells across untreatedand IFN-y and/or IFN-p treatedHBMECI—+I-+Ia-i— .+ + + +. - ?-I—I— .+ ++ +H”I00)04UUJ4D8J1193Figure 39: Migration of restingT lymphocytes across untreated HBMEC monolayers(a - d). a) A lymphocyte (L) initiates its migrationacross the endothelialcell (EC) monolayers by directing cytoplasmicprocesses between twoadjacent EC. N, nucleus; C, cellagen membrane. Bar= 1 m. b and c) Partof the cytoplasm and nucleus movesbetween two adjacent EC. The cellmembranes of the T cell and EC are closely apposed.Bars= 2imfor (b)and 1 tm for (c). d) At the end of the migrationperiod, the lymphocytesposition themselves between the overlying ECand underlying cellagenmembrane and become elongated and flattened.The processes of theoverlying EC have been resealed (arrow). Bar = 1 im.-D cit198Figure 40: Transendothelial migration of aresting lymphocyte (L) across untreatedHBMEC (EC). This lymphocyte is considered moving through rather thanbetween adjacent EC because the EC cytoplasm surrounds the lymphocyte.C, collagen membrane. Bar = 1 tm.200Figure 41: The integrity of the ECmonolayers is reestablished once restinglymphocytes (L) have completed their migration across the IFN-y (150units/mi, 3 days) treated EC(a, b). Bars = 2 rim.N00203Figure 42: Nonspecific activation of Tlymphocytes enhances their migration acrossuntreated HBMEC monolayers. Migrated T cells (arrows) remain betweenEC and cellagen membranes (C). Bar = 10 pm.204205Figure 43: Migration of activated lymphocytes acrossuntreated (a) and IFN-y treatedHBMEC (b). a) An activated T lymphocyte (L) begins to migratebetweenadjacent endothelial cells (EC). Close contact betweenEC and thelymphocyte is maintained (arrows). C, collagen membrane. b)At the endof the migration period, monolayers resume their continuityover themigrated lymphocytes (L). EC, endothelial cells; C, collagen membrane.Bars = 2 m.0IzoqA208Figure 44: Migration of anti-CD3 stimulated lymphocytesacross IFN-y treatedHBMEC. The lymphocytes (L) that havecompleted their migration acrossEC of the top layer, proceeded to migrate acrossthe next layer of EC. Bar2 tm.0C..SLLZLr‘-.,,‘—C210Figure 45: Effect of (A) Ca2ionophore, (B) EGTA, and (C) IFN-y on the release ofFVIIIR:Ag from human brain microvessel endothelial cells. After a 10 mmincubation with 10 pMCa2+ionophore (A), the cytoplasmic vesicles aredepleted of FVIIIR:Ag, as indicated by their lack of staining. N,nucleus.(B) After 10 mm treatment with 1 mM EGTA, mostof the vesicles containgold particles in small aggregates. N, nucleus. (C) Incubationwith 200U/mi IFN-y for 24 hours is associated withvariable staining of a largenumber of vesicles. Bars: A = 1 aim; B,C = 1.5’•••‘••‘:s1•‘••.*•*•*J*•---•:—,•...--%••..--y:.:-‘i1--tvkt-•_;•—‘‘:‘tc‘-•-1,•is4’•*n’---L•§Ig1-%.•K-;*I4D:::e*-•w_*:--:•—,•-•*_+-**cst..%c%vtS.t4*It)3flt4—1-ici’C (F’V4i-‘PY#r”$.%_*;*,.-Ar.7*4W--•-1-•.j:wc:”**-O-’•;,cNiiqØf’$I;c-,*egn.t.P;-v---i-•*•*It•..*Ifl•4*.*¾t%*4.j15•frç--*bc9•I-.,-.-t-$t)4-s-.,t4-½-çh—%:4”•:1--.“—---,----n!-,.1-.t-$Z*•e.•-•.-••-,•--4’3,.-.e:1,--i,,.*-c--.•.-cj•••:*T--.•-‘i:\4”_i%’—‘%‘-3***-*‘*42*-‘----*t$••2•‘.-•*•r(••.--,-•;4•.•43t<----“:--I*---4$.-r:H6—.•-S‘4id•-t1&i•-•-*---H-A‘4*-r-t$’----?-•--*-•---I—1’.--4%;%—.••A4‘¾•H’**-Ip-*:,7it%tt*E:‘•*---t.a212Figure 46: Percentage of immunostained vesicles in untreated and treated cultures.There is a slight increase in the number of labeled vesicles after incubationwith EGTA (55%) vs the untreated cultures (45%), p 0.21. The numberof stained vesicles increased significantly after pre-incubation with IFN-y(72%),p= 0.000. Staining was largely abolished after treatment withCa2+ionophore. 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