1. Autoimmune reactions against heat shock proteins (HSPs), particularly HSP60, may play an important role in the early development of atherosclerosis by attracting immune cells into the arterial wall. 2. HSPs released from dying cells in advanced atherosclerotic plaques can stimulate the innate immune response and promote inflammation, attracting more immune cells and linking to complications like plaque rupture. 3. Both humoral and cellular immune responses against HSP60 work together with classical risk factors to develop and progress atherosclerosis.

1. Atherosclerosis, an Autoimmune Disease?
What could be the culprit antigen(s)? A brief appraisal of the role
of heat shock proteins.
Mohammad Madjid, MD
Center for Vulnerable Plaque Research
University of Texas-Houston Health Science Center and
Texas Heart Institute

2.  In1856 Virchow described atherosclerosis as
“endarteritis”. A century later Russel Ross named
atherosclerosis “an inflammatory disease”. Ross
likened atherosclerosis to other chronic inflammatory
diseases such as rheumatoid arthritis and
glomerulonephritis. 1
 The central role of immune system in atherosclerosis
and its clinical complications is now widely accepted.
Many investigators are searching to find out what
antigens attract immune cells into the arterial wall and
possibly later on into atherosclerotic plaques. 2,3,4
 Autoantibodies against oxidized low-density lipoprotein
(oxLDL), cardiolipin, beta2-glycoprotein-I and heat-
shock protein 60/65 have been suggested. 2

3.  Georg Wick, Qingbo Xu, and colleagues have
hypothesized that an autoimmune reaction
against heat shock protein 60s, expressed by
endothelial cells in areas that are subject to
increased hemodynamic stress, is the initiating
event in atherogenesis.5,6
 The hypothesis indicates that because a high
degree of antigenic homology exists between
microbial (bacterial and parasitic) and human
HSP60, the 'cost' of immunity to microbes
might be the danger of cross-reactivity with
human HSP60 expressed by the endothelial
cells of stressed arteries subjected to classical
risk factors.7

4.  Two major families of HSPs (60s and 70s) have
been related to atherosclerosis. Unlike HSP60s,
HSP70s are not reported as strong triggers of
autoimmune reactions, however, Bond, Johnson
and colleagues have suggested certain role for
HSP70s in atherosclerosis. 8,9
 Chen et al described autologous hsp60 as a danger
signal to the innate immune system.10
 Xu et al. showed induction of arteriosclerosis in
normocholesterolemic rabbits by immunization with
heat shock protein 65. 5
 George, Afek, and colleagues reported induction of
arteriosclerosis in normocholesterolemic rabbits by
immunization with heat shock protein 65. 11,12

5.  A number of other experimental and observational
studies have shown a significant relationship between
heat shock proteins and atherosclerosis. 9,11,13,14
 In humans, expression of HSP60 is correlated
positively with atherosclerotic severity, with the highest
levels of expression seen in the shoulder regions and
around the necrotic core of atherosclerotic plaques. 15

6.  In addition to its antigenic properties, bacterial HSP60
product may stimulate macrophages by production of
cytokines such as TNF-α and also MMPs. It may as
well interfere with innate immunity by binding to CD14
and activating monocytes and/or macrophages and
endothelial cells. 8, 21, 22
 Bocharov et al reported that HSP60 is a high-affinity
high-density lipoprotein binding protein suggesting a
potential mechanism to explain the known association
between immunity developed against HSP60 and the
development of atherosclerosis. 16

7.  Comparing the similarities between atherosclerosis
and other autoimmune disorders such as
rheumatoid arthritis (as indicated by Ross in the
following slide) can also give some hints about the
potential role of autoimmune mechanisms in
atherosclerosis and it’s complications. 1
 Interestingly, recent studies have uncovered an
important role for heat shock proteins in
pathogenesis of rheumatoid arthritis. 17,18
 Like in rheumatoid arthritis, the suggested role of
HSPs in atherosclerosis may also in part explain
the missing link between infectious agents and
atherosclerosis where a high degree of antigenic
homology between human and microbial HSPs can
cause cross-reaction. 17,7

8. DiseaseDisease Monocytes &Monocytes &
MacrophageMacrophage
LymphocyteLymphocyte GranulocyteGranulocyte Connective-Connective-
Tissue CellsTissue Cells
ExtracellularExtracellular
MatrixMatrix
Pathogenetic MechanismsPathogenetic Mechanisms
AtherosclerosisAtherosclerosis
++ ++ -- SMCsSMCs Collagen typeCollagen type
I, III, IV,I, III, IV,
elastin,elastin,
fibronectin,fibronectin,
proteoglycanproteoglycan
Endothelial-cell injury andEndothelial-cell injury and
dysfunction; fibrous cap;dysfunction; fibrous cap;
new matrix formation &new matrix formation &
degeneration; necrotic coredegeneration; necrotic core
CirrhosisCirrhosis
++ ++ -- FibroblastsFibroblasts Collagen typeCollagen type
I, IIII, III
Parenchymal cell injury, newParenchymal cell injury, new
matrix and scarringmatrix and scarring
replacing necroticreplacing necrotic
parenchymaparenchyma
RheumatoidRheumatoid
arthritisarthritis ++ ++ +/-+/- SynovialSynovial
fibroblastsfibroblasts
Collagen typeCollagen type
I, III,I, III,
fibronectin,fibronectin,
proteoglycanproteoglycan
Synovial-cell injury; erosionSynovial-cell injury; erosion
of cartilage; new matrixof cartilage; new matrix
scarring (pannus)scarring (pannus)
Glomeruloscle-Glomeruloscle-
rosisrosis ++ ++ -- MesangialMesangial
cellscells
Collagen typeCollagen type
I, IV,I, IV,
fibronectinfibronectin
Epithelial- and endothelial-Epithelial- and endothelial-
cell injury and dysfunction;cell injury and dysfunction;
decrease in glomerulardecrease in glomerular
filtration; new matrixfiltration; new matrix
formation;formation;
PulmonaryPulmonary
fibrosisfibrosis ++ ++ +/-+/- SMCs,SMCs,
FibroblastsFibroblasts
Collagen typeCollagen type
III, IV,III, IV,
fibronectinfibronectin
Inflammatory exudate inInflammatory exudate in
alveoli & bronchi; organizedalveoli & bronchi; organized
by extensive matrixby extensive matrix
deposition and scarringdeposition and scarring
ChronicChronic
pancreatitispancreatitis ++ ++ -- FibroblastsFibroblasts Collagen,Collagen,
fibronectin,fibronectin,
proteoglycanproteoglycan
Epithelial injury; periductalEpithelial injury; periductal
inflammation; interstitial fatinflammation; interstitial fat
necrosis; new matrixnecrosis; new matrix
formationformation
Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med. 1999 Jan 14;340(2):115-26

9.  Kanwar, Krissansen, et al. found that
expression of HSP60 and HSP70 was strongly
upregulated very early at lesion-prone sites in
the aortas of young apoE-/- knockout mice and
then dramatically down-regulated in the chronic
lesions of aged mice. 20
 They showed that HSP60 and HSP70 were
detectable in the aortas of 3-week-old apoE-/-
mice and were highly expressed in the aortas
of 8-week-old mice. 20

10.  Kanwar et al. indicated that in 8-week-old
apoE-/- mice, HSP60 and 70 were strongly
expressed at valve commissures of the aortic
sinus, extending to the free aortic wall and
including expression by endothelial and intimal
cells. 20
 They concluded that HSP60 and HSP70 were
heterogeneously expressed in lesions of 20-
week-old mice. HSP60 and HSP70 were
strongly expressed in advanced plaques of the
abdominal aorta of 20-week-old mice, whereas
medial layers lack expression. 20

11.  In 69-week-old mice, there was complete loss
of HSP60 and HSP70 in advanced
complicated collagen-rich plaques of the
aortic sinus. (down-regulated in aged mice) 20
 As a result of this study, lesion-prone sites
displayed strong endothelial HSP60
expression, whereas non–lesion-prone sites
of the distal abdominal aorta lacked hsp
expression. 20
 Monocytes/macrophages expressing HSP70
and hsp60 (data not shown) were the most
prominent cell type in lesions. 20

12. Summary:Summary:
1- Autoimmune reactions (cellular and humoral) against
HSPs particularly HSP60s may play an important role
in early stage development of atherosclerosis.
2- HSP60s and HSP70s released from necrotic cells in
the core area of advanced plaques may stimulate the
innate immune response to promote inflammation and
attract new inflammatory cells thereby may link to
complications of plaque such as rupture and or
thrombosis.
3- Humoral and cellular reactions against HSP60 work in
conjunction with classical proven CVD risk factors.

13. Debates:Debates:
I. According to our current body of knowledge, the
development of atherosclerosis seems to have two
major preceding components, metabolic disorder
(lipid abnormality etc.) and inflammatory disorder
(enhanced immune or autoimmune response). The
question is which one comes first?
II. Since the complication of atherosclerosis (vulnerable
plaque) is more important than it’s development
(stable plaque), the question is which one of the two
(1-metabolic, 2-Immune) components of
atherosclerosis plays a more important role?

14. Debates:Debates:
III. How feasible is the idea of vaccination against HSPs
or oxidized-LDL or other suggested antigens? Can
we induce tolerance against HSPs without damaging
the innate immune system?
IV. Which one is more feasible? Eradication of
atherosclerosis by vaccination against triggers of
plaque development, or, eradication of vulnerable
plaque by vaccination against triggers of plaque
vulnerability?

15. 1. Ross R. Atherosclerosis--an inflammatory disease.
N Engl J Med. 1999 Jan 14;340(2):115-26. Review
2. Shoenfeld Y, Sherer Y, George J, Harats D. ;Autoantibodies associated with
atherosclerosis. Ann Med. 2000 Dec;32 Suppl 1:37-40. Review.
3. Hansson, G.; Immunological markers of atherosclerosis.
Lancet. 1993 Jan 30;341(8840):278.
4. Witztum JL, Palinski W. ; Are immunological mechanisms relevant for the
development of atherosclerosis? Clin Immunol. 1999 Feb;90(2):153-6. Review.
5. Xu Q, Dietrich H, Steiner HJ, Gown AM, Schoel B, Mikuz G, Kaufmann SH, Wick
G. ; Induction of arteriosclerosis in normocholesterolemic rabbits by
immunization with heat shock protein 65. Arterioscler Thromb. 1992
Jul;12(7):789-99.
6. Wick G, Schett G, Amberger A, Kleindienst R, Xu Q.; Is atherosclerosis an
immunologically mediated disease?; Immunol Today. 1995 Jan;16(1):27-33.
Review.
References

16. 7. Wick G, Perschinka H, Millonig G. ; Atherosclerosis as an autoimmune disease:
an update.; Trends Immunol. 2001 Dec 1;22(12):665-669.
8. Johnson AD, Berberian PA, Tytell M, Bond MG. ; Differential distribution of 70-
kD heat shock protein in atherosclerosis. Its potential role in arterial SMC
survival.; Arterioscler Thromb Vasc Biol. 1995 Jan;15(1):27-36.
9. Berberian PA, Myers W, Tytell M, Challa V, Bond MG.; Immunohistochemical
localization of heat shock protein-70 in normal-appearing and atherosclerotic
specimens of human arteries.; Am J Pathol. 1990 Jan;136(1):71-80.
10. Chen W, Syldath U, Bellmann K, Burkart V, Kolb H.; Human 60-kDa heat-shock
protein: a danger signal to the innate immune system.; J Immunol. 1999 Mar
15;162(6):3212-9.
11. George J, Shoenfeld Y, Afek A, Gilburd B, Keren P, Shaish A, Kopolovic J, Wick
G, Harats D.; Enhanced fatty streak formation in C57BL/6J mice by
immunization with heat shock protein-65. Arterioscler Thromb Vasc Biol. 1999
Mar;19(3):505-10.
References

17. 12. Afek A, George J, Gilburd B, Rauova L, Goldberg I, Kopolovic J, Harats D,
Shoenfeld Y.; Immunization of low-density lipoprotein receptor deficient (LDL-
RD) mice with heat shock protein 65 (HSP-65) promotes early atherosclerosis.;
J Autoimmun. 2000 Mar;14(2):115-21.
13. Hansen PR, Chew M, Zhou J, Daugherty A, Heegaard N, Jensen P, Mouritsen
S, Falk E.; Freunds adjuvant alone is antiatherogenic in apoE-deficient mice and
specific immunization against TNFalpha confers no additional benefit.
Atherosclerosis. 2001 Sep;158(1):87-94.
14. George J, Afek A, Gilburd B, Shoenfeld Y, Harats D.; Cellular and humoral
immune responses to heat shock protein 65 are both involved in promoting fatty-
streak formation in LDL-receptor deficient mice.
J Am Coll Cardiol. 2001 Sep;38(3):900-5.
15. Kleindienst R, Xu Q, Willeit J, Waldenberger FR, Weimann S, Wick G.
Immunology of atherosclerosis. Demonstration of heat shock protein 60
expression and T lymphocytes bearing alpha/beta or gamma/delta receptor in
human atherosclerotic lesions.; Am J Pathol. 1993 Jun;142(6):1927-37.
References

18. 16. Bocharov AV, Vishnyakova TG, Baranova IN, Remaley AT, Patterson AP,
Eggerman TL.; Heat shock protein 60 is a high-affinity high-density lipoprotein
binding protein.; Biochem Biophys Res Commun. 2000 Oct 14;277(1):228-35.
17. Gaston, JS.; Heat shock proteins and arthritis--new readers start here.
Autoimmunity. 1997;26(1):33-42. Review.
18. Schett G, Tohidast-Akrad M, Steiner G, Smolen J.; The stressed synovium.;
Arthritis Res. 2001;3(2):80-6. Review.
19. Gaston, JS. ; Heat shock proteins and arthritis--new readers start here.;
Autoimmunity. 1997;26(1):33-42. Review.
20. Rupinder K. Kanwar, Jagat R. Kanwar, Dongmao Wang, Douglas J. Ormrod,
and Geoffrey W. Krissansen Temporal Expression of Heat Shock Proteins 60
and 70 at Lesion-Prone Sites During Atherogenesis in ApoE-Deficient Mice
Arterioscler Thromb Vasc Biol 2001 21: 1991-1997.
References

19. 21.21. Kol A, Sukhova GK, Lichtman AH, Libby P.Kol A, Sukhova GK, Lichtman AH, Libby P. Chlamydial heat shock protein 60Chlamydial heat shock protein 60
localizes in human atheroma and regulates macrophage tumor necrosis factor-localizes in human atheroma and regulates macrophage tumor necrosis factor-
alpha and matrix metalloproteinase expression. Circulation. 1998 Jul 28;98(4):300-alpha and matrix metalloproteinase expression. Circulation. 1998 Jul 28;98(4):300-
7.7.
22.22. Kol A, Lichtman AH, Finberg RW, Libby P, Kurt-Jones EA.Kol A, Lichtman AH, Finberg RW, Libby P, Kurt-Jones EA. Cutting edge: heatCutting edge: heat
shock protein (HSP) 60 activates the innate immune response: CD14 is anshock protein (HSP) 60 activates the innate immune response: CD14 is an
essential receptor for HSP60 activation of mononuclear cells. J Immunol. 2000 Janessential receptor for HSP60 activation of mononuclear cells. J Immunol. 2000 Jan
1;164(1):13-7.1;164(1):13-7.
References