Mechanisms of platelet autoimmunity: The role of macrophages

Cytoldne Flow Cytometry: Assessing Cytokhe Productfan at the Single Cell Level Calman Prussin Laboratory @Allergic Diseases,National Institute of Allergy and IIJpcrious Diseases,National Institutes OfHealth, Bethesda,Maryland

cells are the major regulatorycomponentof the vertebrateimmune system.Much of this regulatory functionis mediatedby the elaborationof cytokines,which havepotentbiological activities.NaiveT cells arecapableof limited cytokineexpression,principally secretinginterleukin-2(IL-2). Upon activation anddifferentiationwithin a specific milieu,a naiveT cell differentiatesinto a memoryeffectorT cell andacquiresthe capability to expresscytokinescharacteristic of this memorystats--for example,IL4 IL-5, and interferon-y(EN-y). This differentiationprocessis highly regulated.’ Much of our understandingof these processesoriginatedwith the observations of MosmannandCoffmauzwho initially

T

demonstratedheterogeneityof cytokine productionin murineT cell clones,which weredesignated‘I%,andI&.* Subsequent observationsin both humanand mouse havestrengthenedandelaboratedupon this concept,to thepoint whereit now hasthe statusof paradigm. T cell responsesareclonal in nature: thusthereare numerousbenefitsto studying T cellsat thesinglecell level.However,immunologicstudiesof individual cells arelimited by existingtechnology.3T cell cloningallows oneto circumventthis limitationby clonally expandingindividual T cells in vitro. Insteadof studyinga singlecell, one is ableto studymillions of clonesof that original cell. This allows the useof bulk productionassays,suchas

ELISA andRT-FCR,to studycytokineexpressionby this initial singlecell. Limitations of T Cell Cloning

Thereare severalsignificantliiitations to the studyof T cell clones.First, the generation of T cell clonesis labor intensive; thus,mostof the abovestudiesuponwhich this paradigmhasbeenbuilt haveexamined very limited numbersof clonesand may not be generallyapplicableto all T cells or all subjects.Furtherextensionof the ThdIh2 paradigmto clinical studiesof diseaserequiresa techniquethat is lesslabor intensive,allowing for statisticallysig-

Continued on page 86

Mechanisms of Platelet Autoimmunity: The Role of Macrophages John W. Semple Division of Hematology, St. Michael’s Hospital and the Departmentsof Pharmacology and Medicine, University of Toronto, Toronto, Ontario, Cam.&

utoimmunediseasecanresultfrom the failure of normalself-tolerance mechanisms.’Immunologicaltoleranceis the acquisitionof unresponsiveness to self antigensandis essentialfor thepreservationof thehost.*At present,the two major theoriesof T cell tolerancearethat it is inducedeitherby clonal deletionof selfreactiveT cells within the thymus)or by T

A

cellsbeingrenderedanergicby antigenspecificand nonspecificmechanismsin the periphery.’The breakdownof self tolerancemaybe theresult of a numberof mechanisms,e.g.,abnormalnumbersof self-reactiveT cells, the lack of activeperipheral suppressivemechanisms,and/or environmentalstimuli thatcan mimic self Continued on page 92

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patternsin chronic AITP suggesta Theand Thl activationpattern(lL2, IGlO, and/or Autolmmunlty: The Role of Macrophager IFN-y).mRelatedto this, Garcia-Suarezet Continuedfiom P48e 8s al.” found thatPHA-stimulatedperipheral blood mononuclearcells (PBMC) from patientswith chronic AITP secreteelevated antigens(antigenicmimicry). levels of TNF-a andIPNy, whereas AutoimmuneidiopathicthrombocyNugent et al.” haveshownthatPHAtopenicpmpura(AITP) is a commonorstimulated PBMC from patientswith acute gan-specificautoimmunebleedingdisease AITP secrete significantlylessIL-4 than in which autoantibodiesaredirected normals. Since Th, andThZgenerationis againstthe individual’sown platelets,tecritically dependenton the balancebesultingin enhancedFc-mediateddestructweenIF&y (for Tb, cells) and IL-4 (for tion of theplateletsby macrophagesin the Th2 cells),z7”33it appearsthat the in vitro reticuloendothelialsystem,particularlythe and in vivo T cell activationpatternsassospleen.AITP is characterizedby thrombociatedwith AITP areprimarily mediated cytopeniaincreasedmegakaryocytesin by Th, cells.Thl cell activationhasbeen the marrow,absenceof splenomegaly,and T Cell Abnormalities in AITP implicatedin severalhumanautoimmune oftenthepresenceof platelet-associated Severalabnormalitieswithin T cell popula- d&a&‘” and we havedeterminedthatILimmunoglobulinsand/orC3, in the abtions havebeendescribedin patientswith senceof secondarydisordersassociated 2 andIFN-ytogether with autologousplatewith plateletdestruction.=Both acuteand acuteor chronicAITP and thesehavebeen lets could stimulateplateletautoantibody reviewedJsThe T cell abnormalitiesinchronicformsof AITP occur. productionfrom enrichedCD19+ B cells clude both phenotypicand functionaldefrom patientswith chronic AITP.%What In children,AITP is usuallyacute,prefectsin the effectorcell populations.For triggersand regulatesin vivo Th cell acticededby viral or bacterialinfection and example,severallaboratorieshavedemon- vation,and ultimatelyautoantibodyprogenerallyresolvesspontaneouslywithin 6 strated thatpatientswith chronicAITP duction,in AITP remains,however, weeks;about20% of childrenwith acute have an increaseof CD3+HLA-DR+ T unknown.For Thl cells to becomeactiAITP progressto chronicAITP,8which aplymphocytes,indicatingthatT cells arebe- vated,an antigenpresentingcell (APC), pearstobesimilartochronicAITPin adults.AITP in adultsis generallychronic, ing abnormallyactivatedin vivo.‘6’8 In vi- particularlya macrophage,is requiredto tro, activatedHLA-DR+ T cellscan presentprocessedantigenin association oftenlastingyears.’Althoughboth acute andchronicAITP areimmune-mediated, significantlyactivaterestingCD4+ T cells with moleculesencodedby the majorhistodifferentpathogeneticmechanismsappear and may thusinfluenceautoreactiverecog- compatibilitycomplex(MHC, e.g.,HLAnition in vivo.19 DR in humans).3s to be responsible.9 For example,it has ActivatedCD&positive‘Thcellscanbe beensuggestedthat the thrombocytopenia Macrophage in AITP generallydistinguishedby their secreted in acuteAITP is due to immunecomplex (viru+amivirus)formationandplatelet“in- cytokinepatterns.%%Th, cellsprimarily It is recognizedthat macrophages, as scavengers, are important in the destruction of nocentbystander**destruction.Chronic secreteIL-2, EN-y, GM-CSF,andTNF-a senescent platelets within the spleen.3637 AITPontheotherhandappearstobea and mediatedelayedtypehypersensitivity moreinherentautoimmuneplateletdisorplay reactionsandsomeantibodysynthesis.20J’ Although it is clear that macrophages der in that the antiplateletautoantibodies a critical role at the “end” of the processin Th2cells,on the otherhand,primarily seform without any known extraneousfacAITP, i.e.,phagocytosisof opsonized CreteIL-4, IL-5, IL-6, andIL-10 andare tars (e.g.,virus infection).Currenttherapy superiorin mediatinghumoralimmunity, platelets?- little work hasbeendevoted for chronicAITP focuseson nonspecific to the APC function of macrophagesin particularlyIgE synthesis.~~21 Another reductionof plateletautoantibodyproduc- classof Th cells,termedThe,are thought AITP-i.e., the initiation of the immune tion. Corticosteroidsare the usualfirst-line to be lessdifferentiatedthanThl andTh2 processthat leadsto autoantibodysynthetherapy,followed by splenectomyor more cells,sincethey can secretemostor all of sis.Although the bulk of phagocytosed potentimmunosuppression, e.g.,cyclothe cytokinesmadeby eithercell type,par- proteinsendup in lysosomesand undergo phosphamide.Othertherapieshaveintitularly IL-2 andlL-10.22-26 We havepre- extensivedegradationinto aminoacidsand eludedintravenousgammaglobulin, smallpeptides,3sm4’ it is possiblethat during viously shownthatPBMC from danazol,plasmapheresis, vincristine,vinthe courseof plateletdestruction,macroapproximately60% of patientswith blastine,cyclospotin-A,a-interferon,and chronicAITP canbe stimulatedby platephagesbecomeactivated(e.g.,by plateletascorbicacid. lets in vitro to proliferateandsecreteILderivedmediiors or otherstimuli) and increasethe expressionof HLA classII 2;la this has been confirmed by others.% In Most immunologicstudiesin chronic molecules in associationwith platelet-deAITP havebeendirectedat characterizing addition,consistentwith the in vitro data, the antiplateletautoantibodies.‘~14 Most of we havefound that in vivo serumcytokine rived autoantigenson their surface.This 0197-1859/96/$0.00 + 15.00

the antigenson plateletmembranesrecognizedby autoantibodiesam “public” antigenssharedby normalplatelets.”The mostabundantandbestcharacterizedplatelet membraneglycoproteins(GP)areGPIIblIIa (fibrinogenand fibronectinreceptor) and GPIb-IX (von Willebrandreceptor) and mostof the epitopesrecognizedby autoantibodiesarefound on these,particularly the IIIa chainof GPIIbIIIa,,’*-I4reacting with multipleregions(including occult) of the proteinJ2The antiplateletimmuneresponsein chronicAITP appearsto be platelet-antigendrivenand not the resuit of antigenicmimicry,asit maybe in acuteAITP.13

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CLINICAL

prfxntation event may activateautoim-

chronicAITP. CD68 is a 110kD lysoso-

muneTh cells (ThJTh,) to drive plateletspecific or cross-reactiveB cells into autoantibody production. Sevd reports havedemonstratedan important role for macrophagesin the initiation and/or aggravation of pathology in other autoimmune diseasessuch as rheumatoid arthritis, type 1 diabetes,multiple sclerosisand pemphigus (reviewed in Unanue*‘). Hence, the antigen presenting function of macrophagesmay be important in the activation of autoimmuneT cells in AITP. Little is known regarding the intracellular degmdationand/or processingof platelet membraneglycoproteins-are they terminally degradedwithin lysosomesor can they be re-expressedon the APC surface in the context of MHC class II molecules?Macrophagescan be induced to expressMHC classII molecules on their surfaceby a number of inflammatory agents42 and protein traffic and processing mechanismswithin APC can be altered by cytokines such as IFN-yand inflammatory events(reviewed in Elson et al.43).Soluble factors secretedfrom activated macrophagescan induce platelet structural changesand activation” and there is evidenceto suggestthat activated macrophagesand their secretedproducts are associatedwith AITP. Zeigler et al.45 showedthat patients with chronic AlTP had significantly elevatedsemm levels of macrophage-colonystimulating factor (MCSF), a glycoprotein cytokine that specifitally inducesthe differentiation of cells within the monocytic lineageq4’ and is a potent activator of mature monocytesand macrophages,it enhancesphagocytosis and IL- 1 pmduction.q**9It was suggested that the high M-CSF levels in patients with AITP may contribute to or initiate platelet destruction by affecting macrophagefunction,& but no evidencewas provided. Possibly related to this, Nugent et al.= recently describedthat in 21 of 24 patients with chronic AITP, a correlation existed between in vivo antiplatelet antibodies and in vitro PBMC IL-1 secretion upon GPIIbIIIa stimulation: this may suggestthat activated macrophagescontribute to the immunopathology in chronic AITP. In addition, Nomura et al.51showed i ncreasedCD68 positive microparticles in patients with

mal glycoprotein thought to be involved in endocytosisand internal membranetrafficking and is restricted to macrophages;5233 its surfaceexpressionon macrophagesis significantly enhancedby inflammatory events.%The authors also found elevatedserumlevels of GM-CSF and concluded that in chronic AITP, cytokines such as GM-CSF are releasedfrom T cells that activate macmphagephagocyto8i.sl?Xulting in an increaseof circulating CD68+ microparticles and platelet destruction.” IncreasedserumGM-CSF levels in patients with AITP was also recently reported by Abboud et al.” In 1992, Boshkov et al.* detectedHLA-DR+ platelets in a child with acute AITP. In a large blinded study, we have recently reported that circulating GPIb+ platelets and microparticles from patients with AITP coexpressCD45, CD14, CD80, and HLA-DR molecules.Of potential importance,the platelet HLA-DR expressionwas inversely correlatedto platelet count. We have now found in preliminary experimentsthat HLA-DR expressionon platelets is mediatedby physical contact with adherent macrophagesand pre-activation of the macrophageswith IFJY-ycansignificantly enhanceplatelet HLA-DR expression.n Summary Taken together,the above results suggest that, in All?, activated macrophagesare presentthat could potentially interact with platelets and induce platelet modifications: one of thesemodifications is the expression of macrophage-derivedHLA-DR moleculesnormally restricted to the golgi, endosomal,and membranecompartments. Thesemacrophagemarkerson platelets may have a role in CD4+ Th, cell regulation: a platelet or microparticle expressing a macrophagederived classII MHC molecule (as found in AITP) may be able to interact with CD4+ T cells @rticularly in the presenceof inflammatory-like stimulating factors, i.e., help) and may either initiate autoimmunepathogenesisin AITP or aggravate/perpetuateit. Thus, there is compelling evidenceto suggestthat a) activated macrophagesare presentin patients with AITP, b) macrophageshave enhanced interactions with platelets in AITP, and c) 0 1996 Elsevier Science Inc.

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activated macrophagescan induce platelet abnormalities such as platelet HLA-DR expression. Basedon thesedata and the fact that macrophagesare known to be potent APC stimulators of Th’ cell activation, it could be hypothesizedthat macrophages may play a critical role in the development and maintenanceof autoimmune‘Ih, pathology in AlTP that may lead to autoantibody production, i.e., that marophages, in addition to their role at the “end” stageof platelet destruction in the RES, havean important role in the initiation of the process. In AITP, macrophagesbecomeactivated (e.g., secondaryto infection or other agonists) and processnormal or modified platelets in an altered fashion that relates to platelet HLA-DR expression.These processingevents,particularly platelet HLA-DR expression,may have an influenceon lW cell activation and ultimately autoantibody production. References 1.

Sinha AA, et al.: Autoimmune diseases: the failure of self tolerance. Science 248:138%1386, 1990. 2. Miller A, et al.: Tolerance and suppressor mechanisms in experimental autoimmune encephalomyelitis: implications for immunotbera~ of human autoimmune diseases. Faseb J 5:25602566,199l. 3. Kappler JW, et al.: Self-tnlerancc eliminates T czlls specific for MIS-modified pmducfs of the major histocnrnpatibility complex. Nature 332~35-40.1988. 4. Mueller DL. et al.: Clonal e-ion versus functional clonal inactivation: A cuatimulatory signalling pathway determines the uutcume of T cell antigen receptor occupancy. AM Rev Immunol 7t445-480. 1989. 5. Karpatkin S, et al.: Autoimmune thmmbocylopenic purpura and the compensated thmmbucytolytic state. Am J Med Sl:l, 1971. 6. Nel ID, d al.: Platelet-bound IgM in autoimmune thmmbocytopak Blood61:119.1983. 7. Ware R: Platelets and thmmbocytbpenia. In: Frank MM, et al.. (eds): Samter’s Immunological Diseases, ~012. Little, Brown, Boston, 1995, pp. 919. 8. Blanchette VS. et al.: Role of intravenous immunoglobulin G in autoimmune hematologic disorders. Semin Hematol29:72,1992. 9. Kaplan C, et al.: Virus-induced autoimmune thrombocytopenia and neutropenia. Semin HematoI 1:34, 1992. 10. Beardsky DS: Platelet autuantigens. In: Kunicki TJ, George JN (eds): Platelet Irnmunobiology, Molecular and Clinical Aspects. Lippincutt, Philadelphia, pp. 121-131.1989. 11. Fitzgerald LA: Platelet membmne glycoproteins.

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