1、C1 Inhibitor: Regulation of Vascular Permeability and Inhibition of Leukocyte Migration Across the Vascular EndotheliumAlvin E. Davis III, M.D.Senior Investigator, CBR Institute for Biomedical ResearchProfessor of Pediatrics, Harvard Medical SchoolBoston, Massachusetts, USAC1 Inhibitor: Regulation o

2、f VA serpin (serine proteinase inhibitor).Serpins inactivate proteases by forming a stable complex with the protease.Protease recognizes a substrate-like region of the inhibitor that is referred to as the reactive center loop.C1 INHIBITOR FUNCTIONA serpin (serine proteinase inProteaseSerpinReactive

3、centerThe protease recognizesthe reactive center of theinhibitor. Cleavage of thereactive center loop isfollowed by:covalent bond formaton between the protease active site serine and the reactive center amino acid of the serpin.rearrangement of the serpin with insertion of the reactive center loop (

4、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 the protease. ProteaseSerpinReactive centerTProteases Inactivated byC1 InhibitorComplement SystemClassical pathway: C1r, C1sLectin pathway:

5、 MASP 1 & 2Intrinsic Coagulation/Contact SystemsFactor XIa, Plasma kallikrein, factor XIIaProteases Inactivated byC1 InC4b2a3bC3bBb3bC5C5aC5b + C6, C7, C8, C9C5b-9Membrane Attack ComplexCLASSICAL PATHWAYAntigen-antibodycomplexmicrobeMBL + MASP 1,2LECTIN PATHWAYALTERNATIVE PATHWAYmicrobe+ C3b, B, D,

6、PC3b,Bb,PC1q, C1r, C1sC4C2C4b2aC3C3aC3b + C4b2aC3b + C3bBbPC1INHC1INHFactor IFactor HC1INHC4b2a3bC5C5aC5b + C6, C7, C8, Endothelial cell prolylcarboxypeptidaseFactor XIINegativelychargedsurfaceXIIaC1INHPrekallikreinHigh molecularweight kininogen KallikreinHigh molecularweight kininogenBradykininC1IN

7、HC1INHContact System Activation Endothelial cell prolylcC1 Inhibitor is a MultifunctionalAnti-Inflammatory Protein1. Regulation of vascular permeabilityInhibition of contact system activation.C1 inhibitor knockout mouse.2. Modulation of leukocyte transmigration.Inhibition of complement activation.In

8、teraction with E- and P-selectins onendothelial cells.3. Protection from endotoxin shock.Inhibition of both complement and contactSystems.Interaction with gram negative endotoxinlipopolysaccharide (LPS).C1 Inhibitor is a MultifunctioRecurrent localized edema of skin or mucosa (larynx, gastrointestin

9、al tract)Dominant inheritance: heterozygous for deficiency of C1 inhibitorPathophysiology:Decreased C1INH functionSpontaneous activation of the complement system (C1r, C1s) decreased C4, C2Spontaneous activation of the contact system (plasma kallikrein, factor XIIa) cleaved high molecular weight kin

10、inogenMediator?Hereditary AngioedemaRecurrent localized edema of sPathophysiology of Hereditary AngioedemaBecause 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.Until recently,

11、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.Pathophysiology of HereditaryDefine mediator(s) of angioedemaTest new therapies and define their mechanism of actionPathophysiology of vas

12、cular permeabilityAnalysis of variability of symptoms in HAEC1 Inhibitor Knockout Mouse. Why?Define mediator(s) of angioedeC1 INHIBITOR KNOCKOUT MICEC1INH protein levels:-/- mice: C1INH not detectable.+/- mice: 50% of normal.Decreased C4 levels.Normal in size, appearance and growth.Reproduction, fet

13、al growth and development are normal.Two +/- and six -/- mice intestinal wall edema with obstruction at 10-11 weeks.IV Evans blue dye in +/- & -/- mice revealed enhanced vascular permeability.C1 INHIBITOR KNOCKOUT MICEC1INEvans Blue Dye InjectionWild TypeC1INH +/-Evans Blue Dye InjectionWild TC1INH

14、genotype +/+ +/+ -/- -/- Evans blue dye - + + + C1INH treatment - - - + Does intravenous C1INH reverse theincreased vascular permeability in the C1INH knockout mice?C1INH genotype +/+ +/+C1INH genotype+/+ +/- -/- +/- -/- C1INH treatment 0 0 0 + + Mean .10 .15 .16 .11 .10 S.E. .005 .008 .012 .003 .00

15、6 Vascular Permeability - FootpadsC1INH genotype+/+ +/- -/- C1INH genotype +/+ -/- -/-Bk2R genotype +/+ +/+ -/-Evans blue dye + + + Is Angioedema Mediated by Bradykinin?Bradykinin induces increased vascular permeability via the Bk type 2 receptor (Bk2R).C1INH -/- mice were mated with Bk2R -/- mice.H

16、ypothesis: C1INH -/-, Bk2R -/- mice will be resistant to the development of increased vascular permeability.C1INH genotype +/+C1INH genotype+/+ +/- -/- -/- Bk2R genotype+/+ +/+ +/+ -/- Treatment 0 0 0 0 Mean .10 .15 .16 .10 S.E. .005 .008 .012 .003 Vascular Permeability - FootpadsC1INH genotype+/+ +

17、/- -/- C1INH genotype +/+ -/- -/- -/- -/-Treatment 0 0 Captopril Bk1RA Hoe140 Mean .2577 .4087 .8860 .4477 .0252Standard error .0284 .0806 .0773 .0667 .0252Intestinal Vascular PermeabilityC1INH genotype +/+ Is Angioedema Mediated byBradykinin?In addition, the increased vascular permeability in C1INH

18、 -/- mice was reversed by treatment with the following:A recombinant variant Kunitz domain inhibitor, DX88 (Dyax), that is highly specific for plasma kallikrein.A recombinant C1INH Ala443 Val, that inhibits the contact system but not the complement system.A Bk2R antagonist, Hoe140 (Icatibant).These

19、data all support the hypothesis that the mediator of vascular permeability in C1INH deficiency is bradykinin.Is Angioedema Mediated byIn adSelectins: adhesion molecules responsible for leukocyte rolling on the endothelium.E-selectin: endothelial cells; expression induced by TNF-, IL-1, LPS.P-selecti

20、n: platelets & endothelial cells; mobilized to cell surface from intracellular granules by histamine, thrombin, etc.Selectins bind fucosylated mucin-like glycoproteins.Sialyl Lewisx is a fucose-containing tetrasaccharide expressed by a number of proteins; binds all three selectins.C1 Inhibitor and S

21、electinsSelectins: adhesion moleculesC1INH is extremely heavily glycosylated (35% of its total mass).27% of the N-glycans are fucosylated.Human liver, the primary site of synthesis of C1INH, expresses 1,3-fucosyltransferase-VI which catalyzes attachment of the sialyl Lewisx tetrasaccharide (NeuNAc2-

22、3Gal1-4(Fuc1-3)GlcNAc).Hypothesis: C1INH expresses the Lewisx moiety.C1 Inhibitor and SelectinsC1INH is extremely heavily glyC1 Inhibitor Expresses Sialyl LewisxHECA452: monoclonal anti-sLewx and sLewaLanes 1-4: C1INH 8, 4, 2, 1 gLane 5: U937 lysateLane 6: LEC11 lysateLane 7: CHO-K1 lysateLane 8: BS

23、ACSLEX-1: monoclonal anti-sLewxLane 1: BSALanes 2-3: C1INH 5 & 2.5 gLane 4: U937 lysateDeglycosylation experiments: sLewx isexpressed on N-linked carbohydrate.C1 Inhibitor Expresses Sialyl Recombinant C1INH Expressed in LEC11 Cells Expresses the Sialyl Lewisx EpitopeLEC 11 cells express a (1,3)-fuco

24、syltransferaseCHOK1 cells do notRecombinant C1INH Expressed inEndothelial 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, Western blot probed with antis

25、erum to E or P selectin.E SelectinP selectinBinding of C1INH to CHO cells thatexpress either E or P selectin alsowas demonstrated by FACS.Endothelial Cell E and P SelecC1INH Suppresses U937 Cell Adhesion to Endothelial CellsHUVEC monolayers treated with TNF- & H2O2, then incubated with or withoutC1I

26、NH (31.5 500 g/ml). BCECF-AM labelled U937 cells added & incubated for60 min at 37oC, then washed & bound cells quantitated by analysis of fluorescenceintensity.C1INH Suppresses U937 Cell AdhHUVEC (3x104) were seeded onto an insert and grown in 24-well plates for 4 days, then stimulated with TNF alp

27、ha (50 ng/ml, 18 h). The integrity of the monolayer was tested with FITC-BSA. U937 cells were labeled 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 6

28、00l medium in the lower chamber. The fluorescence intensity of 100 l medium from the lower chamber of each well was measured.C1INH Inhibits U937 Cell Transmigration Across the EndotheliumHUVEC (3x104) were seeded ontoC1INH Blocks Carcinoembryonic AntigenBinding to E-selectinC1INH Blocks Carcinoembry

29、onicC1INH Blocks HL-60 Cells from Rolling on E- and P-Selectin Under Flow Conditions In VitroImmobilizedE-selectinCHO-PCellsC1INH Blocks HL-60 Cells fromC1INH Inhibits TNF- Induced Leukocyte Rolling In VivoC1INHiC1INHdN-C1INHPBSC1INH Inhibits TNF- Induced C1INH Blocks Leukocyte Infiltration inThioglycollate Induced PeritonitisC1INH Blocks Leukocyte InfiltC1INH expresses the Lewisx moiety on one or more of its N-linked sugars.C1INH binds to both E- and P-selectins.In