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Immunisatie1

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Immunisatie1

  1. 1. Toll-like receptors inToll-like receptors in cardiovascular diseasecardiovascular disease Experimental Cardiology, UtrechtExperimental Cardiology, Utrecht AEHA, New Orleans 2004
  2. 2. Toll-like receptorsToll-like receptors • Innate immune systemInnate immune system • First line of defenseFirst line of defense • Receptors for pathogen-associated patternsReceptors for pathogen-associated patterns • Family of 10 receptors in humanFamily of 10 receptors in human • Toll-like receptor 2 and 4 most attention inToll-like receptor 2 and 4 most attention in the cardiovascular fieldthe cardiovascular field
  3. 3. ThisThis PathPath wayway :: OthOth erer SpeSpe ciescies :: Toll-like receptor pathway
  4. 4. Toll-like receptor 2Toll-like receptor 2 • Ligands associated with gram-positiveLigands associated with gram-positive bacteriabacteria • Peptidoglycan.Peptidoglycan. • Peptidoglycan is associated with anPeptidoglycan is associated with an unstable plaque phenotypeunstable plaque phenotype (Laman et al Am. J.(Laman et al Am. J. Cardiol. 2002)Cardiol. 2002)
  5. 5. Toll-like Receptor 4Toll-like Receptor 4 • Toll-like receptor 4 is the receptor of exogenous LPS andToll-like receptor 4 is the receptor of exogenous LPS and endogenous EDA (fibronectin) and Hsp60.endogenous EDA (fibronectin) and Hsp60. • Activation results:Activation results: Cytokines and chemokinesCytokines and chemokines Matrix metallo proteases, elastasesMatrix metallo proteases, elastases • Endogenous ligands highly expressed in artritic andEndogenous ligands highly expressed in artritic and oncological specimens (Inflammation & matrix turn-over)oncological specimens (Inflammation & matrix turn-over)
  6. 6. Infection Injury Ischemia Oxidative stress Exo. Ligands Endo. Ligands Toll-like Receptor 2&4 Matrix turn- over & Inflammation Atherosclerosis* Plaque stability Neointima formation* Aneurysms Heart Infarction Heart dysfunction remodeling* Hypothetical pathway with a central role for Tlr2 & 4
  7. 7. Evidence atherosclerosisEvidence atherosclerosis • Present in atherosclerotic lesionsPresent in atherosclerotic lesions (Edfeldt et al. Circulation 2002)(Edfeldt et al. Circulation 2002) • TLR4 polymorphism is associated with carotid intimaTLR4 polymorphism is associated with carotid intima thickness in humansthickness in humans (Kiechl et al. NEJM 2002)(Kiechl et al. NEJM 2002) • TLR4 is involved in neointima formationTLR4 is involved in neointima formation in vivoin vivo (Vink et al. Circulation(Vink et al. Circulation 2002)2002) • TLR4 is involved in outward remodelingTLR4 is involved in outward remodeling (Hollestelle et al, Circulation 2004)(Hollestelle et al, Circulation 2004)
  8. 8. TLRs, ligands and atherosclerosisTLRs, ligands and atherosclerosis • PeptidoglycanPeptidoglycan • ED-AED-A
  9. 9. Peptidoglycan (PGN)Peptidoglycan (PGN) • bacterial wall antigen (gram + bacteria)bacterial wall antigen (gram + bacteria) • mucosal sites (intestinal flora)mucosal sites (intestinal flora) • promotes chronic inflammation at non mucosal sitespromotes chronic inflammation at non mucosal sites (functional analog of LPS)(functional analog of LPS) • stimulates production of proinflammatory cytokinesstimulates production of proinflammatory cytokines and matrix metalloproteinases, can activateand matrix metalloproteinases, can activate complement and upregulates adhesion molecules oncomplement and upregulates adhesion molecules on endothelial cellsendothelial cells • Furthermore, IgM, IgA and IgG antibodies specificFurthermore, IgM, IgA and IgG antibodies specific for PGN have been found in human serafor PGN have been found in human sera
  10. 10. PeptidoglycanPeptidoglycan Bacterium Cytoplasmic membrane Peptidoglycan Capsule Immune response against peptidoglycan in atherosclerotic patients
  11. 11. PeptidoglycanPeptidoglycan
  12. 12. Presence of PGN is associated withPresence of PGN is associated with vulnerable plaque phenotypevulnerable plaque phenotype MacrophagesMacrophages ↑↑ Smooth muscle cellsSmooth muscle cells ↓↓ AtheromaAtheroma ↑↑ PGNPGN J.D. Laman et al. Am J Cardiol. 2002 Jul 15;90(2):119-23.
  13. 13. Antibody response against peptidoglycanAntibody response against peptidoglycan Immune response against peptidoglycan in atherosclerotic patients 0,0 0,2 0,4 0,6 0,8 1,0 1,2 IgM IgG IgA ratio(patientserum/standardserum) control patients atherosclerotic patients * *: P=0.02 OudeNijhuis et al Atherosclerosis 2004
  14. 14. Peptidoglycan andPeptidoglycan and intimaintima-media thickness-media thickness *: p=0.02 **:p=0.004 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 2 3 4 IMT in quartiles IgMlevelsagainstPGN (patientserum/standardserum) * ** peptidoglycan and atherosclerotic disease
  15. 15. • Immunoglobulin M Type of Autoantibodies toImmunoglobulin M Type of Autoantibodies to Oxidized Low-Density Lipoprotein Has an InverseOxidized Low-Density Lipoprotein Has an Inverse Relation to Carotid Artery AtherosclerosisRelation to Carotid Artery Atherosclerosis (Karvonen et al Circulation 2003)(Karvonen et al Circulation 2003) • Antibodies to oxidized LDL in relation to carotidAntibodies to oxidized LDL in relation to carotid atherosclerosis, cell adhesion molecules, andatherosclerosis, cell adhesion molecules, and phospholipase A.phospholipase A. (Hulthe et al. ATVB 2002)(Hulthe et al. ATVB 2002)
  16. 16. Cuff experiment in Apo E-/- miceCuff experiment in Apo E-/- mice 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 intima/mediaratio gelatin gelatin + peptidoglycan
  17. 17. Flow Chamber Model VCR Video Recorder Pump 37 C Incubation Chamber Heater Camera Flow Chamber Cell Reservoir Microscope Monitor
  18. 18. TLR2 stimulation decreases totalTLR2 stimulation decreases total monocyte adhesion (2)monocyte adhesion (2) Control Pam3Cys-SK4 PGN Anti L-selectin Ab 0 100 200 300 400 500 600 700 800 #adherentmonocytes/mm2 0 100 200 300 400 500 600 700 800 #adherentmonocytes/mm2 * * * Shear rate 0.8 dyn/cm2 * ** TNFα-activated HUVEC E-selectin and ICAM-1 expressing L-cells *: p<0.05
  19. 19. Monocyte-endothelium interactions Endothelium Monocyte Rolling adhesion Firm adhesion Transmigration Activation Selectins Integrins Chemoattractants
  20. 20. TLR2 stimulation increases firmTLR2 stimulation increases firm monocyte adhesionmonocyte adhesion Control Pam3Cys-SK4 PGN Anti L-selectin Ab E-selectin and ICAM-1 expressing L-cells 0 20 40 60 80 100 120%firmlyadherentmonocytes 40 60 80 100 120 mlyadherentmonocytes * * * *: p<0.05
  21. 21. Migration assayMigration assay Boyden chamber Monocytes Migration filter => staining Stop filter Chemoattractant Cell count per 10 µm 170 µm
  22. 22. Monocyte migration 0 5 10 15 20 25 30 35 40 45 50 HEPES3+ C5a 10e-8 C5a 10e-9 PAF 10e-8 meandistance(um) Control PGN
  23. 23. ED-AED-A • Fibronectin variants are generated from aFibronectin variants are generated from a single gene by alternative RNA splicing ofsingle gene by alternative RNA splicing of the V, EIIIA, and EIIIB segments, whichthe V, EIIIA, and EIIIB segments, which are also known as CS-1, ED-A, and ED-Bare also known as CS-1, ED-A, and ED-B segments, respectively.segments, respectively. • ED-A is an endogenous ligand for Toll LikeED-A is an endogenous ligand for Toll Like receptor 4.receptor 4.
  24. 24. EDA and Hsp60 upregulated 30 days after the ligation
  25. 25. The natural history of atherosclerosis in the Apo E KO mouse.The natural history of atherosclerosis in the Apo E KO mouse. EDA in apoE 0 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0 1 2 3 4 5 6 group EDA(pg) apo 5 apo 11 apo 15 apo25 apo 40
  26. 26. Tan MH, Blood. 2004 Jul 1;104(1):11-8.
  27. 27. ConclusionsConclusions • TLR ligation can induce an inflammatoryTLR ligation can induce an inflammatory response and subsequently acceleratedresponse and subsequently accelerated plaque formation and intima formationplaque formation and intima formation • (The adaptive immune reponse upon)(The adaptive immune reponse upon) endogenous and exogenous ligands forendogenous and exogenous ligands for TLRs may be considered as targets forTLRs may be considered as targets for interventionintervention
  28. 28. AcknowkedgementsAcknowkedgements • Many people but in particular:Many people but in particular: – Dominique de Klein (project leader TLRs)Dominique de Klein (project leader TLRs) – Manon OudeNijhuisManon OudeNijhuis – Arjan SchoneveldArjan Schoneveld

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