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(Circulation. 2000;101:2568.)
© 2000 American Heart Association, Inc.
Clinical Investigation and Reports |
Relationship of Chlamydia pneumoniae Infection to Severity of Human Coronary Atherosclerosis
From the Unit of Forensic Medicine, Department of Surgery (K.E., T.G.P.S., O.L.) and Unit of Infectious Diseases and Clinical Microbiology, Department of Medicine (C.P.), University of Uppsala, Uppsala, Sweden; and the Department of Medicine (J.L.M.), University of Florida, College of Medicine, Gainesville, Fla.
Correspondence to J.L. Mehta, MD, PhD, University of Florida College of Medicine, PO Box 100277, Gainesville, FL 32610-0277. E-mail mehta{at}medmac.ufl.edu
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Abstract |
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Background—Infection with Chlamydia pneumoniae has been postulated to play a pathogenic role in atherosclerosis. We examined the role of infection with C pneumoniae in relation to the extent of coronary atherosclerosis.
Methods and Results—Coronary atherosclerosis was graded microscopically on a postmortem basis in a blinded fashion in 60 subjects as mild (n=18) or severe (n=42) atherosclerosis. Serum antibodies to C pneumoniae were measured by microimmunofluorescence test. Paraffin-embedded coronary artery specimens were examined for the presence of chlamydia by use of a genus-specific direct immunofluorescence monoclonal antibody. Frozen coronary artery specimens were examined by immunoperoxidase for the presence of C pneumoniae by use of a specific monoclonal antibody RR-402. Direct immunofluorescence was reactive in 86% of cases with severe atherosclerosis but in only 6% of cases with mild atherosclerosis (P<0.01), whereas immunoperoxidase staining was reactive in 80% and 38% of cases with severe and mild atherosclerosis, respectively (P<0.01). Elevated IgG and IgA levels against C pneumoniae were not different in cases with severe and mild atherosclerosis (61% and 30% for severe atherosclerosis and 67% and 42% for mild atherosclerosis, respectively).
Conclusions—This study supports the hypothesis that intracellular infection with C pneumoniae may relate to the severity of atherosclerosis in some subjects. Serum antibody titers against C pneumoniae do not differentiate between severe and mild atherosclerosis.
Key Words: atherosclerosis • pathology • infection
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Introduction |
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There has been a resurgence of interest in the infectious basis of coronary atherosclerosis. Several recent reviews1 2 have suggested that coronary atherosclerosis may be an autoimmune process triggered by an infectious agent, most likely Chlamydia pneumoniae. This proposition is based on the observation of raised antibody titers to C pneumoniae in patients with coronary artery disease in some studies.3 4 Experimental studies have demonstrated that C pneumoniae can replicate and maintain infection in human macrophages, endothelial cells, and aortic smooth muscle cells.5 6 These cell types show particular susceptibility to infection with C pneumoniae.7 Recently, Muhlestein et al8 showed that intranasal inoculation of C pneumoniae accelerates the development of atherosclerosis in a rabbit model of cholesterol-induced atherosclerosis. They also showed that treatment with azithromycin prevented atherosclerosis in rabbits infected with C pneumoniae. In earlier clinical studies, Gupta et al9 and Gurfunkel et al10 found that antibiotic treatment of patients with high titers of antibody against C pneumoniae significantly reduced the number of cardiac events. However, other studies11 12 have failed to show similar seroepidemiological association between infection with C pneumoniae and presence and extent of coronary atherosclerosis.
The aim of the present study was to correlate the severity of coronary atherosclerosis with intracellular infection with C pneumoniae and with seroposivity against C pneumoniae.
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Methods |
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Autopsy Material
Between December 1995 and June 1996, tissues from 300 consecutive autopsy cases were collected at the Department of Forensic Medicine, University of Uppsala, Uppsala, Sweden. Tissues from 60 of these cases (45 men and 15 women) were examined in the present investigation. Thirty of these cases had a diagnosis of cardiac death. In the remaining 30 cases, the cause of death was noncardiac (suicide, 10; accident, 8; gastrointestinal bleeding, 4; hypothermia, 1; epilepsy, 1; diabetes, 1; aortic aneurysm, 1; bronchial asthma, 1; uremia, 1; cerebral bleeding, 1; and pulmonary embolism, 1). The mean (±SD) age of these cases was 61±17 years. In these cases, serum samples were also available for serological testing (described below).
At autopsy, a 1x1-cm section of heart muscle was frozen at -70°C. Coronary arteries were dissected, and a 2-cm-long segment of the left anterior descending coronary artery (LAD) proximal to the first diagonal branch was removed in each case and fixed in 4% buffered formalin. Pieces from the rest of the coronary arteries were frozen at -70°C. Postmortem blood was collected via a femoral vein. Sera were frozen at -70°C for measurement of lipoprotein(a) and other lipid fractions.
Formalin-fixed and paraffin-embedded coronary arteries were cut in 4-µm-thick serial sections for direct immunofluorescence examination and for May-Grünwald-Giemsa staining. Frozen 10-µm-thick tissue sections were also prepared for immunoperoxidase staining.
All LAD segments were examined under light microscope, and the degree of atherosclerosis was quantified by 2 independent investigators as follows: grade 0, no atherosclerosis; grade 1, arteries with intima plus media (I+M) thickness <0.25 mm; grade 2, arteries with I+M thickness 0.25 to 0.60 mm; grade 3, arteries with I+M thickness between 0.61 and 1.0 mm; grade 4, arteries with I+M thickness between 1.01 and 1.5 mm; and grade 5, arteries with I+M thickness >1.5 mm.
Atherosclerosis in the LAD was then divided into the following groups for the purpose of the study: no atherosclerosis, grade 0; mild atherosclerosis, grade 1 to 2; and severe atherosclerosis, grade 3 to 5.
Direct Immunofluorescence
The sections of the paraffin-embedded and formalin-fixed
arteries were deparaffinized with xylene and alcohol. They were stained
by use of a direct fluorescent Chlamydia
genus–specific monoclonal antibody (Pathfinder, Kallestad
Diagnostics), and the sections were examined under a UV
microscope. The presence of apple-green fluorescent particles
was recorded. Positive control was Imagen control slides (Dakopatts
AB).
Immunoperoxidase Staining
Frozen sections of the coronary arteries were fixed in
acetone and stained with C pneumoniae–specific monoclonal
antibody RR-402 (Dakopatts AB) against the major outer membrane protein
and then stained by the avidin-biotin complex immunoperoxidase method
with the Dako-LSAB kit (Dakopatts AB). The sections were then
counterstained by Mayer’s hematoxylin. Coronary arteries
positive for C pneumoniae by polymerase chain reaction
served as positive controls. Adjacent sections stained without primary
antibody were used as negative controls.
Serology
IgG and IgA antibodies to C pneumoniae were measured
by a microimmunofluorescence test (Dakopatts AB).
An IgG titer of 
1/64 and IgA titer of 1/16 were considered
positive.
Statistical Analysis
All data are expressed as mean±SD. Data in the 2 groups were
corrected for multiple comparisons. Results of direct
immunofluorescence and immunoperoxidase positivity
were compared by 
2 test. The relationship
between IgG and IgA levels was examined by linear regression
analysis. A P value of 
0.05 was considered
significant. Software from InStat 2.01 for McIntosh was used for these
analyses.
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Results |
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Degree of Atherosclerosis
On examination of multiple LAD sections, 18 cases were classified as mild atherosclerosis, and the remaining 42 cases were classified as severe atherosclerosis. The mean age of the 2 groups was 54±18 and 64±16 years, respectively (P=NS). All 60 cases had coronary atherosclerosis.
Data on immunofluorescence were available in all 60 cases, but other data were not available in some cases. The precise number of cases with the relevant study (and positivity, in percent) is expressed in the appropriate sections.
Direct Immunofluorescence
By direct immunofluorescence, 36 (86%) of 42
cases with severe atherosclerosis were reactive for
chlamydia. In contrast, only 1 (6%) of 18 cases with mild
atherosclerosis was reactive for chlamydia. The
difference in the presence of Chlamydia genus–specific
antibodies was highly significant between the 2 groups
(P<0.01). An example of
immunofluorescence reactivity to C
pneumoniae in a case with severe atherosclerosis
is shown in the Figure
(left panel).
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Immunoperoxidase Staining
Immunoperoxidase staining for the presence of C
pneumoniae was reactive in 32 (80%) of 40 cases with
severe atherosclerosis. In contrast, only 6 (38%) of
16 cases with mild atherosclerosis were reactive for
C pneumoniae. The difference in
immunopositivity was significant
(P<0.01). An example of immunoperoxidase reactivity to
C pneumoniae in a case with severe
atherosclerosis is shown in the Figure
(right
panel).
Serology
Serological results were available in 59 of 60 subjects. Serum
antibodies to C pneumoniae (IgG titer 1/64) were
present in 40 cases. Nine of these 40 were also positive for
Chlamydia trachomatis and/or Chlamydia psittaci.
These sera were excluded due to the possibility of cross-reactivity.
Positive antibody levels to C pneumoniae were present in
23 (61%) of 38 subjects with severe atherosclerosis
and 8 (67%) of 12 subjects with mild
atherosclerosis.
Serum antibodies to C pneumoniae (IgA titers 1/16) were
present in 20 cases. Three cases were also positive for C
trachomatis and/or C psittaci. These sera were excluded
due to the possibility of cross-reactivity. Positive antibody levels to
C pneumoniae were present in 12 (30%) of 39 subjects
with severe atherosclerosis and 5 (42%) of 12
subjects with mild atherosclerosis. The difference in
seropositivity was not significant between the 2 groups.
IgG levels correlated with IgA levels with a high degree of correlation coefficient (P<0.001).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In the present carefully conducted study in cases of autopsy-proven coronary atherosclerosis, several key points associated with C pneumoniae became evident.
Importantly, this study did not identify an association between seropositivity to C pneumoniae and the degree of atherosclerosis. In the past, the causative relationship between C pneumoniae and atherosclerosis was speculated on the basis of seropositivity in previous studies.2 3 Clinical trials to examine the causative role of C pneumoniae in cardiac events were designed exclusively on the basis of seropositivity. Two recent clinical trials have shown reduction in acute cardiac events in a small number of patients with coronary artery disease treated with macrolide antibiotics.9 10 However, a recent trial of azithromycin13 in a relatively large number of patients did not show any benefit (in terms of reduction of acute cardiac events), although the therapy decreased markers of infection and inflammation. The results of the latter large clinical trial and the present study indicate that seropositivity to a ubiquitous organism, such as C pneumoniae, is not an index of severity of atherosclerosis in coronary arteries. Results of several other large studies have also yielded data that do not show a direct correlation between seropositivity and extent of coronary atherosclerosis and its complications.11 12 14 15 16
Infection with C pneumoniae is common, especially in the Scandinavian countries, where populations live in a closed environment for a large part of the year. This may well explain the high incidence of seropositivity (62% for IgG and 33% for IgA) in our patients with coronary atherosclerosis.
The absence of correlation between atherosclerosis and serum markers of prior infection with C pneumoniae, however, does not exclude a causative role of C pneumoniae in coronary atherosclerosis. Experimental studies have indicated that nasal inoculation with this organism causes localization of this bacteria in arteries and accelerates the process of atherosclerosis in coronary arteries.17 C pneumoniae has also been identified in human atherosclerotic specimens.18 19 20 21 22 23 A variety of techniques, including immunocytochemistry, polymerase chain reaction, electron microscopy, and bacterial culture, have been used to confirm the presence of C pneumoniae. All these techniques have limitations in terms of identification of the presence of C pneumoniae. The choice of method used to identify C pneumoniae may explain why some investigators have failed to identify C pneumoniae in coronary atherectomy specimen,12 whereas others have found positivity rates as high as 80% in similar tissues.22 We used 2 different methods to define the presence of C pneumoniae: direct immunofluorescence using a genus-specific probe and immunoperoxidase stain with a highly specific monoclonal antibody. Both methodologies revealed a high rate of reactivity (86% and 80%) in severe atherosclerosis and a much lower rate (6% and 38%) in mild atherosclerosis. These observations clearly suggest that C pneumoniae exists and grows in the atherosclerotic regions and that its growth correlates with the degree of atherosclerosis. Fryer et al24 showed that C pneumoniae can infect human vascular endothelial cells and stimulate a 4-fold increase in the expression of tissue factor and platelet adhesion. C pneumoniae can also accumulate, replicate, and maintain infection in human macrophages and smooth muscle cells5 6 ; these cells show particular susceptibility to infection with C pneumoniae.7 It may be speculated that bacteria precipitate local thrombosis and lipid peroxidation in vascular tissues,25 26 27 and the bacterial load determines the extent of atherosclerosis.
There is much emphasis on genetic predisposition to development of coronary atherosclerosis. In related unpublished data, we found a greater prevalence of immunoperoxidase staining for C pneumoniae and certain HLA-DR genotypes (13, 15, and 17) in subjects with severe atherosclerosis than in those with mild atherosclerosis. These observations obviously raise the possibility of genetic predisposition to accumulation of bacteria in arterial tissues and development of atherosclerosis.
These data suggest a link between localization of common bacteria, such as C pneumoniae, in the arterial tissues and degree of atherosclerosis. Once the vessel wall is infected, the process of atherosclerosis is accentuated by activation of clotting system and inflammatory mediators. However, the central role of infection in initiating atherosclerosis in most patients with atherosclerosis remains far from clear. The activation of inflammation and an immune response locally in the coronary arteries may be a response to infection in some patients. Nonetheless, the degree of immune response in circulation (antibody titers) is not a predictor of the degree of infection or the extent of atherosclerosis.
Received September 7, 1999; revision received December 6, 1999; accepted December 22, 1999.
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