Chlamydia pneumoniae (TWAR) in Atherosclerosis of the Carotid Artery
Background Chlamydia pneumoniae has been demonstrated in atherosclerotic lesions of coronary arteries and aorta. A seroepidemiological study found C pneumoniae–specific antibody more frequently in persons with significant carotid artery wall thickening than in matched control subjects.
Methods and Results Fresh-frozen or formalin-fixed tissue obtained at carotid endarterectomy was examined by immunocytochemistry (ICC) and the polymerase chain reaction (PCR) for the presence of C pneumoniae. Five of five fresh-frozen and formalin-fixed carotid endarterectomy specimens were positive for C pneumoniae by ICC (three of five by PCR). A total of 56 archival formalin-fixed, paraffin-embedded carotid endarterectomy tissues from three hospitals were examined by ICC. Thirty-two were positive. Thirteen normal carotid artery tissue sections from six patients were negative for C pneumoniae.
Conclusions C pneumoniae organisms are frequently found in the advanced carotid atherosclerotic lesions of persons undergoing endarterectomy. Although these findings do not establish causality for C pneumoniae in carotid artery atherosclerosis, they should stimulate investigation of a possible causal or pathogenic role for the organism in the disease.
The chlamydia isolates called TWAR (after the laboratory designations of the first two isolates, TW-183 and AR-39) were first isolated from respiratory infections a decade ago1 and have subsequently been shown to be a common cause of pneumonia, bronchitis, sinusitis, and pharyngitis in both adults2 and children.3 DNA homology studies and electron microscopy established these organisms as a new third species of chlamydia named Chlamydia pneumoniae.4
The three chlamydia species that infect humans (C trachomatis, C psittaci, and C pneumoniae) are known to cause disease of the heart (myocarditis) and blood vessels (endocarditis).5 Saikku and colleagues6 showed an association of C pneumoniae antibody and coronary artery disease. These seroepidemiological observations were extended by the Finnish group and others.7 8 9 10 In addition, an association of C pneumoniae antibody and carotid artery thickening has been reported.11
The presence of C pneumoniae (TWAR) in atheroma of the coronary artery has been established in several populations by ICC, PCR, and electron microscopy.12 13 14 By ICC techniques, TWAR was demonstrated in macrophages and smooth muscle cells of atheromatous plaques of the aorta.15
This study was undertaken to explore the presence of the organism in other arteries of the body. The finding of C pneumoniae in a high proportion of atheromatous plaque material removed by surgery from carotid arteries led to an expansion of the data by use of archival tissue. As in our other studies, the organism was not found in normal arteries.
The tissue studied was obtained at the Dameron Hospital Heart Institute, Stockton, Calif, during surgery. The first group of tissues examined was frozen in dry ice and shipped to Seattle. The specimens were divided and used for culture and PCR. Formalin-fixed tissue in paraffin blocks from the same patients (although a different part of the removed tissue) was available for ICC study. The tissue blocks from carotid endarterectomy typically had several pieces of plaque material embedded together. Serum from a blood sample obtained before surgery was available from the prospectively studied patients. The study was approved by the Institutional Review Committee of the Dameron Hospital, and all patients studied prospectively signed consent forms.
Additional formalin-fixed tissue blocks from carotid endarterectomy were studied. These tissues were from the surgical pathology archives of the Dameron Hospital, the HMC, and the UWMC, Seattle.
The formalin-fixed portion of each carotid artery specimen was paraffin-embedded and processed according to conventional techniques. Serial sections 4 μm thick were cut and placed on glass slides for hematoxylin-eosin and ICC staining.
ICC Staining for Detection of Chlamydia-Specific Antigens
To determine the presence of chlamydia, one slide from each specimen was stained with CF-2, a genus-specific monoclonal antibody directed against chlamydial lipopolysaccharide. Immunoperoxidase staining was done by the avidin-biotin-peroxidase method as described previously.13 Briefly, it involved deparaffinizing sections, blocking endogenous peroxidase activity with H2O2, and applying the primary CF-2 antibody (1:6000 dilution) for 60 minutes. A biotinylated anti-mouse secondary antibody was then applied for 30 minutes, followed by an avidin-biotin-peroxidase conjugate (ABC Elite, Vector Laboratories) for 30 minutes at room temperature. Standard peroxidase enzyme substrate, 3,3′-diaminobenzidine with nickel chloride, was then added to yield a black reaction product. The slides were counterstained with methyl green. For each experiment, a slide with C pneumoniae–infected HL cells was used as a positive control, and a tissue section stained with mouse ascitic fluid from mice inoculated with NS-O myeloma cells was used as a negative control. Ten of the carotid artery tissue sections that stained positive with CF-2 were stained with TT-401 (1:2000 dilution), a C pneumoniae species–specific monoclonal antibody, and KK-12 (1:6000 dilution), a C trachomatis species–specific monoclonal antibody.15
Detection of C pneumoniae–Specific DNA by PCR
PCR was done with the HL-1, HR-1 primer set as described by Campbell et al.16 DNA was purified from tissues either as described previously13 or by Qiagen columns according to the directions of the manufacturer (Qiagen). Mock extractions of buffer were done and amplified to ensure that no contamination occurred during DNA isolation. Controls consisting of PCR reagents without any specimen and with various dilutions of purified C pneumoniae DNA were done in each PCR run as the negative and positive controls, respectively. Confirmation of presumptive positive specimens and detection of products below the sensitivity of agarose gels were done by immunochemiluminescence using the 474-bp Pst I C pneumoniae–specific fragment as a probe of Southern transfers as described by Kuo et al.13 If the specimen was negative, aliquots were seeded with 100 fg of C pneumoniae DNA and reamplified to rule out that the negative results were due to inhibition of PCR. If inhibition was observed, drop dialysis against sterile water was performed and PCR was repeated.
To minimize the risk of contamination, tissue homogenization, DNA extraction, sample preparation, PCR amplification, and product analysis were done in separate rooms. For tissue homogenization, separate pairs of forceps, scissors, and mortar and pestle were used for each tissue. The rooms used for tissue homogenization, DNA extraction, and sample preparation were equipped with germicidal lamps. Before and after use, the laboratory benches were wiped with Absolve (DuPont NEN) and the UV lights were turned on for 30 minutes.
Cell Culture for C pneumoniae
The HL cell line was used for isolation of C pneumoniae.17
The chlamydia microimmunofluorescence technique18 with whole elementary bodies of TWAR strain AR-39 as antigen was used for serology. Antibodies in the IgG and IgA serum fractions were measured.
Surgical specimens from five patients undergoing carotid artery surgery were studied prospectively. The results are shown in Table 1⇓. All had advanced atherosclerotic plaques, as shown on hematoxylin-eosin–stained sections; four were calcified. All had chlamydia genus–specific antigen demonstrated by ICC, although the number of positive fragments varied among the patients. The density of antigen staining in two of these patients was greater than had been seen in our previous studies of coronary arteries and aorta.12 13 14 15 PCR for TWAR-specific DNA was positive in three patients (two of three fresh-frozen and one of two formalin-fixed specimens). All the patients had some TWAR antibody, although the titers varied. None of the four isolation attempts by culture were successful.
To obtain more information on carotid artery tissue and C pneumoniae, we studied formalin-fixed blocks of tissue from carotid endarterectomies from the archives of the Department of Pathology of the Dameron Hospital, HMC, and UWMC. Table 2⇓ summarizes the results of the ICC studies of these tissues. Tissue from 56 patients was examined, and 31 were positive for chlamydia antigen.
The difference in percent positive between the archival specimens and the fresh specimens was not statistically significant. The majority of tissue pieces (lesion fragments) examined from the positive patients of the Dameron Hospital and HMC were positive (61 of 100). Two of 25 positive patients had only one piece positive.
The Figure⇓ shows photomicrographs of the ICC stain of a positive section with control.
Five of the Dameron Hospital ICC positive carotid tissues and five of the HMC positive tissues were stained with C pneumoniae and C trachomatis species–specific monoclonal antibodies to demonstrate the species specificity of the ICC positive tissues with the genus-specific antibody. All 10 stained with the C pneumoniae–specific antibody but not with the C trachomatis antibody.
PCR was attempted with these fixed carotid tissues with limited success. Most of the HMC specimens had been decalcified with formic acid. Of the five that were not decalcified, two were ICC positive, and one of the two was PCR positive. No other HMC specimen was PCR positive. Four of the 20 Dameron Hospital archival tissues were PCR positive; three of these were ICC positive, and one was not. EDTA was used for decalcification, and each of the positive tissues had been treated. The PCR-positive, ICC-negative patient brings the number of Dameron Hospital archival specimens positive for TWAR to 14 and the total for the archival specimens to 32. PCR was not attempted with the UWMC specimens, all of which had been decalcified with formic acid.
Thirteen specimens of normal carotid arteries (8 specimens from one person) were obtained at autopsy from six patients, 21 to 46 years old, and examined for TWAR antigen by ICC. All were negative.
The major finding of this investigation is that C pneumoniae can frequently be demonstrated in atherosclerotic plaques removed from carotid arteries. This extends the finding of C pneumoniae in atherosclerotic lesions in coronary arteries and aorta to another major artery in which atherosclerotic disease causes an important health hazard. Finding the organism in the arterial lesion does not prove that C pneumoniae is the cause of or plays a role in the progression of atherosclerosis. However, having a known pathogen replicating in the viable cells of atheromata raises the possibility that C pneumoniae contributes to the pathogenesis of atherosclerosis.
Most of the persons studied were consecutive patients undergoing endarterectomy in the three hospitals during the years indicated. We have no information on how representative they are of persons with carotid atherosclerosis in the population at large.
The ICC findings in our series of studies of coronary artery and aorta atheromata12 13 14 15 were confirmed and supported in a number of ways. Two monoclonal antibodies, a genus- and a species-specific antibody, were used. Whenever the genus antibody, which is more avid, was positive, the C pneumoniae–specific antibody was positive. Multiple observers, including pathologists with extensive experience with ICC, have confirmed the findings.12 13 14 15 Double immunocytochemical staining showed that the C pneumoniae antigen is associated with macrophages and smooth muscle cells.15 Because of the very small (4-μm) area of an atheroma represented on a microscopic slide, it is more likely that the ICC results are an underestimation than an overestimation of the frequency of C pneumoniae organisms in atheromata.
Because all previous ICC-positive results for chlamydia in arteries have proved to be C pneumoniae, in this study not all ICC genus–positive specimens were stained with C pneumoniae–specific antibody. Five carotid artery positive specimens each from Dameron Hospital and HMC were confirmed as C pneumoniae with the species-specific antibody.
Our PCR technique for identifying C pneumoniae–specific DNA has been successful with throat swab specimens. PCR has been more sensitive than culture and been closely associated with serological evidence of infection.19 20 Although the PCR results with arterial tissue have confirmed that C pneumoniae is present in atheromata, PCR has been less sensitive than ICC in identifying the organism.13 14 In fixed tissues, formalin may degrade DNA.21 With fresh tissue, there is still some lack of sensitivity that may be due to inhibitors but remains to be explained.
The normal carotid artery tissue specimens examined were not age-matched to the case specimens. Obtaining appropriate comparison of normal artery tissue has been a problem throughout our studies.13 14 15 It is virtually impossible to find older adults without atherosclerosis. Data suggesting that the organism is not found in normal-appearing arteries have been restricted to studies of arteries of young persons or normal parts of arteries with atherosclerosis elsewhere. We also failed to demonstrate C pneumoniae in arteries not expected to develop atherosclerosis, such as the internal mammary artery. Recently we studied age- and sex-matched normal coronary artery segments with material provided from the multicenter study entitled “Pathobiological Determinants of Atherosclerosis in Youth,”22 in which forensic autopsies on persons 15 to 34 years old were studied. Although most of the segments with atheromatous plaques were positive for C pneumoniae, none of 31 normal control coronary artery segments were positive.23
Although C pneumoniae infection can be effectively treated with tetracyline and macrolide drugs, it is not known whether the organisms in atheromata, in which there is probably a low level of replication, could be eradicated by treatment or whether eradication of the organisms would have a favorable effect on the lesions. Nevertheless, because of the lack of effective medical treatment of carotid artery narrowing, a controlled treatment trial with an effective antibiotic might be considered for patients ineligible for surgery or before the need for surgery.
Selected Abbreviations and Acronyms
|HMC||=||Harborview Medical Center|
|PCR||=||polymerase chain reaction|
|UWMC||=||University of Washington Medical Center|
This study was supported in part by a grant (RO1-AI-21885) from the National Institutes of Health.
- Received July 5, 1995.
- Revision received August 23, 1995.
- Accepted September 11, 1995.
- Copyright © 1995 by American Heart Association
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