SHAPE Society

1. Immunomodulation to treat
atherosclerotic disease
G. Pasterkamp, UMCU, Utrecht,
the Netherlands

2. Atherosclerosis and inflammation
Hansson GK
N Engl J Med
2005;352:1685-95

3. Immunomodulation and
atherosclerosis
• Immunosuppression
– Cyclosporin (Emeson et al. Am J path 1993)
– Transfer of CD4+ cells in Apo E mice

4. Zhou X et al.
Circulation 2000

5. Immunomodulation and
atherosclerosis
• Immunosuppression
– Cyclosporin (Emeson et al. Am J path 1993)
– Transfer of CD4+ cells in Apo E mice
– Transfer of B2GP-I lymphocytes in LDL-R
mice (George J et al. Circulation 2000)
– Antibodies against CD40 and CD40 L

6. Anti CD40L in LDL-R
mouse.
Mach F et al,
Nature 1998
CD154 -/-/ ApoE -/-
Lutgens et al
Nature Med 1999

7. Immunomodulation and
atherosclerosis
• Active immunization
– LDL modified epitopes

8. Palinski et al. PNAS 1995, LDL-R rabbit
MDA =malondialdehyde-LDL

9. Hypercholesterolemic
rabbit,
Ameli S et al.
ATVB 1996

10. Immunomodulation and
atherosclerosis
• Active immunization
– LDL modified epitopes
• Passive immunisation
– IgG
– Antibodies Against Aldehyde-Modified
Apolipoprotein B-100 Peptide Sequences

11. ivIg reduces fatty streak formation in apo E KO mice
Nicoletti A et al
J Clin Invest 1998

12. Antibodies Against Aldehyde-Modified Apolipoprotein B-100
Schiopu et al.
Circulation 2004
Mice were treated with different doses of IEI-E3 (red) or FITC-8 (blue)
antibodies. Values on y axis represent oil red O–stained areas as percentage
of total descending aorta area..

13. Immunomodulation and
atherosclerosis
• Active immunization
– LDL modified epitopes
• Passive immunisation
– IgG
– Antibodies Against Aldehyde-Modified Apolipoprotein B-100
Peptide Sequences
• Interleukin-12 (IL-12) has been identified as a key
inducer of a type 1 T-helper cell cytokine pattern.
Blockade of interleukin-12 function by protein vaccination
attenuates atherosclerosis. (Hauer et al. Circulation 2005)
• Cytokine network manipulation

14. Immunomodulation and
atherosclerosis
• Induction of tolerance
– Influencing Toll Like Receptor Responsiveness.

15. Toll-like receptors
• Innate immune system
• First line of defense
• Receptors for pathogen-associated
patterns
• Family of 10 receptors in human
• Toll-like receptor 2 and 4 most attention in
the cardiovascular field

16. Toll-like receptor pathway
NODs: intracellular proteins involved in inflammation
Inflammatory cytokines
TLR2
TLR1
or
TLR6
MyD88
TIRAP/Mal
IRAK
TRAF6
NEMO/IKKκ
IKKα
IKKβ
NF- κB
NF- κB

17. Immunomodulation and
atherosclerosis
• Induction of tolerance
– Influencing Toll Like Receptor Responsiveness.
• Cross-tolerance TLR2 and TLR4
• Surgery influences endotoxin responsiveness (Lemaire et al J Clin
Imunology 1998)

18. Pretreatment with LPS results in impaired infarct
size in animal experimental model
Eising et al
Cardiovasc Res 1996

19. cTnT release in a model of myocardial ischemia of the LAD
in the anesthetized rat
Zacharowski et al
ATVB 1999

20. 1a- patients with a history of UA and persisting complaints
1b- patients with a history of UA who were free of symptoms
2- patients with chronic angina
3- healthy volunteers
Liuzzo et al
Circulation 2001

21. Methods
• Patients scheduled for percutaneous coronary
intervention (PCI) in the morning included after
informed consent was signed
• Bloodsamples drawn immediately after sheath
insertion and 2 hours after procedure
• Clinical questionnaires, data from patient file and
angiographic data

22. Results
• 100 patients included
• 20 patients excluded:
– 5 patients without 2nd bloodsample
– 2 patients with intravenous corticosteroids excluded
– In 13 patients no balloon dilatation was performed
due to negative FFR, unpassable lesion or
impossibility to visualize coronary lesion
• Flowcytometry of TLR2 and TLR4

23. TLR2 response after 5000 ng/ml Pam3Cys
0
200
400
600
TNF-αconcentration(pg/ml)
p < 0.01
before
PTCA
2h after
PTCA

24. TLR4 response after 100 ng/ml LPS
0
1000
2000
3000
4000
5000
TNF-αconcentration(pg/ml)
p < 0.01
before
n=80
after
n=80

25. TLR2 response (Pam3Cys)
mean TNF-α (±SEM)
n=80
stimulatory
ligand before after p
Pam3Cys
5000 ng/ml
159
(±20)
68
(±10)
<0,01
Pam3Cys
500 ng/ml
68
(±9)
32
(±5)
<0,01
Pam3Cys
50 ng/ml
41
(±5)
20
(±4)
<0,01

26. TLR4 response (LPS)
mean TNF-α (±SEM)
n=80
stimulatory
ligand before after p
LPS 1000
ng/ml
2338
(±161)
1957
(±147)
<0,01
LPS 100
ng/ml
1723
(±128)
1271
(±112)
<0,01
LPS 10
ng/ml
893
(±83)
519
(±51)
<0,01

27. TLR2 expression
before after p
TLR2 on
granulocytes
0,73
(±0,02)
0,70
(±0,02)
0,01
TLR2 on
monocytes
3,30
(±0,19)
2,90
(±0,12)
<0,01

28. TLR4 expression
before after p
TLR4 on
granulocytes
0,61
(±0,02)
0,59
(±0,01)
0,65
TLR4 on
monocytes
3,31
(±0,19)
2,87
(±0,19)
0,05

29. TLR4 response without dilatation
0
1000
2000
3000
4000
5000
TNF-αconcentration(pg/ml)
before,
n=13
after,
n=13

30. TLR2 response without dilatation
0
200
400
600
TNF-αconcentration(pg/ml)
*
before,
n=13
after,
n=13

31. TLR2 response without dilatation
before after p
TLR2 on
granulocytes
0,82
(±0,08)
0,79
(±0,08)
0,26
TLR2 on
monocytes
4,16
(±0,57)
3,82
(±0,52)
0,08

32. Summary
• Coronary balloon dilatation decreases
responsiveness of TLR2 and 4
• Coronary balloon dilatation decreases
expression of TLR2 and TLR4 on individual
granulocytes and monocytes
• These effects were also evident but less
pronounced in patients with less traumatic
intervention

33. TNF-αconcentrationafter10ng/mlLPS
percentage diameter stenosis
percentage diameter stenosis in relation to TLR4 response
51-70% 71-90% 91-99% 100%
0
1000
2000
3000
4000
5000
6000






34. Inhibition of TLR receptor and cytokine signaling-
A unifying theme in ischemic tolerance
(Kariko et al J Cerebral Blood flow &Metabolism, 2004)
• Protection against effects (inflammatory responses) of
acute ischemia by TLR tolerance induction.

35. Conclusion
Baseline responsiveness or tolerance of the
innate immune system upon ligand
stimulation differs among patients.
Understanding the mechanisms of tolerance
development of the innate immune system
may provide new targets for intervention

36. Tracey KJ, Nature. 2002 Dec 19-26;420(6917):853-9
The Immune reflex

37. Cholinergic
anti-inflammatory pathway
• ACh prevents release of pro-inflammatory
cytokines like TNF in macrophages
• Direct electrical vagal stimulations inhibits
TNF synthesis in RES
• Vagotomy exacerbates TNF response to
inflammatoy stimuli