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(Stroke. 2001;32:1966.)
© 2001 American Heart Association, Inc.

Original Contributions

Increased CD8⁺ T Cells Associated With Chlamydia pneumoniae in Symptomatic Carotid Plaque

Zurab G. Nadareishvili, MD, PhD; Deloris E. Koziol, PhD; Brian Szekely; Christl Ruetzler, BA; Ronald LaBiche, PhD; Richard McCarron, PhD Thomas J. DeGraba, MD

From the Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Md.

Correspondence to Thomas J. DeGraba, MD, Stroke Branch, NINDS/NIH, 36 Convent Dr, MSC 4128, Bldg 36, Room 4A-03, Bethesda, MD 20892-4128. E-mail degrabat{at}ninds.nih.gov

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References Chlamydia pneumoniae: A Role... References

 
Background and Purpose—— The presence of Chlamydia pneumoniae has been reported in carotid atheroma, but its causative effect in the activation of an atherosclerotic plaque to a prothrombotic state remains unproved. Antigen- mediated activation of T lymphocytes within plaque may represent a mechanism by which infection can result in plaque conversion. The goal of the present study was to characterize the T-cell subtype profile related to the presence of C pneumoniae in patients with symptomatic versus asymptomatic carotid atherosclerosis.

Methods—— We studied 14 plaques (5 symptomatic and 9 asymptomatic) positive for C pneumoniae confirmed by polymerase chain reaction and 14 plaques (6 symptomatic and 8 asymptomatic) from age- and stenosis-matched patients negative for C pneumoniae by polymerase chain reaction. T-cell subpopulations of T-helper, T-cytotoxic, and T-memory lymphocytes were identified through indirect enzyme immunohistochemistry with anti-CD3+, anti-CD4+, anti-CD8+, and anti-CD45RO+ monoclonal antibodies, respectively. Results are expressed as the number of positive cells per millimeter squared.

Results—— In the absence of C pneumoniae, symptomatic plaques had a modest but significant increase of CD3+ (89.6 versus 55.3, P=0.013), CD4+ (57.3 versus 32.7, P=0.01), and CD45RO+ (82.8 versus 43.7, P=0.007), but not CD8+ T cells (28.5 versus 25.5, P=0.245) compared with asymptomatic. However, in the presence of C pneumoniae, there was significant increase of all T-lymphocyte subtypes in symptomatic plaques, including CD8+ (76.8 versus 30.3, P=0.03), CD3+ (192.1 versus 80.4, P=0.004), CD4+ (111.9 versus 37.9, P=0.003), and CD45RO+ (120.2 versus 72.9, P=0.003) cells compared with asymptomatic plaques. With use of 2-way ANOVA, both the presence of chlamydia and symptoms were associated with significantly higher T-cell counts (P<0.005 for all subtypes).

Conclusions—— Although all patients with symptomatic disease show a modest elevation in the concentration of intraplaque lymphocytes, a preferential increase in CD8+ class I-restricted T cells is observed in symptomatic carotid plaque positive for C pneumoniae. These data provide incentive to further explore the role of Chlamydia in the modification of immune-mediated mechanisms in active atherosclerotic plaque.

Key Words: carotid artery diseases • Chlamydia pneumoniae • leukocytes • plaque

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References Chlamydia pneumoniae: A Role... References

 
The role of inflammation is emerging as a pivotal component in the initiation, maturation, and activation of atherosclerotic plaque.^1,2 Evidence indicates that plaques associated with thrombotic and thromboembolic events in the coronary and carotid vessels have an increased expression of inflammatory mediators, including leukocyte adhesion molecules,³ and of inflammatory cells, including macrophages and T lymphocytes.^4,5 Activation of these inflammatory cells is believed to result in plaque wall destabilization and increased prothrombotic character of the plaque, leading to increased risk for stroke and heart attack.^4–8

Recently, attention has been given to T lymphocytes as a mediator of activation of the atherosclerotic plaque. T cells can be recruited to the inflammatory environment of the arterial atherosclerotic plaque wall regardless of their antigenic

See Editorial Comment, page 1972

specificity.⁹ The majority of the T cells are believed to be a heterogeneous population made up predominantly of memory T cells that are quiescent, but can be activated by exposure to specific antigen or mimic antigens to T-cell receptors.^9–11 Oligoclonal expansion of T cells in atherosclerotic plaque suggests the occurrence of an antigen driven T-cell proliferation in atherosclerosis.¹² T lymphocytes that can be stimulated with antigens such as oxidized, low-density lipoprotein¹³ have been suggested as possible triggers for plaque activation. However, to date, evidence is insufficient to demonstrate specific antigen activation of an atherosclerotic plaque in association with symptomatic disease.

Chlamydia pneumoniae, a common pathogen in human respiratory tract infections, is a potential candidate antigen that has been repeatedly identified in atherosclerotic plaque of the coronary and carotid vessels.^14–20 C pneumoniae has been identified as an antigen that can activate T cells cultured from the peripheral blood of patients with atherosclerotic disease in addition to T cells cultured from carotid atherosclerotic plaque.^9,11,21 In addition, C pneumoniae has been reported to stimulate a cell-mediated immune (CMI) response^9,11,22 that could potentially activate both the CD4+ and CD8+ T cells through antigenic peptide binding to HLA class II and class I molecules, respectively.^23–25 The predominant T-cell type that has been reported from examination of cultured cells from plaques are CD4+ T cells,²⁴ though CD8+ T lymphocytes also have been reported.²³ Despite the current information regarding the presence of chlamydia-specific T cells and the potential for CMI, the role of chlamydia in the conversion of a plaque to a prothrombotic symptomatic state and its affect on immune expression remains undetermined.

The goal of the present study was to examine and characterize the T-cell subtype profile in atherosclerotic plaques from symptomatic and asymptomatic patients with and without Chlamydia, as identified by polymerase chain reaction (PCR), to determine whether an association exists between the presence of Chlamydia and immune-mediated symptomatic disease. We hypothesized that (1) CD4+ and CD8+ T cells will be increased in symptomatic plaques with C pneumoniae compared with symptomatic plaques without C pneumoniae, and (2) that CD4+ and CD8+ T-cell increase will occur preferentially in symptomatic carotid plaque with C pneumoniae presence compared with plaques that are asymptomatic with Chlamydia. Analysis of T-cell subsets in the carotid plaques of 14 patients identified with C pneumoniae by PCR compared with those from 14 stenosis-matched patients without C pneumoniae will be performed.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References Chlamydia pneumoniae: A Role... References

 
Ninety-four patients undergoing carotid endarterectomy consented in an institutional review board-approved protocol to allow the atherosclerotic plaque specimen to be analyzed, which included histological analysis, as well as identification of C pneumoniae organisms by PCR.¹⁴ From the cohort, 14 patients were identified by PCR to have C pneumoniae. Fourteen plaques from the 80 patients without demonstrable C pneumoniae were matched by degree of stenosis with the 14 plaques identified to have Chlamydia. Plaques from the C pneumoniae-positive group were matched by decile with the C pneumoniae-negative group. Each group of 14 plaques had 2 plaques from patients with 90% to 99% stenosis, 5 from those with 80% to 89% stenosis, 2 from those with 70% to 79% stenosis, and 2 from those with 50% to 59% stenosis. To examine whether the 14 stenoses-matched C pneumoniae-negative plaques were representative of the entire C pneumoniae negative cohort, analysis was performed to compare the 14 matched plaques with the remaining 66 patients with respect to age, sex, symptoms, hypertension, diabetes, smoking, and hypercholesterolemia. No significant differences were identified between the matched group and the chlamydia negative population as a whole (Table 1). No histological information was known about individual plaques prior to selection. The ratio of symptomatic and asymptomatic plaque used for the C pneumoniae-negative group, which approximates the ratio in the entire group, allowed sufficient comparison with the C pneumoniae-positive plaques and allowed for comparison of T cells in the symptomatic versus asymptomatic plaque of C pneumoniae-negative patients.

View this table: [in this window] [in a new window]   Table 1. Stenosis-Matched vs Total Chlamydia-Negative Group

Comparison between C pneumoniae-positive and C pneumoniae-negative groups revealed no differences in the medication profile with respect to aspirin, angiotensin-converting enzyme inhibitors, ß-blockers, or statins. No patient was taking steroids.

Carotid plaques were obtained from patients undergoing carotid endarterectomy at the National Naval Medical Center (Bethesda, Md) who had stenotic atheromatous lesions of 50%, as measured by North American Symptomatic Carotid Endarterectomy Trial criteria.²⁶ All patients gave consent for use of their plaque and blood samples for research analysis per the hospital’s institutional review board.

History and neurological examination were obtained by a neurologist from the Stroke Branch of the National Institute of Neurological Disorders and Stroke before surgery for all patients to determine symptomatic and asymptomatic group classification. Stroke risk factors of hypertension (blood pressure >140/90 mm Hg for 1 year), past history of smoking (5 pack-year history), diabetes (oral agent or insulin dependent for >1 year), and hypercholesterolemia (LDL >160 mg/dL untreated, fasting triglycerides >200 mg/dL, or on cholesterol-lowering medication for >1 year), current medications including use of antiplatelet agents, and time from last ischemic event were recorded. Patients with atrial fibrillation were excluded from the symptomatic group to avoid the possible confusion between a cardiac and carotid source of embolism. A CT scan of the head was obtained for all patients and used as supporting evidence of the history and physical examination in the asymptomatic population to rule out silent infarction. Patients were classified as symptomatic if they had a prior transient ischemic attack (TIA) or stroke within 4 months or asymptomatic if they had no prior history of cerebral ischemic event (TIA or stroke) and no evidence of ischemic lesion on the CT scan performed before surgery. All 5 patients with C pneumoniae-positive symptomatic plaques had TIAs, and of the 6 symptomatic patients with C pneumonia-negative plaques, 4 had strokes and 2 had TIAs.

Carotid plaque specimens were collected in the operating room, with care to remove the plaque in a single piece. The highest-grade region of the plaque was identified on angiography. Using a correction factor of 15% magnification of the angiographic image, measurements from the bifurcation to the area of highest-grade stenosis was performed and a 3- to 5-mm cross-sectional segment was obtained at that point, with visual inspection of the dissected specimen to confirm that the high-grade region of the plaque was obtained. All preparation of the tissue was performed under sterile conditions. These frozen specimens were snap-frozen in liquid nitrogen and stored at -70°C and used for PCR analysis. Cryostat sections directly adjacent to the material used for PCR analysis were mounted for immunohistochemical staining.

PCR Protocol
The presence of C pneumoniae-specific DNA within the carotid plaque was confirmed by PCR according to previously described protocol.^14,27

DNA was isolated from approximately 5 to 10 serial sections with the QIAamp Tissue Kit (Qiagen) according to the manufacturers instructions, except that DNA was eluted in 2 steps of 50 µL each.

Care was taken to maintain aseptic handling of the tissue samples. Patient samples and primary PCR reaction assembly were kept isolated in a separate laboratory to eliminate contamination of DNA samples by primary or secondary PCR products. This primary laboratory was supplied separately from the main laboratory. Each sample was analyzed in triplicate to insure reliability.

To detect C pneumoniae DNA at the required level of sensitivity, a 2-step nested PCR protocol was implemented. Both the inner and outer sets of PCR primers were designed with the Lasergene software (DNAstar) on an LCIII computer (Apple) to maximize PCR performance. The sequence of the 474-bp PstI fragment was used as the basis for this PCR since it has previously been shown to have little sequence similarity to the other members of this genus by dot blot hybridization. The outer primers for the primary PCR step were designated CP1-L (5'-TTATTCACCGTCCTACAGCAGAAA-3'), and CP2-R (5'-GGGGGTTCAGGGATCATTTGT-3') and produce a 404-bp sequence that corresponds to chlamydial polymerase. The inner primers (nested) contained within the 404 bp sequence were designated CPN1-L (5'-TTACGAAACGGCATTACAACGGCTA-GAAATCAAT-3') and CPN1-R (5'-TATGGCATATCCGCT-TCGGGAACGAT-3'), which result in a 214-bp product. After an initial 9 minutes at 95°C to activate the PCR enzyme, the outer PCR was carried out using 40 cycles of: 95°C for 30 seconds to melt, 60°C for 50 seconds to anneal, and 72°C for 30 seconds to extend the primers in 25 µL of 100 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 2 mmol/L MgCl₂, 100 µmol/L concentration of each dNTP, and 50 nmol/L of each Chlamydia primer (5'-ATCG-3', 5'-ATCG-3'), 0.25 U AmpliTaq Gold (Perkin Elmer), templated with 1 µL of the DNA isolated from the patient samples. This first-step PCR was carried out in individual thin-wall reaction tubes (Molecular Bio-Products), and when the reaction tubes were inoculated with the template DNA, only 1 tube was opened at a time in a further step to avoid cross-contamination of samples.

The inner, nested PCR was carried out with 25 to 30 cycles at 94°C for 15 seconds to melt, 60°C for 1 minute to anneal, and 72°C for 15 seconds to extend the primers in the same PCR buffer as given except that the primers were 5'-ATCG-3' and 5'-ATCG-3', and the enzyme used was AmpliTaq Gold. The nested reaction was templated using 1 µL of the primary PCR product hot started at 50°C.

PCR products were visualized under 300-nm UV transillumination (Fotodyne) after electrophoresis for 20 minutes at 5 V/cm in 2% ultraPURE agarose (Life Technologies), 45 mmol/L Tris, 45 mmol/L borate, and 5 mmol/L EDTA, pH 8.3 (Biofluids), and 1:10 000 SYBR Green II (Molecular Probes). Images were photographed using Polapan type 57 film (Polaroid Corp). Specific nested PCR product was identified using 5 to 15 ng X174 DNA restricted with HaeIII as a size marker. All PCRs were carried out in a Perkin Elmer 9700 Gene Amp PCR System (PE Applied Biosystems).

Immunohistochemistry
In sections adjacent to the regions of C pneumoniae identification, total T-lymphocyte count (CD3+), T-helper cells (CD4+), T-cytotoxic cells (CD8+), and T-memory cells (CD45RO+) were studied using single indirect enzyme immunohistochemistry according to previously described method by Frosterard et al.²⁸ Sections of tissue directly adjacent to the plaque material used for PCR determination was used to optimize immunohistological correlation and characterization with the regions of the plaque identified to have C pneumoniae.

Cryostat sections of 16 µµ were mounted on gelatin-coated glass slides and fixed for 20 minutes in 2% formaldehyde in PBS. Endogenous peroxidase activity was blocked, and membranes were permeabilized through incubation for 1 hour at room temperature with 1% hydrogen peroxide and 2% sodium nitride (Sigma Chemical Co) dissolved in 0.1% saponin (Sigma), in Earle’s balanced salt solution (EBSS; Life Technologies) with Ca²⁺, Mg²⁺, and 10 mmol/L HEPES (EBSS-Saponin). Endogenous biotin and avidin-binding activity were blocked with avidin and biotin incubators for 30 minutes each (Vector Laboratory). After 3 additional washes in EBSS-Saponin, the slides were incubated overnight at room temperature in a humid chamber with 100 µL of a panel of T-cell-type specific monoclonal antibodies. Cell-specific monoclonal antibodies against CD3+ (T cell, concentration 1:3) and CD4+ (T-helper cell, concentration 1:4) were obtained from Becton Dickinson; antibodies against CD8+ (T-cytotoxic cell, concentration 1:20) and CD45RO+ (T-memory cell, concentration 1:5) were obtained from DAKO. After incubation with primary antibodies, the slides were washed 3 times with EBSS-Saponin, incubated for 15 minutes at room temperature with 1% normal goat serum in EBSS-Saponin to reduce background caused by Fc interactions between primary and second-step antibodies, and incubated for 30 minutes at room temperature with biotinylated goat anti-mouse IgG1 (Vector Laboratories) at a concentration of 1:500. The slides were washed 3 times with EBSS-Saponin and incubated with an avidin-biotin-horseradish peroxidase complex (Vectastain ABC-HP-kit; Vector) for 1 hour at room temperature. The reaction was developed with 0.5 mg/mL diaminobenzidine (Vector; Figure 1).

View larger version (141K): [in this window] [in a new window]   Figure 1. A, Monoclonal antibody immunohistochemical staining of CD8+ T-cells shows an increase in cell counts in tissue from a symptomatic patient positive by PCR for Chlamydia pneumoniae. B, Representative of CD8+ T-cell staining in an asymptomatic patient positive by PCR for C pneumoniae (magnification x200).

T-Cell Counting
T lymphocytes were counted individually using MetaMorph imaging processing system (Universal Imaging Corp). Optical images were obtained from a transmitted light microscope (Axioplan, Zeiss) in digitized form at magnification x200 with a color video camera (Sony CCD-IRIS). Video signals from the small detection area (0.07 mm²) of this camera were fed into an image processor linked to a personal computer (IBM).

Fifty random images were obtained per section for each monoclonal antibody. Areas were outlined manually, and positive cells were counted within each image. T-lymphocyte counts were expressed as a number of positive cells per millimeter squared of section area.

To validate this method of cell counting, we obtained images that covered the total section area from 8 different slides (total of 2512 images, 314±49 magnification x200 images per slide), and the number of positive cells per mm² from each section was compared with the counting results of 50 randomly obtained images from the same sections. Regression analysis showed a high degree of correlation with a coefficient of 0.99798, confirming validity of the method.

Statistical Analysis
Statistical analysis was performed with StatView software (SAS Institute Inc, 1999). Differences in proportions between the groups were evaluated with the Fisher exact test. We used the Student 2-sample t test to compare means for continuous variables, but if normality and/or equal variance tests failed, the Mann-Whitney U test was used for comparison of median values. We used 2-way ANOVA, including an interaction term, to compare means between 2 classifications. Logistic regression was used to model symptomatic outcome as the dependent variable, with other possible risk factors as covariates; the logistic likelihood ratio test was used to determine significance. T-cell counts were log transformed for 2-way ANOVA analyses to compare means and for use in logistic regression analyses. For all tests, a 2-tailed P value of 0.05 was considered significant.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References Chlamydia pneumoniae: A Role... References

 
We analyzed T-cell subsets in 14 plaques (5 symptomatic and 9 asymptomatic) from patients positive for C pneumoniae-specific DNA confirmed by PCR and 14 plaques (6 symptomatic and 8 asymptomatic) from stenosis-matched patients negative for C pneumoniae. Table 2 shows demographic characteristics of patients with C pneumoniae-positive and C pneumoniae-negative carotid atherosclerotic plaques. There was no significant difference between the groups with respect to age, sex, degree of stenosis, occurrence of ischemic symptoms, and risk-factor profile, except for the presence of diabetes. A total of 8293 images (a median of 53 images per section for each monoclonal antibody) that covered an area 556.2 mm² of 112 sections from 28 plaque specimens were analyzed.

View this table: [in this window] [in a new window]   Table 2. Demographics and Distribution of Risk Factors in the Study Population

To explore the coeffect of symptoms and the presence of Chlamydia on counts of T-cell populations, 2-way ANOVA was performed, including an interaction term. For each T-cell subset, CD3+, CD4+, CD8+, and CD45RO+, both presence of chlamydia and symptoms were significantly associated with higher cell counts (all P<0.005).

In carotid atherosclerotic plaques, in the absence of C pneumoniae, there was a significant increase in the total T-lymphocyte count (median, cells per millimeter squared) in symptomatic versus asymptomatic patients (CD3+ 89.6 versus 55.3, P=0.013). The predominant T-cell subtypes in symptomatic plaque were CD4+ helper T cells (57.3 versus 32.7, P=0.01) and CD45RO+ memory T cells (82.9 versus 43.7, P=0.007). CD8+ cytotoxic T cells were at relatively lower levels and not significantly different between symptomatic and asymptomatic patients (28.6 versus 25.5, P=0.25; Figure 2).

View larger version (32K): [in this window] [in a new window]   Figure 2. T cells in symptomatic and asymptomatic patients with C pneumoniae-negative carotid atherosclerotic plaque.

However, in the presence of C pneumoniae, a significant elevation of CD8+ cytotoxic T cells was observed in the symptomatic versus asymptomatic patients (76.8 versus 30.3, P=0.03). In addition, CD4+ cells (111.9 versus 37.9, P=0.003), CD45RO+ cells (120.2 versus 72.9, P=0.003), and total T-cell count (CD3+ T cells 192.1 versus 80.4, P=0.004) were elevated in C pneumoniae-positive symptomatic plaque (Figure 3).

View larger version (40K): [in this window] [in a new window]   Figure 3. T cells in symptomatic and asymptomatic patients with C pneumoniae-positive carotid atherosclerotic plaque.

Comparison of symptomatic plaques with and without evidence of C pneumoniae presence revealed a significant increase in all T-cell populations in plaque positive for C pneumoniae (Figure 4).

View larger version (43K): [in this window] [in a new window]   Figure 4. T cells in symptomatic patients with C pneumoniae-positive [Chlam (+)] and C pneumoniae-negative [Chlam (-)] carotid atherosclerotic plaques.

Logistic regression was used to explore the effect of presence of Chlamydia and each T-cell subset count (log transformed) on the presence of symptoms. Each T-cell subset count was statistically significant as a predictor of symptoms (all P=0.003), and chlamydia presence was also significant for all subsets P=0.008), except for CD8+ (P=0.08). Other demographic and risk factors were explored in a univariate logistic regression to see whether they were associated with symptoms. Smoking, high cholesterol, and diabetes were significant (all P=0.04). When these covariates were entered into the logistic models singly and in any combination, with presence of Chlamydia and each of the T-cell subsets, each T-cell subset still remained a significant predictor of symptoms (all P=0.04).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References Chlamydia pneumoniae: A Role... References

 
In this study, we examined the association between the cellular immune response and the presence of C pneumoniae in carotid plaque specimens from patients with symptomatic and asymptomatic atherosclerotic disease. Our study confirms the growing body of literature that reports symptomatic atherosclerotic disease is associated with a proinflammatory profile, including an increase in T lymphocytes.^2,4 In the absence of C pneumoniae in our study, a significant increase in CD3+, CD4+, and CD45RO+ cells was seen in patients with symptomatic versus those with asymptomatic plaque. No increase in CD8+ cells was seen, and the HLA class II-dependent CD4+-restricted cell immune response appears to be the dominant cell type. Previous studies^21,29 have reported CD4+ cells as the dominant type in atherosclerotic plaque, although the studies did not compare patients with symptomatic to those with asymptomatic plaque. In the present study, there is an increase in CD4+ and CD45RO+ subsets in the symptomatic versus the asymptomatic patients. This CD4+ predominance could represent either a T-cell recruitment to the site of inflammatory tissue in the atherosclerotic plaque or a major histocompatibility complex class II response to antigen, such as oxidized LDL¹³ or heat-shock protein-60.²¹ These antigens are capable of initiating an inflammatory cascade within the carotid atherosclerotic plaque via the CD4+ pathway.²⁸

However, our data show a strong association between the presence of C pneumoniae and the accumulation of T lymphocytes, including CD8+ cytotoxic T cells in addition to CD4+ T cells in symptomatic versus asymptomatic patients. These data raise the possibility that in the presence of C pneumoniae, an antigen-specific CD8+-restricted activation of the cellular immune response occurs within symptomatic plaque. Further, the findings support our hypothesis that a specific antigen can activate resting atherosclerotic plaque via both the CD4+ and CD8+ pathways of cell-mediated immunity. These data have to be justified against data which suggest that the simple presence of C pneumoniae alone is insufficient to result in plaque destabilization.^14,30 Given that not all plaques with C pneumoniae became symptomatic, our data also suggest that the presence of that organism is insufficient for symptomaticity and that another factor, such as reactive Chlamydia-specific T cells, is also required. Memory T-lymphocyte predisposition for a specific antigen may play a role in the differential activation of a plaque.

It is known that Chlamydia spp can induce both CD4+ and CD8+ T-cell response.^23,31 The CD4+-recognized antigens are derived from vacuolar content bound to HLA class II molecules expressed by antigen presenting cells.³² The CD8+ T cells recognized antigen bound to HLA class I molecules expressed on nucleated cells in the cytoplasmic compartments.³³ Induction of HLA class I CD8+ cytotoxic T-lymphocyte response can be induced by several potential antigens expressed by C pneumoniae.^23,31 The lack of increased CD8+ T-cell levels in asymptomatic patients with Chlamydia indicates that Chlamydia can be present without necessarily triggering a cytotoxic T-cell response. This could be due either to an insufficient antigen load or a potential lack of resting memory T cells for Chlamydia in the plaque. Either possibility could explain the findings seen in previously reported studies that failed to identify a correlation between PCR-defined C pneumoniae presence and symptoms.^14,30

CD8+ T-cell activation results in the increase in interferon (IFN)- that can be stimulated by C pneumoniae.³⁴ IFN- can inhibit collagen synthesis and smooth muscle proliferation, which could facilitate plaque disruption. Increased levels of IFN- can also activate tumor necrosis factor- expression by plaque macrophages, increasing the plaque thrombogenicity as well as increasing intracellular adhesion molecule-1 expression on the endothelial cells, which can recruit further inflammatory cells to the atherosclerotic lesion.^3,28,35

Although an association between C pneumoniae and elevated CD8+ T-cell count in symptomatic plaque was identified, the limitation of this study is that it remains to be proved that the T-cell response was C pneumoniae antigen-specific and that this cellular response was causative for plaque destabilization. Work is ongoing in our laboratory to examine the antigen-specific T-cell response in the setting of symptomatic and asymptomatic atherosclerotic disease. Additionally, the labor-intensive nature of this study prevents examination of the entire plaque, and therefore the sampling may not represent all regions of a heterogeneous plaque. However, the intent of this study was to characterize T-cell subtype in regions of the plaque conventionally identified as being associated with symptoms and in which the status of C pneumoniae presence was known.

In conclusion, CD4+ T lymphocytes were significantly elevated in all patients with symptomatic atherosclerotic plaque versus those with asymptomatic plaque. However, there was a significant and preferential increase of CD8+ class I-restricted T cells in symptomatic plaque positive for C pneumoniae. These data provide intriguing information that warrants further exploration of C pneumoniae as a potential modifier of the immune system in atherosclerotic plaque. Further proof is needed to draw a direct link to C pneumoniae presence and plaque destabilization.

	Acknowledgments

 
Dr Nadareishvili is supported by NIH grant 1F05-TW-05469-01, which is an International Research Fellowship Award from the Fogarty International Center.

Received April 18, 2001; revision received June 4, 2001; accepted June 25, 2001.

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Editorial Comment

Sebastian Jander, MD; Guest Editor

	Chlamydia pneumoniae: A Role in Carotid Artery Plaque Destabilization?

Top Abstract Introduction Subjects and Methods Results Discussion References Chlamydia pneumoniae: A Role... References

 
The annual risk of stroke due to high-grade stenoses of the internal carotid artery can be as high as 13% after the recent occurrence of transient cerebral or retinal ischemia or as low as 1% to 2% in clinically asymptomatic patients.^1,2 The mechanisms that convert a stable plaque into "unstable internal carotid artery disease" are incompletely understood but have obvious implications for the treatment of cerebrovascular disease patients at risk of stroke. Advanced atherosclerotic plaques exhibit abundant infiltration by immune cells such as macrophages and T cells.³ This local inflammatory response has been proposed to play an important role in the process of plaque destabilization manifesting clinically as unstable angina or ischemic stroke.^4–6 In symptomatic patients with high-grade carotid stenosis, increased expression of matrix metalloproteinase-9 and thrombogenic tissue factor at sites of plaque inflammation may provide key effector mechanisms underlying plaque rupture and luminal thrombosis, respectively.^7,8

Numerous studies have recently reported the detection of C pneumoniae in atherosclerotic plaques, raising the possibility that this microorganism may be one of the factors driving T-cell-mediated inflammation during atherogenesis.⁹ In their interesting study, Nadareishvili and colleagues show a preferential increase of CD8+ T cells in symptomatic carotid artery plaques positive for C pneumoniae. The results of this study are important because they suggest a potential mechanism by which C pneumoniae may modulate the local inflammatory microenvironment in the plaques. However, it should be stressed that at the present stage, conclusions with respect to a causal role of C pneumoniae in plaque destabilization are still circumstantial and not directly supported by the data presented. Of note, previous findings reported by both LaBiche et al¹⁰ and Gibbs et al¹¹ showed that the presence of chlamydial DNA in plaque material is not directly correlated with clinical features of plaque destabilization. Thus, specific T-cell-mediated immune responses rather than the mere presence of the microorganism itself may predispose to plaque rupture and ischemic complications. In this regard, future studies addressing the antigen specificity of the observed CD8+ T-cell response will be of particular importance.

Department of Neurology

Heinrich Heine University

Düsseldorf, Germany

	References

Top Abstract Introduction Subjects and Methods Results Discussion References Chlamydia pneumoniae: A Role... References

 

1. North American Symptomatic Carotid Endarterectomy Trial Collaborators. Beneficial effect of carotid endarterectomy in symptomatic patients with high-grade carotid stenosis. N Engl J Med. 1991; 325: 445–453.
2. European Carotid Surgery Trialists Collaborative Group. MRC European Carotid Surgery Trial: interim results for symptomatic patients with severe (70–99%) or with mild (0–29%) carotid stenosis. Lancet. 1991; 337: 1235–1243.[Medline] [Order article via Infotrieve]
3. Ross R, Atherosclerosis: an inflammatory disease. N Engl J Med. 1999; 340: 115–126.
4. Moreno PR, Falk E, Palacios IF, Newell JB, Fuster V, Fallon JT. Macrophage infiltration in acute coronary syndromes: implications for plaque rupture. Circulation. 1994; 90: 775–778.[Abstract/Free Full Text]
5. van der Wal AC, Becker AE, van der Loos CM, Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation. 1994; 89: 36–44.
6. Golledge J, Greenhalgh RM, Davies AH. The symptomatic carotid plaque. Stroke. 2000; 31: 774–781.
7. Loftus IM, Naylor AR, Goodall S, Crowther M, Jones L, Bell PR, Thompson MM. Increased matrix metalloproteinase-9 activity in unstable carotid plaques: a potential role in acute plaque disruption. Stroke. 2000; 31: 40–47.
8. Jander S, Sitzer M, Wendt A, Schroeter M, Buchkremer M, Siebler M, Müller W, Sandmann W, Stoll G. Expression of tissue factor in high-grade carotid artery stenosis: association with plaque destabilization. Stroke. 2001; 32: 850–854.[Abstract/Free Full Text]
9. Mosorin M, Surcel HM, Laurila A, Lehtinen M, Karttunen R, Juvonen J, Paavonen J, Morrison RP, Saikku P, Juvonen T. Detection of Chlamydia pneumoniae-reactive T lymphocytes in human atherosclerotic plaques of carotid artery. Arterioscler Thromb Vasc Biol. 2000; 20: 1061–1067.
10. LaBiche R, Koziol D, Quinn TC, Gaydos C, Azhar S, Ketron G, Sood S, DeGraba TJ. Presence of Chlamydia pneumoniae in human symptomatic and asymptomatic carotid atherosclerotic plaque. Stroke. 2001; 32: 855–860.[Abstract/Free Full Text]
11. Gibbs RG, Sian M, Mitchell AW, Greenhalgh RM, Davies AH, Carey N. Chlamydia pneumoniae does not influence atherosclerotic plaque behavior in patients with established carotid artery stenosis. Stroke. 2000; 31: 2930–2935.

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