C-抑制剂在血管通透性调节和抑制白细胞内皮迁移的作用课件.ppt

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1、C1 Inhibitor:Regulation of Vascular Permeability and Inhibition of Leukocyte Migration Across the Vascular EndotheliumAlvin E.Davis III,M.D.Alvin E.Davis III,M.D.Senior Investigator,CBR Institute for Biomedical Research Senior Investigator,CBR Institute for Biomedical ResearchProfessor of Pediatrics

2、,Harvard Medical School Professor of Pediatrics,Harvard Medical SchoolBoston,Massachusetts,USA Boston,Massachusetts,USAC1Inhibitor:RegulationofV A serpin(serine proteinase inhibitor).Serpins inactivate proteases by forming a stable complex with the protease.Protease recognizes a substrate-like regio

3、n of the inhibitor that is referred to as the reactive center loop.C1 INHIBITOR FUNCTIONAserpin(serineproteinaseinProteaseSerpinReactive centerThe protease recognizesthe reactive center of theinhibitor.Cleavage of thereactive center loop isfollowed by:(1)covalent bond formaton between the protease a

4、ctive site serine and the reactive center amino acid of the serpin.(2)rearrangement of the serpin with insertion of the reactive center loop(in yellow)into sheet A(in red),which results in movement of the protease from one pole of the molecule to the other.This results in structural distortion of th

5、e protease.ProteaseSerpinReactivecenterTProteases Inactivated byC1 Inhibitor Complement SystemClassical pathway:C1r,C1sClassical pathway:C1r,C1sLectin pathway:MASP 1&2Lectin pathway:MASP 1&2 Intrinsic Coagulation/Contact SystemsFactor XIa,Plasma kallikrein,factor XIIaFactor XIa,Plasma kallikrein,fac

6、tor XIIaProteasesInactivatedbyC1InC4b2a3bC3bBb3bC5C5aC5b+C6,C7,C8,C9C5b-9MembraneAttackComplexCLASSICALPATHWAYAntigen-antibodycomplexmicrobeMBL+MASP1,2LECTINPATHWAYALTERNATIVEPATHWAYmicrobe+C3b,B,D,PC3b,Bb,PC1q,C1r,C1sC4C2C4b2a C3C3aC3b+C4b2aC3b+C3bBbPC1INHC1INHFactorIFactorHC1INHC4b2a3bC5C5aC5b+C6,

7、C7,C8,EndothelialcellprolylcarboxypeptidaseFactorXIINegativelychargedsurfaceXIIaC1INHPrekallikreinHighmolecularweightkininogenKallikreinHighmolecularweightkininogenBradykininC1INHC1INHContactSystemActivationEndothelialcellprolylcC1 Inhibitor is a MultifunctionalAnti-Inflammatory Protein1.Regulation

8、of vascular permeability1.Regulation of vascular permeabilityInhibition of contact system activation.Inhibition of contact system activation.C1 inhibitor knockout mouse.C1 inhibitor knockout mouse.2.Modulation of leukocyte transmigration.2.Modulation of leukocyte transmigration.Inhibition of complem

9、ent activation.Inhibition of complement activation.Interaction with E-and P-selectins onInteraction with E-and P-selectins onendothelial cells.endothelial cells.3.Protection from endotoxin shock.3.Protection from endotoxin shock.Inhibition of both complement and Inhibition of both complement and con

10、tactcontactSystems.Systems.Interaction with gram negative endotoxinInteraction with gram negative endotoxinlipopolysaccharide(LPS).lipopolysaccharide(LPS).C1InhibitorisaMultifunctioRecurrent localized edema of skin or Recurrent localized edema of skin or mucosa(larynx,gastrointestinal tract)mucosa(l

11、arynx,gastrointestinal tract)Dominant inheritance:heterozygous for Dominant inheritance:heterozygous for deficiency of C1 inhibitordeficiency of C1 inhibitorPathophysiology:Pathophysiology:Decreased C1INH function Decreased C1INH function Spontaneous activation of the complement Spontaneous activati

12、on of the complement system(C1r,C1s)system(C1r,C1s)decreased C4,C2 decreased C4,C2 Spontaneous activation of the contact system Spontaneous activation of the contact system(plasma kallikrein,factor XIIa)(plasma kallikrein,factor XIIa)c cleaved high leaved high molecular weight kininogen molecular we

13、ight kininogen Mediator?Mediator?Hereditary AngioedemaRecurrentlocalizededemaofsPathophysiology of Hereditary Angioedema Because there is clearcut evidence for both complement and contact system activation,the question arises as to which is responsible for generation of the mediator of angioedema.Un

14、til recently,some data suggested that the mediator was a product of complement system activation while other data suggested that it was derived from contact system activation.PathophysiologyofHereditary Define mediator(s)of angioedema Test new therapies and define their mechanism of action Pathophys

15、iology of vascular permeability Analysis of variability of symptoms in HAEC1 Inhibitor Knockout Mouse.Why?Definemediator(s)ofangioedeC1 INHIBITOR KNOCKOUT MICEC1INH protein C1INH protein levels:levels:-/-mice:C1INH not detectable.-/-mice:C1INH not detectable.+/-mice:+/-mice:50%of normal.50%of normal

16、.Decreased C4 levels.Decreased C4 levels.Normal in size,appearance and growth.Normal in size,appearance and growth.Reproduction,fetal growth and development Reproduction,fetal growth and development are normal.are normal.Two+/-and six-/-mice Two+/-and six-/-mice intestinal wall intestinal wall edema

17、 with obstruction at 10-11 weeks.edema with obstruction at 10-11 weeks.IV Evans blue dye in+/-&-/-mice revealed IV Evans blue dye in+/-&-/-mice revealed enhanced vascular permeability.enhanced vascular permeability.C1INHIBITORKNOCKOUTMICEC1INEvans Blue Dye InjectionWild Type C1INH+/-EvansBlueDyeInje

18、ctionWildTC1INHgenotype+/+/+-/-/-Evansbluedye-+C1INHtreatment-+Does intravenous C1INH reverse theincreased vascular permeability in the C1INH knockout mice?C1INHgenotype+/+/+C1INHgenotype+/+/-/-+/-/-C1INHtreatment 000+Mean.10.15.16.11.10S.E.005.008.012.003.006Vascular Permeability-FootpadsC1INHgenot

19、ype+/+/-/-C1INHgenotype+/+-/-/-Bk2Rgenotype+/+/+-/-Evansbluedye+Is Angioedema Mediated by Bradykinin?Bradykinin induces increased vascular permeability via the Bk type 2 receptor(Bk2R).C1INH-/-mice were mated with Bk2R-/-mice.Hypothesis:C1INH-/-,Bk2R-/-mice will be resistant to the development of in

20、creased vascular permeability.C1INHgenotype+/+C1INHgenotype+/+/-/-/-Bk2Rgenotype+/+/+/+-/-Treatment 0000Mean.10.15.16.10S.E.005.008.012.003Vascular Permeability-FootpadsC1INHgenotype+/+/-/-C1INHgenotype+/+-/-/-/-/-Treatment00 CaptoprilBk1RAHoe140Mean.2577.4087.8860.4477.0252Standarderror.0284.0806.0

21、773.0667.0252Intestinal Vascular PermeabilityC1INHgenotype+/+Is Angioedema Mediated byBradykinin?In addition,the increased vascular permeability in C1INH-/-mice was reversed by treatment with the following:A recombinant variant Kunitz domain inhibitor,DX88(Dyax),that is highly specific for plasma ka

22、llikrein.A recombinant C1INH Ala443 Val,that inhibits the contact system but not the complement system.A Bk2R antagonist,Hoe140(Icatibant).These data all support the hypothesis that the mediator of vascular permeability in C1INH deficiency is bradykinin.IsAngioedemaMediatedbyInad Selectins:adhesion

23、molecules responsible for Selectins:adhesion molecules responsible for leukocyte rolling on the endothelium.leukocyte rolling on the endothelium.E-selectin:endothelial cells;expression induced by E-selectin:endothelial cells;expression induced by TNF-TNF-,IL-1,LPS.,IL-1,LPS.P-selectin:platelets&endo

24、thelial cells;mobilized to P-selectin:platelets&endothelial cells;mobilized to cell surface from intracellular granules by histamine,cell surface from intracellular granules by histamine,thrombin,etc.thrombin,etc.Selectins bind fucosylated mucin-like glycoproteins.Selectins bind fucosylated mucin-li

25、ke glycoproteins.Sialyl Lewis Sialyl Lewisx x is a fucose-containing tetrasaccharide is a fucose-containing tetrasaccharide expressed by a number of proteins;binds all three expressed by a number of proteins;binds all three selectins.selectins.C1 Inhibitor and SelectinsSelectins:adhesionmoleculesC1I

26、NH is extremely heavily glycosylated(35%C1INH is extremely heavily glycosylated(35%of its total mass).of its total mass).27%of the N-glycans are fucosylated.27%of the N-glycans are fucosylated.Human liver,the primary site of synthesis of Human liver,the primary site of synthesis of C1INH,expresses C

27、1INH,expresses 1,3-fucosyltransferase-VI 1,3-fucosyltransferase-VI which catalyzes attachment of the sialyl which catalyzes attachment of the sialyl LewisLewisxx tetrasaccharide(NeuNAc tetrasaccharide(NeuNAc2-3Gal2-3Gal1-1-4(Fuc4(Fuc1-3)GlcNAc).1-3)GlcNAc).Hypothesis:C1INH expresses the LewisHypothe

28、sis:C1INH expresses the Lewisxx moiety.moiety.C1 Inhibitor and SelectinsC1INHisextremelyheavilyglyC1 Inhibitor Expresses Sialyl LewisxHECA452:monoclonalanti-sLewxandsLewaLanes1-4:C1INH8,4,2,1gLane5:U937lysateLane6:LEC11lysateLane7:CHO-K1lysateLane8:BSACSLEX-1:monoclonalanti-sLewxLane1:BSALanes2-3:C1

29、INH5&2.5gLane4:U937lysateDeglycosylation experiments:sLewx isexpressed on N-linked carbohydrate.C1InhibitorExpressesSialylRecombinant C1INH Expressed in LEC11 Cells Expresses the Sialyl Lewisx EpitopeLEC 11 cells express(1,3)-fucosyltransferaseCHOK1 cells do notRecombinantC1INHExpressedinEndothelial

30、 Cell E and P SelectinCo-precipitate with C1 InhibitorHUVEC treated with TNF-&H2O2,then incubated with C1 inhibitor.Cells lysed&immunoprecipitated with rabbit anti-C1 inhibitor antiserum.SDS-PAGE,Western blot probed with antiserum to E or P selectin.ESelectinPselectinBinding of C1INH to CHO cells th

31、atexpress either E or P selectin alsowas demonstrated by FACS.EndothelialCellEandPSelecC1INH Suppresses U937 Cell Adhesion to Endothelial CellsHUVEC monolayers treated with TNF-&H2O2,then incubated with or withoutC1INH(31.5 500 g/ml).BCECF-AM labelled U937 cells added&incubated for60 min at 37oC,the

32、n washed&bound cells quantitated by analysis of fluorescenceintensity.C1INHSuppressesU937CellAdhHUVEC(3x104)were seeded onto an insert and grown in 24-well plates for 4 days,then stimulated with TNF alpha(50 ng/ml,18 h).The integrity of the monolayer was tested with FITC-BSA.U937 cells were labeled

33、with BCECF-AM,5x105 cells(100l)in the absence or presence of either native or reactive center cleaved C1INH at the indicated concentrations were incubated with the HUVEC at 37oC for 45 min with 600l medium in the lower chamber.The fluorescence intensity of 100 l medium from the lower chamber of each

34、 well was measured.C1INH Inhibits U937 Cell Transmigration Across the EndotheliumHUVEC(3x104)wereseededontoC1INH Blocks Carcinoembryonic AntigenBinding to E-selectinC1INHBlocksCarcinoembryonicC1INH Blocks HL-60 Cells from Rolling on E-and P-Selectin Under Flow Conditions In VitroImmobilizedE-selecti

35、nCHO-PCellsC1INHBlocksHL-60CellsfromC1INH Inhibits TNF-Induced Leukocyte Rolling In VivoC1INHiC1INHdN-C1INHPBSC1INHInhibitsTNF-InducedC1INH Blocks Leukocyte Infiltration inThioglycollate Induced PeritonitisC1INHBlocksLeukocyteInfiltC1INH expresses the LewisC1INH expresses the Lewisxx moiety on one m

36、oiety on one or more of its N-linked sugars.or more of its N-linked sugars.C1INH binds to both E-and P-selectins.C1INH binds to both E-and P-selectins.Interaction with selectins does not require Interaction with selectins does not require protease inhibitory function.protease inhibitory function.Thi

37、s interaction inhibits adherence of U937 This interaction inhibits adherence of U937 cells to endothelial cells,prevents cells to endothelial cells,prevents transmigration across HUVEC monolayers,transmigration across HUVEC monolayers,and suppresses leukocyte rolling and suppresses leukocyte rolling

38、 in vivoin vivo.Data are consistent with previous data Data are consistent with previous data showing binding of C1INH to endothelial cells showing binding of C1INH to endothelial cells incubated in the cold(Bergamaschini,et al.incubated in the cold(Bergamaschini,et al.2001.Clin Exp Immunol 126:412)

39、.2001.Clin Exp Immunol 126:412).C1INH and Selectins:SummaryC1INHexpressestheLewisxmoiC1INH may play a role in the normal down-C1INH may play a role in the normal down-regulation of the acute inflammatory regulation of the acute inflammatory response.response.Inhibition of complement system activatio

40、n.Inhibition of complement system activation.Suppression of leukocyte transmigration.Suppression of leukocyte transmigration.Furthermore,the C1INH-selectin Furthermore,the C1INH-selectin interaction may explain the utility of interaction may explain the utility of C1INH in a variety of animal models

41、C1INH in a variety of animal models Hyperacute transplant rejection.Hyperacute transplant rejection.Cardiopulmonary bypass.Cardiopulmonary bypass.Ischemia-reperfusion injury(heart,brain).Ischemia-reperfusion injury(heart,brain).What is the Relevance of thisC1 Inhibitor-Selectin InteractionC1INHmayplayaroleinthenShenghe CaiDongxu LiuEun HanJennifer ScafidiCort CramerShengheCai