Chlamydial Heat Shock Protein 60 Localizes in Human
Atheroma and Regulates Macrophage Tumor Necrosis
Factor-a and Matrix Metalloproteinase Expression
Amir Kol, MD; Galina K. Sukhova, PhD; Andrew H. Lichtman, MD, PhD; Peter Libby, MD
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Background—Recent evidence has implicated Chlamydia pneumoniae in the aggravation of atherosclerosis. However, the
mechanisms by which this agent affects atherogenesis remain poorly understood. Chlamydiae produce large amounts of
heat shock protein 60 (HSP 60) during chronic, persistent infections, and C pneumoniae localizes predominantly within
plaque macrophages. Several studies have furnished evidence that endogenous (human) HSP 60 may play a role in
atherogenesis. We tested here the hypothesis that atheroma contains chlamydial HSP 60 and that this bacterial product
might stimulate macrophage functions considered relevant to atherosclerosis and its complications, such as production
of proinflammatory cytokines as tissue necrosis factor-a (TNF-a) and matrix-degrading metalloproteinases (MMPs).
Methods and Results—Surgical specimens of human carotid atherosclerotic arteries (n519) and normal arterial wall
samples (n57, 2 carotid arteries and 5 aortas) were tested immunohistochemically for the presence of chlamydial HSP
60 and human HSP 60. Macrophage localization of these antigens was assessed by double immunostaining. Murine
peritoneal macrophages, maintained in serum-free conditions for 48 hours after harvesting, were incubated with
C pneumoniae, chlamydial HSP 60, human HSP 60, or Escherichia coli lipopolysaccharide (LPS). Culture supernatants,
collected at 24 hours for concentration-dependence experiments and at up to 72 hours for time-dependence experiments,
were analyzed for TNF-a by ELISA and for MMP by gelatin zymography. Atherosclerotic lesions showed
immunoreactive chlamydial HSP 60 in 47% (9 of 19) of the cases and human HSP 60 in 89% (17 of 19) of the cases.
Chlamydial HSP 60 colocalized with human HSP 60 within plaque macrophages in 77% (7 of 9) of the cases.
Nonatherosclerotic samples contained neither HSP. Both C pneumoniae and recombinant chlamydial HSP 60 induced
TNF-a production by mouse macrophages in a concentration- and time-dependent fashion. E coli LPS and human HSP
60 produced similar effects. Similarly, C pneumoniae and HSPs induced MMPs in a concentration- and time-dependent
manner. Heat treatment abolished the effect of C pneumoniae and HSPs on both TNF-a and MMP production, but it
did not alter the ability of E coli LPS to induce these functions.
Conclusions—Chlamydial HSP 60 frequently colocalizes with human HSP 60 in plaque macrophages in human
atherosclerotic lesions. Chlamydial and human HSP 60 induce TNF-a and MMP production by macrophages.
Chlamydial HSP 60 might mediate the induction of these effects by C pneumoniae. Induction of such macrophage
functions provides potential mechanisms by which chlamydial infections may promote atherogenesis and precipitate
acute ischemic events. (Circulation. 1998;98:300-307.)
Key Words: proteins n ischemia n atherosclerosis
S
ubstantial seroepidemiologic and some experimental evidence links Chlamydia pneumoniae with the pathogenesis and natural history of atherosclerosis.1 Airborne infection
with this agent, often chronic and asymptomatic, is prevalent
in the general population: An antibody titer against C pneumoniae, detectable in 40% to 50% of the adult population,2
correlates positively with the occurrence of coronary artery
disease.3–5 C pneumoniae infection does not appear limited
to the coronary arteries6 – 8 but can involve other segments
of the vascular tree as well.9 Atheromatous lesions of the
carotid arteries10 and of the lower extremities11 also contain
C pneumoniae. Preliminary reports show that short courses of
macrolide antibiotic therapy can reduce recurrent coronary
events in patients with recent myocardial infarction or unstable angina and elevated anti–C pneumoniae antibody titers.12,13 Despite the various lines of evidence linking atheroma and C pneumoniae, the mechanisms by which this agent
may affect vascular wall cells and atherogenesis remain
poorly understood.14
Atherosclerosis is largely viewed as a chronic inflammatory disease.15 In this respect, the life cycle of Chlamydiae,
obligate intracellular pathogens, appears particularly interest-
Received January 5, 1998; revision received March 10, 1998; accepted March 26, 1998.
From the Vascular Medicine and Atherosclerosis Unit, Cardiovascular Division (A.K., G.K.S., P.L.), and the Immunology Research Division,
Department of Pathology (A.H.L.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass.
Correspondence to Peter Libby, MD, Cardiovascular Division, Brigham and Women’s Hospital, 221 Longwood Ave, Boston, MA 02115. E-mail
[email protected]
© 1998 American Heart Association, Inc.
300
Kol et al
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Figure 1. Western blotting showing selective recognition of
chlamydial and human heat shock protein (HSP) 60 by their
respective antibodies. Recombinant chlamydial and human HSP
60 (1 mg/lane) were subjected to SDS/PAGE under reducing
conditions and blotted with anti-human HSP 60 antibody (top)
or with anti-chlamydial HSP 60 antibody (bottom). Each antibody recognized only its specific protein, without
cross-reactivity.
ing. During the usual infective cycle generating new infectious progeny, Chlamydiae express basal levels of two major
antigens: the major outer membrane protein (MOMP) and the
heat shock protein 60 (HSP 60; 60 stands for 60 kDa).2 Under
some conditions, however, such as in the presence of
interferon-g, a product of activated T cells within atheroma,16
certain Chlamydiae can achieve a state of intracellular
chronic, persistent infection in which they remain viable but
metabolically quiescent and do not replicate.17 During such
chronic, persistent infections HSP 60 production increases
substantially, whereas MOMP becomes almost
undetectable.18
Expression of HSPs, also called chaperonins, a ubiquitous
family of highly conserved proteins, increases during a
variety of conditions such as heat shock, nutrient deprivation,
infections, and inflammatory reactions, functioning to stabilize cellular proteins.19 Atheromatous vessels contain endogenous human HSP 60.20 Human HSP 60, when expressed by
heat-shocked endothelial cells, can provoke an autoimmune
reaction mediating endothelial cytotoxicity.21 Microbial HSP
60, abundantly produced during a chronic chlamydial infection of the vessel wall, might augment atherosclerosis and/or
stimulate humoral and cellular immunity in atheroma. Previous studies investigating the presence of C pneumoniae
within atheroma have mainly addressed the detection of
antigens such as the MOMP or the genus-specific lipopolysaccharide.6 – 8 The presence, localization, and functions of
chlamydial HSP 60 with regard to the pathophysiology of
atheroma remain unexplored.
Although C pneumoniae can infect most cells present in
atheroma,22,23 it localizes mainly in plaque macrophages.6
Mediators elaborated by these phagocytic leukocytes probably contribute importantly to atherogenesis. Tumor necrosis
factor-a (TNF-a) provides one example of a cytokine produced by macrophages within atheroma.24 C pneumoniae
infection induces TNF-a secretion by peripheral human
monocytes.25 This cytokine can induce a number of vascular
cell functions relevant to atherogenesis, including expression
July 28, 1998
301
of endothelial leukocyte adhesion molecules and synthesis of
interleukin-1 mRNA by endothelial cells and smooth muscle
cells.26 Lesional macrophages can also produce matrix metalloproteinases (MMPs),27 enzymes now accorded a major
role in the degradation of connective tissue.28 Thus macrophage-derived MMPs might promote plaque rupture and
consequent thrombosis, the ultimate causes of acute coronary
syndromes.29
This study addressed two hypothesis in this regard: (1)
chlamydial HSP 60, which indicates a chronic, persistent chlamydial infection, localizes within human atheroma; (2) chlamydial HSP 60 can activate macrophage TNF-a and MMP
production, two functions relevant to atherogenesis and to
lesional complications.
Methods
Reagents
Specific monoclonal antibodies against chlamydial HSP 60 and
human HSP 60 were purchased from Affinity Bioreagents, Inc.
Escherichia coli lipopolysaccharide (LPS) was purchased from
Sigma. Formalin-inactivated C pneumoniae organisms were obtained from Washington Research Foundation. The inactivated
C pneumoniae were used to provide a noninfectious source of
chlamydial products. This preparation is antigenically intact and can
elicit a specific immune response in mice (Kol, Libby, Lichtman,
unpublished observations, 1997). Recombinant Chlamydia trachomatis HSP 60 was a generous gift of Dr Ying Yuan (Rocky Mountain
Laboratories, National Institute of Allergy and Infectious Diseases,
Hamilton, Mont). Recombinant human HSP 60 was purchased from
StressGen Biotechnologies Corporation, Victoria, British
Columbia, Canada.
Immunohistochemistry
Surgical specimens of human carotid atherosclerotic arteries (n519)
and normal arterial wall samples (n57, 2 carotid arteries and 5
aortas) were obtained by protocols approved by the Human Investigation Review Committee at the Brigham and Women’s Hospital.
The studied samples were all from different subjects. Serial cryostat
sections (5 mm) were cut, air dried onto microscope slides (Fisher
Scientific), and fixed in acetone at 220°C for 5 minutes. Sections
were preincubated with 0.3% hydrogen peroxidase for 20 minutes.
One serial cross section from each lesion was used for each antibody:
one for staining with anti-CD68 (Dako), one for staining with
anti-chlamydial HSP 60 antibody, one for staining with anti-human
HSP 60 antibody, and one for staining with control antibody (mouse
IgG2, PharMingen; mouse IgG1 myeloma protein MOMP-21, Sigma). Staining was performed with LSAB Kit, Peroxidase (Dako),
according to manufacturer’s instructions with light modifications;
antibody binding was then visualized with 3-amino-9-ethylcarbazole
(Dako). For single immunostaining, nuclear counterstaining was
performed with hematoxylin (Sigma). For double immunostaining,
sections stained with anti-chlamydial or anti-human HSP 60 were
preincubated with avidin and biotin (Vector blocking kit) to block
nonspecific binding of avidin/biotin complex. To identify macrophages within lesions, sections were then incubated overnight with
primary antibody against CD68, followed by biotinylated secondary
antibody (45 minutes; Vector laboratories) and avidin/biotin complex linked to alkaline-phosphatase (Vectastain ABC kit, Vector
laboratories); antibody binding was visualized with fast
blue (Sigma).
The number of positive samples are expressed in the “Results”
section as percentages of the total number of samples examined per
group, followed by the 95% confidence interval limits (CI). Fisher’s
exact test was used for statistical comparison between unpaired data.
A value of P#0.05 was considered significant.
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Chlamydial HSP 60 Induces Macrophage TNF-a and MMP Release
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Figure 2. Chlamydial heat shock protein (HSP) 60 colocalizes with human HSP 60 within atherosclerotic plaque macrophages.
A, Human atherosclerotic plaque (magnification 3100) stained for macrophages (CD68, red). Rectangle indicates the macrophage-rich
region (intimal plaque shoulder) sampled in high power views (3400, B, C, and D) of serial sections adjacent to the one depicted in A.
B, Section stained with mouse IgGs as negative control yielded no staining. C, Double staining for chlamydial HSP 60 (red) and macrophages (CD68, blue). D, Double staining for human HSP 60 (red) and macrophages (CD68, blue). Arrowheads in C and D indicate macrophages (CD681) that stain positively for either chlamydial or human HSP 60. Analysis of adjacent sections showed that both human
and chlamydial HSP 60 colocalized within macrophage clusters. Lumen of the artery is at the top of each photomicrograph. Analysis of
7 of 9 samples (77%) showed similar results.
Western Blotting
Because HSPs are well conserved among various species and share
considerable homology, Western blotting was performed to ascertain
specific recognition of chlamydial HSP 60 and human HSP 60 by
their respective antibodies and to assess potential cross-reactivity.
Recombinant human and chlamydia HSP 60 (1 mg/lane) were
subjected to SDS/PAGE under reducing conditions and blotted to a
polyvinylidene difluoride membrane (Millipore) in semidry conditions (Bio-Rad blotting apparatus). PBS containing 5% defatted dry
milk and 0.1% Tween 20 was used to block membranes and to dilute
primary antibodies and horseradish peroxidase-conjugated goat antimouse secondary antibodies (Santa Cruz Biotechnology). Antibody
binding was visualized by enhanced chemiluminescence (NENDupont). The antibody against chlamydial HSP 60 recognized the
chlamydial but not the human protein, whereas the anti-human HSP
60 antibody recognized the human but not the chlamydial protein
(Figure 1).
Macrophage Isolation and Culture
C57BL/6 female mice (Sprague-Dawley) were maintained on normal
chow diet in pathogen-free facilities approved by the American
Association for Accreditation of Laboratory Animal Care and in
accordance with regulations and standards of the United States
Department of Agriculture, Department of Health and Human
Services and National Institutes of Heath. Mice 8 to 12 weeks old
were injected intraperitoneally with 1.5 mL of 4% thioglycollate
broth (DIFCO). After 4 days, macrophages were collected by
peritoneal lavage, washed with Hanks’ Balanced Salt Solution
(Sigma), and resuspended in RPMI-1640 medium (BioWhittaker)
supplemented with 10% fetal calf serum (HyClone) for plating at a
density of 43105 cells/cm2. After 2 hours, nonadherent cells were
removed by washing with RPMI-1640, and the resultant monolayer
was incubated in serum-free conditions for 48 hours before being
used for experiments. The macrophage monolayer was .99% pure,
as detected by specific immunostaining.
Experimental Conditions and Preparation of
Conditioned Medium
After 48 hours in serum-free conditions, cells were incubated with C
pneumoniae, E coli LPS, recombinant chlamydia HSP 60, human
HSP 60, or with medium alone as a negative control. In the same
experiments, before incubation, an aliquot of each reagent was
heat-treated by boiling for 20 minutes. Concentrations used and
incubation periods are indicated in individual experiments. Concentrations of C pneumoniae are expressed as U/mL, which correspond
to the number of microorganisms per milliliter of culture medium.
Conditioned medium was collected at different time points and
frozen for further analysis. For analysis of gelatinolytic activity,
samples were first centrifuged (500g, 10 minutes, 4°C) and concentrated 35 (Ultrafree centrifugal filter-4; Millipore).
Kol et al
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Figure 3. Chlamydia pneumoniae induce tumor necrosis
factor-a (TNF-a) production by mouse macrophages in a timeand concentration-dependent fashion. Mouse macrophages
were maintained in serum-free conditions for 48 hours and then
incubated with C pneumoniae at a concentration of 107 U/mL
for up to 72 hours (A) or with increasing concentrations for 24
hours (B). Unstimulated serum-free medium was used as negative control. Conditioned medium was collected at the specified
time points and analyzed for TNF-a by ELISA. Three independent experiments showed similar results.
TNF-a Assay
TNF-a levels in culture supernatants were measured with a sandwich
ELISA kit (R&D Systems). Samples were assayed in triplicate.
Absorbance was measured in a Dynatech plate reader at 450 nm.
Data are expressed as mean6SD. Differences between experimental conditions were assessed by ANOVA with Bonferroni correction.
A value of P#0.05 was considered significant.
Gelatin Zymography
Gelatinolytic activity was assessed by SDS/PAGE of concentrated
conditioned medium under nonreducing conditions in gels containing 8% polyacrylamide (Bio-Rad) and 2 mg/mL gelatin (Bio-Rad).
To renature proteins after electrophoresis, SDS was removed from
gels by washing at room temperature in 2.5% Triton X-100 (VWR
Scientific). Gels were then incubated overnight in a buffer containing
50 mmol/L Tris-HCl (pH 7.6), 15 mmol/L NaCl, 10 mmol/L CaCl2,
0.02% NaN3, and 0.1% Brij 35 (Sigma). To detect bands of
gelatinolytic activity, gels were stained with 0.25% Coomassie
brilliant blue R-250 (Sigma). For molecular weight standardization,
prestained (Bio-Rad) or nonprestained (Gibco-BRL) molecular
weight markers were used.
Results
Localization of Chlamydial and Human HSP 60 in
Atherosclerotic Plaque
Immunohistochemical analysis of human carotid atherosclerotic lesions showed colocalization of chlamydial and human
HSP 60 within plaque macrophages. Atherosclerotic lesions
July 28, 1998
303
Figure 4. Chlamydial heat shock protein (HSP) 60 induces
tumor necrosis factor-a (TNF-a) production by mouse macrophages in a time- and concentration-dependent fashion. Mouse
macrophages were maintained in serum-free conditions for 48
hours and then incubated with chlamydial HSP 60 at a concentration of 1 mg/mL for up to 72 hours (A) or with increasing concentrations for 24 hours (B). Unstimulated serum-free medium
was used as negative control. Conditioned medium was collected at the specified time points and analyzed for TNF-a by
ELISA. Three independent experiments showed similar results.
(n519) showed immunoreactive human HSP 60 in 17 of the
cases (89%; 95% CI 66.9% to 98.7%) and immunoreactive
chlamydial HSP 60 in 9 of the cases (47%; 95% CI 24.5% to
71.1%). The two samples that were negative for human HSP
60 were negative also for chlamydial HSP 60. Incubation of
tissue samples with control IgGs yielded no staining. Analysis of nonatherosclerotic tissue (n57) showed no immunoreactivity with either antibody (0%; 95% CI 0% to 41%).
Positivity ratios were significantly different between atherosclerotic and nonatherosclerotic tissues for both human
(P#0.001) and chlamydial (P50.022) HSP 60. As both C
pneumoniae and endogenous HSPs localize mainly within
plaque macrophages,6,30 cellular association of both antigens
was defined by double immunostaining with macrophagespecific antibody. Both chlamydial HSP 60 and human HSP
60 localized mainly within macrophage-rich areas. Little HSP
60 was found outside macrophage-rich areas. Analysis of
serial double-stained sections showed that chlamydial HSP
60 colocalized with human HSP 60 in 7 out 9 specimens
examined (77%; 95% CI 40.0% to 97.2%) (Figure 2).
Induction of TNF-a Expression in Macrophages
by Chlamydial Products
We explored the functional consequences of HSP 60 localization
within macrophages by monitoring the production of TNF-a, a
304
Chlamydial HSP 60 Induces Macrophage TNF-a and MMP Release
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Figure 5. Induction of tumor necrosis
factor-a (TNF-a) production in mouse
macrophages by Chlamydia pneumoniae, chlamydial heat shock protein
(HSP) 60, human HSP 60, and Escherichia coli lipopolysaccharide (LPS):
Effect of heat treatment. A, Mouse macrophages were incubated without serum
for 48 hours and then incubated for 24
hours with serum-free medium only
(unstimulated control), or with E coli LPS
(1 mg/mL), C pneumoniae (107 U/mL),
chlamydial HSP 60 (1 mg/mL), and
human HSP 60 (1 mg/mL). Samples were
collected and analyzed for TNF-a by
ELISA. C pneumoniae, chlamydial HSP
60, and human HSP 60 had a similar
effect on TNF-a production as E coli
LPS. B, Before incubation, reagents
were heat-treated by boiling for 20 minutes. Heat treatment abolished the effect
on TNF-a production of C pneumoniae,
chlamydial HSP 60, and human HSP 60
but did not modify the effect of thermostable E coli LPS. Results shown represent mean6SD of 3 independent experiments. *Statistically significant versus
control (P#0.001, two-sided).
cytokine known to be produced by these cells in atheroma.24 Both
C pneumoniae (Figure 3) and purified recombinant chlamydial HSP
60 (Figure 4) induced a time- and concentration-dependent increase
in TNF-a elaboration by macrophages; maximal induction by
both these stimuli occurred after 6 hours and lasted up to 72 hours.
C pneumoniae induced TNF-a elaboration by macrophages to the
same extent as maximally effective concentrations of E coli LPS.
Chlamydia HSP 60 had nearly the same stimulatory effect on
TNF-a production as did E coli LPS (Figure 5A). Human HSP 60
also induced TNF-a to a similar extent (Figure 5A). Chlamydiae
express their own genus-specific endotoxin, thus C pneumoniae
could induce TNF-a because of its own endotoxin or because of its
HSP 60. To distinguish among these possibilities, we heat-treated
the C pneumoniae, the HSP 60s (both chlamydia and human), and
the control E coli endotoxin. Bacterial lipopolysaccharides exhibit
thermostability, whereas most proteins are thermolabile. Heat treatment of C pneumoniae or HSP 60 abrogated their ability to induce
TNF-a but did not alter the effect of E coli endotoxin (Figure 5B),
thus indicating that the C pneumoniae effect was mainly mediated
by a heat-labile component rather than its lipopolysaccharide.
Induction of MMP Expression in Macrophages by
Chlamydial Products
In addition to production of cytokines such as TNF-a,
macrophages may contribute to plaque evolution and instability by elaborating matrix metalloproteinases capable of
Kol et al
July 28, 1998
305
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Figure 6. Chlamydia pneumoniae induce
matrix-degrading metalloproteinase
(MMP) production by mouse macrophages in a time- and concentrationdependent fashion. Mouse macrophages
were maintained in serum-free conditions for 48 hours and then incubated
with C pneumoniae at a concentration of
107 U/mL for up to 72 hours (A) or with
increasing concentrations for 24 hours
(B). Escherichia coli lipopolysaccharide
(LPS, 1 mg/mL) and unstimulated serumfree medium (O) were used, respectively,
as positive and negative controls at the
different time points. Conditioned
medium was collected at the specified
time points and analyzed for MMP by
gelatin zymography. The 105-kDa band
corresponds to MMP-9, the 72 kDa
band to MMP-2. Three independent
experiments yielded similar results.
degrading the plaque’s fibrous cap.28 We therefore tested
whether C pneumoniae or chlamydial HSP 60 might regulate
MMP expression by macrophages. C pneumoniae induced a
time-dependent elaboration of gelatinases by macrophages
(Figure 6A). A 105-kDa band, corresponding to MMP-9
appeared early (6 hours); a fainter 72-kDa band, corresponding to MMP-2 appeared later (48 hours), increasing up to 72
hours. Chlamydia HSP 60 also induced a time-dependent
increase in MMP-9 (Figure 7A), reaching a peak at 12 to 24
hours and gradually decreasing after 48 and 72 hours;
however, the HSP did not induce MMP-2 to same extent as
C pneumoniae. The highest concentration of C pneumoniae
or Chlamydia HSP 60 tested actually decreased MMP-9
activity (Figures 6 and 7B). This decrease in gelatinolysis
might be due to generation of an inhibitor or to protein
degradation by macrophages, although the mechanism is not
clear.31
As in the case of TNF-a induction, heat treatment reduced
MMP-9 induction by C pneumoniae and chlamydial HSP 60,
but not by E coli LPS, thus indicating that this property of the
chlamydial components also does not depend on endotoxin.
In addition, human HSP 60 also induced MMP-9 in a
heat-sensitive manner (Figure 8).
Discussion
Increased chlamydial HSP 60 expression characterizes
chronic, persistent chlamydial infections. The ongoing inflammatory response in atherosclerosis, including macro-
Figure 7. Chlamydial heat shock protein
(HSP) 60 induces matrix-degrading metalloproteinase (MMP) production by
mouse macrophages in a time- and
concentration-dependent fashion. Mouse
macrophages were kept in serum-free
conditions for 48 hours and then incubated with chlamydial HSP 60 at a concentration of 1 mg/mL for up to 72 hours
(A) or with increasing concentrations for
24 hours (B). Escherichia coli lipopolysaccharide (LPS) (1 mg/mL) and unstimulated serum-free medium (O) were used,
respectively, as positive and negative
controls at the different time-points.
Conditioned medium was collected at
the specified time points and analyzed
for MMP by gelatin zymography. The
105-kDa band corresponds to MMP-9,
the 72 kDa band to MMP-2. Three independent experiments yielded similar
results.
306
Chlamydial HSP 60 Induces Macrophage TNF-a and MMP Release
Figure 8. Induction of matrix-degrading
metalloproteinase (MMP)-9 production in
mouse macrophages by Chlamydia
pneumoniae, chlamydial heat shock protein (HSP) 60, human HSP 60, and Escherichia coli lipopolysaccharide (LPS):
Effect of heat-treatment. Mouse macrophages were maintained in serum-free
conditions for 48 hours and then incubated with serum-free medium only (O,
unstimulated control), nontreated (2), or
heat-treated (1) E coli LPS (1 mg/mL),
C pneumoniae (107 U/mL), chlamydial
HSP 60 (1 mg/mL), and human HSP 60
(1 mg/mL). Conditioned medium was collected after 24 hours and analyzed for
MMP by gelatin zymography. Heat treatment abolished the effect on MMP-9
(105-kDa band) production of C pneumoniae, chlamydial HSP 60, and human
HSP 60 but did not modify the effect of
thermostable E coli LPS. Three independent experiments yielded similar results.
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phage activation, now widely recognized, can result from
traditional risk factors such as the consequences of hyperlipidemia. However, the potential contribution of nontraditional
risk factors, such as infectious agents, has recently garnered
considerable interest. This study examined the localization of
chlamydia HSP 60 within atheroma and tested the potential
role of this specific molecular component of Chlamydiae in
modulating macrophage functions linked to complications of
atheroma, such as TNF-a and MMP expression.
This article reports 3 novel findings: (1) chlamydia HSP 60
colocalizes with human HSP 60 within atherosclerotic plaque
macrophages; (2) HSP 60, either chlamydial or human, potently
stimulates TNF-a and MMP-9 production by macrophages; and
(3) when these effects are elicited by C pneumoniae, they are
mediated by a heat-labile component, possibly HSP 60, rather
than a thermostable lipopolysaccharide.
Wick et al32 have proposed that HSP 60/65 might promote
atherosclerosis by stimulating autoimmunity. Our finding that
chlamydial HSP 60 colocalizes with its homolog, human HSP
60, within plaque macrophages in the majority of cases
(77%), suggests that bacterial HSP might play such a role.
The homology between human and chlamydial HSP 60
suggests the possibility of antigenic mimicry. Our approach
cannot distinguish whether chlamydial HSP 60 found in
plaques was produced by those C pneumoniae that were
actively replicating within plaque macrophages or by those in
a chronic, persistent infective state. However, chronic infection with C pneumoniae, through the expression of HSP 60,
might provoke an autoimmune reaction against human HSP
60. Indeed, patients with carotid atherosclerosis or coronary
artery disease have high titers of antibodies against human
HSP 60.33,34 HSP 60, like other heat shock proteins, has been
previously considered to act intracellularly to maintain cellular protein stability during stressful conditions.19 We report
here the surprising finding that HSP 60 itself can activate
macrophage stimulation, an observation with potentially important pathologic implications in atheroma formation and
evolution. Human HSP 60 shares with the chlamydial protein
the ability to stimulate TNF-a and MMP-9 production by
macrophages. This study used mouse rather than human
macrophages to facilitate the development of an animal
model. Preliminary experiments in our laboratory have shown
similar results using human monocytes-derived macrophages
(Kol, Lichtman, Libby, unpublished observations, 1998).
When endothelial cells or macrophages express HSP 60 on
their surface and are exposed to antibodies against HSP 60,
they are susceptible to complement-mediated or antibodydependent cellular cytotoxicity.21,35,36 If this were the case in
vivo, this mechanism of cell injury might contribute to the
pathobiology of atherogenesis.
Although it might be tempting to consider C pneumoniae
infection as a possible primary cause of atherosclerotic lesion
formation in some cases, the data currently available do not
justify this conclusion. Infection of the vascular wall with
C pneumoniae is generally focal and does not affect all
lesions examined, raising legitimate questions about the
specificity and the biological significance of the detection of
this agent within atheroma. However, a recent autopsy study
showed greater frequency of chlamydial antigens in the
cardiovascular tissue of patients who died of ischemic heart
disease than in patients who died of noncardiac causes (64%
versus 38%).8 Moreover, the effects of a focal infection might
influence the pathobiology of the surrounding atherosclerotic
environment.
This study sheds new light on the potential molecular
triggers to macrophage activation during atherogenesis. Because HSP 60 is mainly expressed during chronic, persistent
chlamydia infection,17 chronic stimulation of macrophages by
bacterial products might promote inflammatory aspects of
atherogenesis and hence the development of acute coronary
syndromes. Certainly, local infections with agents such as
C pneumoniae will most likely potentiate the evolution of
preexisting atheroma, to which macrophages have already
been recruited by traditional risk factors such as hypercholesterolemia. However, one cannot exclude a priori that
macrophage infiltration in response to a chronic arterial
infection might in some cases instigate lesion formation. The
use of antibiotics to treat chlamydial infection might remove
this stimulus for lesion complication and thus diminish the
likelihood of acute ischemic events. The positive results of
Kol et al
recent preliminary secondary prevention trials with macrolide
antibiotics are intriguing in this regard.12,13
In conclusion, this study shows that chlamydial HSP 60
colocalizes with human HSP 60 within plaque macrophages
and that HSP 60 from both species can induce macrophage
production of TNF-a and matrix-degrading metalloproteinases, two mediators of atherosclerosis complications. Chlamydial HSP 60 might produce such effects in macrophages
harboring C pneumoniae infection. These findings help to
understand the molecular pathways by which C pneumoniae
might participate in atherogenesis and to explain the mechanisms of the epidemiologic and pharmacologic links between
this infectious agent and the clinical manifestations of
atherosclerosis.
Acknowledgments
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This work was supported in part by grants from the National Heart,
Lung, and Blood Institute to Dr Libby (HL-48743) and to Dr
Lichtman (HL-56985). Dr Kol is a Fulbright research scholar. The
authors thank Eugenia Shvartz and Elissa Simon-Morrissey (Vascular Medicine and Atherosclerosis Unit, Cardiovascular Division) and
Lori Henault (Immunology Research Division, Department of Pathology) for their valuable and skillful assistance.
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Macrophage Tumor Necrosis Factor- α and Matrix Metalloproteinase Expression
Amir Kol, Galina K. Sukhova, Andrew H. Lichtman and Peter Libby
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Circulation. 1998;98:300-307
doi: 10.1161/01.CIR.98.4.300
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