Skip Navigation

Cardiovascular Research 1997 36(2):256-267; doi:10.1016/S0008-6363(97)00129-6
© 1997 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Oksenberg, J. R
Right arrow Articles by Erusalimsky, J. D
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oksenberg, J. R
Right arrow Articles by Erusalimsky, J. D
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1997, European Society of Cardiology

Analysis of the T-cell receptor repertoire in human atherosclerosis

Jorge R Oksenberga,*, George T Stavrib, Matthew C Jeonga, Natara Garovoya, Jonathan R Salisburyc and Jorge D Erusalimskyd

aDepartment of Neurology, School of Medicine, University of California at San Francisco, 505 Parnassus St., San Francisco, CA 94143-0435, USA
bDepartment of Medicine, King's College School of Medicine and Dentistry, London, UK
cDepartment of Histopathology, King's College School of Medicine and Dentistry, London, UK
dThe Cruciform Project and Department of Medicine, University College, London, UK

* Corresponding author. Tel.: +1 (415) 476 1335; fax: +1 (415) 476 5229; e-mail: oksen@itsa.ucsf.edu

Received 25 February 1997; accepted 6 May 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Analysis of T-cell receptor (TCR) β-chain gene expression in atherosclerotic lesions of human aorta. Methods: TCR diversity was studied using non-radioactive polymerase chain reaction for quantitative assessment of TCRBV gene transcripts, together with size and sequence analysis of the β-chain third complementarity-determining region (CDR3). Samples represent a wide range of atheromatous histology, allowing evaluation of the T-cell repertoire at different stages of disease. Results: Diverse TCRBV family usage was observed in the majority of the samples, as the 25 different TCRBV products were detected at levels exceeding background. The data also showed that TCRBV transcripts expressed in the diseased aorta tissue displayed considerable size heterogeneity and no repetition of CDR3 nucleotide motifs. Conclusions: The early presence of T-lymphocytes in the atheromatous blood vessel has been interpreted as an indication of specific immunological reactions operating during the course of the atherosclerotic process. Although a T-cell infiltrate characterized by limited usage of TCRAV genes cannot be excluded, the unrestricted usage of TCRBV genes argues against a local T-cell clonal expansion in atherogenesis.

KEYWORDS Atherosclerosis; T-lymphocytes; T-cell receptor; Gene expression; Human, aorta


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The progression of human atherosclerosis from the early fatty streak, through the intermediate fibrofatty lesions, to the advanced complex fibrous plaque, is accompanied by infiltration and accumulation of inflammatory and immune cells. Substantial numbers of T-lymphocytes and macrophages are found within the intimal lesions at all stages of disease progression, whereas B lymphocytes and plasma cells can be detected mainly in the adventitia, adjacent to advanced plaques [1, 2]. Immunohistological studies have shown that T-cells are recruited early during atherogenesis, and that in the transition zone between the normal intima and the fatty streak, they are more abundant than macrophages, suggesting that lymphocytes precede the monocytes in the infiltration of the arterial intima [3]. The expression of major histocompatibility complex (MHC) class II molecules in lesions indicates that the local microenvironment is enriched in MHC-activating factors such as interferon (IFN)-{gamma}, and that antigen is possibly presented to T-cells [3, 4]. Accordingly, cell surface marker analysis shows that many T-cells within atherosclerotic lesions are in a state of immunological activation [2–7].

The presence of activated T-lymphocytes within atherosclerotic lesions may suggest the involvement of antigen-driven immunological mechanisms in the onset and/or progression of the disease [8, 9]. The nature of the antigen(s) that may trigger such response is unknown. It might be a mutant structural gene product, a stress protein, a modified lipoprotein, an unmasked autoantigen or a pathogen that provokes a secondary autoimmune response with a major role in the ultimate destruction of the target tissue. For example, a correlation between anti-heat shock protein (hsp) 65 serum antibodies and carotid atherosclerosis has been recently reported [10]. In addition, a response to oxidized LDL was detected in CD4+ T-cell clones derived from atherosclerotic plaques but not from the peripheral blood of the same individuals [11]. A significant association between dental infections and severe coronary atherosclerosis has also been observed in a group of 88 Finish male patients [12]. Furthermore, Chlamydia pneumoniae [13], cytomegalovirus [14], herpes virus [15]and other infectious agents have also been associated with the development of atherosclerosis. Alternatively, the T-cell response can be a secondary epi-phenomenon, induced as a consequence of the abundant chemoattractants released by macrophages, endothelial and vascular smooth muscle cells. Indeed, it has not been formally demonstrated that a local T-cell clonal expansion takes place in response to putative athero-antigens.

We have previously shown that the polymerase chain reaction (PCR) primed with T-cell receptor variable (TCRV) region sequence-specific oligonucleotide primers and a common constant (C)-region primer, can be used to study the identity and composition of T-lymphocyte populations infiltrating inflamed tissues [16]. Since then, numerous studies have used PCR-based strategies to study the dynamics and specificity of the immune response in infectious, autoimmune and neoplastic diseases [17, 18]. Constrained TCR gene usage has been shown for some, but not for other conditions [18]. This type of approach has also shown that the degree of detectable TCRV gene segments may change with the development of the inflammatory process [19]. Modeling of the trimolecular interaction between the TCR, the MHC-antigen presenting molecules and bound antigenic peptide, suggests that the complementary-determining regions (CDR)1 and CDR2 of the TCR provide the structural framework and topology for the interaction of the junctional CDR3 region, (N)J{alpha} and (N)Dβ(N)Jβ, with the peptide bound in the MHC cleft [20–22]. Thus, conserved amino acids at the VJ junction of the {alpha}-chain, or the VDJ junction of the β-chain are indicative of antigen specificity among a population of receptors [23].

An early attempt to analyze TCR gene rearrangements in atherosclerosis was based on Southern blot analysis of IL-2 in vitro-expanded T-cells isolated from carotid endarterectomy samples, using cDNA probes for TCRB and TCRG genes [24]. This study suggested that T-lymphocytes within lesions have a polyclonal origin. In a more recent work, the PCR method was used, and the heterogeneous pattern of TCRBV gene usage was observed for advanced atheroma [25]. It is conceivable however, that in in vitro stimulated cell preparations or in the late stages of plaque development, the antigen-specific recruited lymphocyte populations represent just a minor fraction among the non-specific infiltrating T-cells, and as such, are difficult to detect. Adult human aorta may show lesions at all stages of plaque development, and so provides ideal tissue to investigate the dynamics of T-cell infiltration and the immuno-genetic factors involved in the evolution from the early fatty streak to the complex fibrous plaque. Furthermore, previous studies were limited to the analysis of the V region of the TCR genes, while the clonality of a T-cell infiltrate can only be resolved through analysis of TCR-CDR3 sequences. Here, we studied the TCR β-chain repertoire complexity in atherosclerosis, based on quantitative TCRBV gene usage assessment, and on CDR3 length and sequence homogeneity analysis of 10 aortic samples obtained at autopsy. Although individual distinctive patterns of TCRBV gene usage frequency were observed, the repertoire complexity is consistent with a polyclonal T-cell expansion during the early, intermediate and late stages of atherogenesis.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Sample preparation
Segments of 10 human thoracic aortas were obtained from organ donors by the Liver Transplantation Unit of King's College Hospital, London, UK. Tissue was stored in U. Wisconsin solution at 4°C for up to 12 h before being processed. Samples were meticulously washed in ice-cold PBS to remove excess blood. Excess periadventitial tissue was removed, the artery opened longitudinally, and pieces of artery within a diseased area, which is normally evident to the naked eye, were excised in approximately 0.5x1 cm segments. One piece was fixed in neutral-buffered formalin and 4-µm-thick transverse sections were stained for histopathological assessment according to the report from the Committee on Vascular Lesions of the Council on Atherosclerosis, American Hearth Association [26, 27]. Two additional segments which were to be used for PCR analysis were stripped of the adventitia and immediately frozen in liquid nitrogen. Frozen samples were stored at –70°C until processed.

2.2 Preparation of RNA and complementary DNA (cDNA)
Samples for RNA extraction were coded, with the molecular biologist blinded to the origin and nature of the sample. Total RNA was extracted from homogenized samples using a commercially available phenol/guanidinium thiocyanate cocktail (TRIzol reagent, Gibco BRL, Gaithersburg, MD). Synthesis of first cDNA strand templates for PCR analysis was carried out in a final reaction volume of 10 µl containing 0.1 µg of total RNA (OD260/OD280 >1.8), 1 mmol/l dNTPs (Perkin Elmer, Norwalk, CT), 0.25 units random hexamer primers (Pharmacia, Piscataway, NJ), 20 units human placental RNAse inhibitor (Gibco BRL), 100 units Superscript reverse transcriptase (Gibco BRL) and 1 µl 10x PCR buffer (100 mmol/l Tris-HCl, pH 8.3, 500 mmol/l KCl, 15 mmol/l MgCl2, 0.01% (w/v) gelatin) (Perkin Elmer). The reaction mixture was incubated for 10 min at room temperature, then for 45 min at 42°C followed by 5 min at 95°C, and finally chilled on ice.

2.3 PCR reaction
cDNA was subjected to enzymatic amplification by the ‘Hot Start’ PCR method [28]. Ten-microliter aliquots of the reverse transcriptase reaction were mixed in a 50 µl final reaction volume with 4 µl 10x PCR buffer and 0.5 µmol/l of a BV region-specific oligonucleotide primer. Reaction mixtures were brought to 80°C, and 1.25 U Taq polymerase (Perkin Elmer) plus 0.5 µmol/l of a 3'-biotinylated common BC-region primer were immediately added. The reaction was then allowed to proceed for 25 cycles in a DNA Thermal Cycler (Perkin-Elmer), each cycle consisting of denaturation at 95°C for 60 s, annealing at 55°C for 60 s and extension at 72°C for 60 s. Sequences for primers and probes were reported elsewhere [17, 29]. Critical parameters of the reaction such as number of cycles, enzyme and template concentrations were established in preliminary experiments to ensure that the amplification did not reach a plateau.

2.4 Semi-quantitative evaluation of PCR products
A novel approach for semi-quantitation of TCR amplification products was designed [29]. This strategy is based on earlier experiences of HLA genotyping [30]and automated quantification of HIV [31]. Excess primers were removed from the biotinylated PCR products using a Centricon 30 micro-concentrator (Amicon, Beverly, MA). Samples were subsequently denatured at 95°C and 10 µl aliquots were transferred in duplicate to MaxiSorp 96-well microplates (Applied Scientific, San Francisco, CA) pre-coated with avidin (Sigma Chemicals, St. Louis, MO). Ten picomoles of a horseradish peroxidase (HRP)-labeled BC region oligonucleotide probe was added in 65 µl hybridization solution (5x SSPE, 5x Denhardt's solution, 0.1 mg/ml salmon sperm DNA, 0.1% SDS), followed by incubation for 1 h at 42°C to allow for capture and hybridization to take place simultaneously. The reaction was developed by adding 150 µl o-phenylenediamine (Sigma Chemicals) and stopped after 20 min by adding 100 µl 1N H2SO4. Optical density was determined at 492 nm in an ELISA reader.

In addition to the BV-BC specific amplifications, for each sample simultaneous amplifications were performed with serially diluted amounts of cDNA using 3' and 5' reference primers corresponding to the BC segment of the TCR transcripts (Fig. 1). Reference plots of cDNA volume vs. absorbance were then constructed. Results of each BV-BC specific amplification are expressed in terms of the cDNA volume of the reference BC-BC amplification required to give an absorbance equal to that of 10 µl of the undiluted BV-BC PCR product. This method was subjected to validation in extensive preliminary experiments [29]. First, the general reproducibility was demonstrated by similarities in the overall patterns of TCR expression between duplicate amplifications of representative cDNA samples derived from peripheral blood lymphocytes (PBLs) and aorta. Second, the PCR products of each aorta sample and control PBLs were analyzed by Southern blot and hybridization with the HRP-conjugated BC region oligonucleotide probe [17, 29]. These experiments showed a high correlation between the intensity of hybridized PCR bands and the OD values.


Figure 1
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Generic TCR cDNA structure. Schematic representation of a reverse-transcribed TCR β-chain transcript, showing the relative placement of the PCR primers and C-region probe.

 
2.5 CDR3 spectratypes
Conditions for the generation of size spectratypes have been reported elsewhere [32]. Briefly, a new 35 cycles-PCR reaction was performed using a 32P-labeled BC-region primer. Five microliters of amplified products were diluted in a running buffer containing 95% formamide, 10 mM NaOH, and 0.1% bromophenol blue/xylene cyanole, heated at 75°C for 5 min, and electrophoresed in a 5% denaturing polyacrylamide gel (Gibco BRL). Autoradiography was performed on dry gel with overnight exposure.

2.6 Cloning and sequencing of PCR-amplified cDNA
Twenty-five microliter aliquots of PCR products were desalted and concentrated in Centricon 30 columns, and then ligated into the PCR-1000 vector (Invitrogen, San Diego, CA) according to the manufacturer's instructions. After transformation in competent cells, clones containing TCR inserts were identified by transfer to nylon membranes and hybridization to an HRP-labeled TCRBC-region oligonucleotide probe. Single-stranded DNA from positive clones was prepared and VDJC sequences were determined by the dideoxy chain termination method using the AmpliTaq sequencing kit (Perkin-Elmer).

2.7 Statistical analysis
The distribution of TCR transcripts in aorta was compared to that in PBLs using the Mann-Whitney U-test (2-sample Wilcoxon Rank sum test) [33]. Statistical analysis was performed in consultation with the University of California, San Francisco statistical consulting unit.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
Ten aorta specimens were subjected to histopathological characterization in order to assess the degree of atheroma. A summary of this analysis is shown in Table 1. One specimen was of a normal aorta with no detectable CD4 or CD8 positive T-cells (donor 1). One specimen displayed thick but undiseased intima with a small number of CD4 and CD8 positive cells (donor 3). One specimen was classified as type I, with a small number of CD4 and CD8 positive cells (donor 8). Three specimens corresponded to type II lesions of the disease process (fatty streaks) with small numbers of CD4 and CD8 positive cells (donors 4, 6 and 9). One additional sample was also classified as type II (donor 5), but with a more substantial T-cell infiltration. One other specimen (donor 7) was classified as type III lesion (pre-atheroma) with a moderate number of CD4 and CD8 positive cells. The remaining two (donors 2 and 10) had advanced disease (type V lesions), with a moderate to high number of CD4 and CD8 positive cells. T-cell populations showed no major deviations from the normal CD4/CD8 ratio (data not shown).


View this table:
[in this window]
[in a new window]

  		Table 1 Histopathological assessment of aorta samples

 
Total RNA was extracted from these samples and TCRBV gene diversity was analyzed by semiquantitative-PCR of reverse-transcribed VDJC-rearranged TCRβ chain transcripts as schematically shown in Fig. 1. Preliminary experiments showed that PCR replications of the same cDNA samples yielded almost identical results (data not shown). These experiments also indicated that TCRBC PCR-products accumulated exponentially for the first 30 cycles (data not shown). Thus, in subsequent experiments the abundance of TCR transcripts was analyzed after 25 cycles of enzymatic amplification. Fig. 2 illustrates the relative amplification of TCR family-specific transcripts present in PBLs, in two independent cohorts, each consisting of 10 normal controls. There are some BV genes that are consistently expressed at higher levels than others in the peripheral blood of most individuals (BV2, BV4, BV6, BV12 and BV13). These relative frequencies of TCRBV families are consistent with our previous experience and with the patterns observed by other investigators [34].


Figure 2
View larger version (33K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 TCRBV repertoire in control PBLs. Semiquantitative determination of TCRBV gene family utilization in PBLs from 10 unrelated healthy control individuals. For each sample, TCR CB-CB amplifications were performed with serially diluted amounts of cDNA, and a plot of cDNA vs. absorbance was made. Results are expressed in terms of the cDNA volume of the reference CB-CB amplification required to give an absorbance equal to that of 10 µl of the undiluted BV-BC PCR test amplification.

 
Lymphocytes infiltrating the arterial wall were then examined for biased TCR expression in two adjacent tissue sections (Fig. 3). Evaluation of these results against the histopathological characteristics of the corresponding tissue samples revealed no apparent correlation between the pattern of TCR expression and the stage of disease, although a correlation was observed between the magnitude of the inflammatory response and the PCR signal (data not shown). Restricted (oligoclonal) TCRBV family usage was not observed in the studied samples, as all the BV products were usually detected at levels exceeding the background (Fig. 3). With the exception of sample 1, remarkable similarities were observed between two adjacent regions of the same sample (Fig. 3). In sample 1a, significant amplification of TCR transcripts was detected, whereas in the adjacent sample 1b, amplification levels were close to background. The fact that a sample with normal aortic histology had an area that contained enough T-cell-derived messenger RNA for PCR detection is consistent with previous observations showing small accumulations of T-cells in the normal intima of adults and even infants [9].


Figure 3
View larger version (41K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 TCRBV repertoire in aorta. Semiquantitative determination of TCRBV gene family utilization in two adjacent aorta tissue samples obtained at autopsy from 10 different donors. Analysis was performed as described in Fig. 2.

 
Substantial variability in TCRBV gene expression was observed between samples (Fig. 3). Hence, it was conceivable that the individual bias in TCRV gene expression resulted from T-cell activation triggered by local putative athero-antigens that are presented by individual MHC repertoires, according to the phenotype of each donor. Unfortunately, peripheral blood from the tissue donors was not available for comparison in this study. Nevertheless, within each sample the usage frequency of the various BV gene families as a whole was significantly different from that found in the peripheral blood of healthy control volunteers (P = 0.0085, Mann-Whitney U-test). This pattern of skewed expression may be indicative of local clonal proliferation by particular T-cell populations. To test this possibility, we analyzed the TCRB-CDR3 size distribution for the entire infiltrating repertoire. New aliquots of RNA were reverse-transcribed, amplified, and the radioactive PCR products loaded on 5% denaturing polyacrylamide gels for electrophoretic resolution (Fig. 4). For the PBL sample, the analysis showed 8–12 bands with a Gaussian distribution of band intensities. Each of the CDR3 bands seen on the gel are 3 base-pairs apart, indicating that the vast majority of productive transcript rearrangements are in frame. As a control, the PCR product from a BV5+ T-cell clone derived by limiting dilution and PHA+IL-2 stimulation had a clear single band. In aorta tissue, CDR3 length analysis revealed no significant deviations from the Gaussian distribution or dominance of one or several CDR3 lengths. This finding strongly suggests heterogeneity in the infiltrating lymphocytes.


Figure 4
Figure 4
View larger version (117K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 CDR3 size spectratyping. Analysis of CDR3 length in TCR transcripts amplified from aorta and PBLs.

 
The most direct approach to study T-cell clonality is to determine the VDJ sequences at CDR3 regions. New aliquots of RNA were reverse-transcribed, and the two dominant TCR rearrangements of each sample, presumably the proliferating populations, were amplified with the appropriate BV-BC primers and cloned for sequence analysis in order to determine the amino acid structure of the joining CDR3 loops, including the TCRBJ gene segments. Frequent utilization of BJ1.1 and 1.2, and BJ2.1, 2.3, 2.5 and 2.7 was detected in the majority of the samples. The BJ2 family was used more commonly than the BJ1 family (65 vs. 35%), consistent with the increased use of BC2 relative to BC1 [34]. However, the frequency distribution of BJ usage in atherosclerotic lesions was very similar to that found in PBLs isolated from healthy control individuals. The molecular characterization of the NDN sequences at CDR3 regions is shown in Table 2. Alignment of CDR3 coded amino acids failed to detect either conserved amino acids or sequence motifs within or between the TCRBV genes derived from the atherosclerotic lesions. The data are consistent with an heterogeneous T-cell repertoire among the inflammatory infiltrate in the aorta. All together, these results demonstrate that there is no evidence of T-cell clonal expansion at any stage during the progression of atherosclerosis in the aorta.


View this table:
[in this window]
[in a new window]

  		Table 2 TCR β-chain CDR3 rearrangements in atherosclerotic lesions

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The molecular organization of the TCR genes and the rearrangement process that takes place during T-cell differentiation can be used for evaluation of T-cell clonality and therefore of antigen specificity. The presence of T-cells with a restricted pattern of TCR expression may be indicative of a response primarily autoimmune, or of a specific immune response to antigens expressed by infectious agents. The individual distinct pattern of TCRBV expression by infiltrating lymphocytes, detected in our original PCR screening, was indeed suggestive of an antigen-mediated immune response. While the infiltrates contained an heterogeneous population of T-lymphocytes as defined by the V-region usage of the TCR, multiple clones from a few precursors could have undergone local proliferation after being exposed to neo-antigens. Because TCR-CDR3 loops play a significant structural role in the determination of the TCR-antigen specificity [20–22], the accumulation of T-cells within a lesion driven by an immune response to a specific antigen will be characterized by the presence of repeated CDR3 junctional motifs. On the other hand, if the accumulation of T-cells is totally at random, then one might expect a diverse collection of CDR3 sequences with very few repeats. Thus, only CDR3 analysis could definitively resolve the question of T-cell clonality in atherogenesis. To our knowledge, this is the first study to explore this possibility by analyzing the primary structure of the rearranged CDR3 loops. The primary structure of the rearranged CDR 3 sequences was studied by size for the entire repertoire, and by sequencing for TCR families that showed an unusually high relative frequency among other families in the same sample. In all we have sequenced 195 cDNA clones expressing TCR sequences directly amplified from the aortic tissue. Based upon the clear electrophoretic resolution of multiple CDR3 bands, and the extensive diversity in the expression of in-frame CDR3 segments for each of the samples analyzed in this study, the populations of T-cells infiltrating the lesions appear to be heterogeneous in lesions which, from a morphological point of view, spanned the whole history of disease development.

Because it has been suggested that the individual MHC haplotype influences TCR usage [35], it could be argued that in the absence of data from peripheral blood TCR usage of the same individual, this selection is meaningless. However, as shown in Fig. 2, the remarkable modest SD values observed in the semiquantitative determination of TCRBV gene family utilization in PBLs of normal control, and the fact that certain TCRBV families are consistently expressed to a greater degree across histocompatibility differences (Fig. 2 and see also Refs. [34, 36–38]) suggest that the role of the MHC in shaping the T-cell repertoire is quite modest. In this context, it should be emphasized that with the exception of a very few cases (BV2 in samples 6 and 7; BV6 in samples 2 and 6), the TCR families that were highly expressed in the lesions are not among those that are generally highly represented in the peripheral repertoire.

Another issue to consider is that both the V{alpha} and Vβ chains contribute to the peptide and MHC specificity of a T-cell. Although we do not rule out the possibility of restricted {alpha}-chain expression by infiltrating lymphocytes, we focused primarily on the TCRBV genes because it is common to detect two {alpha}-chain transcripts in clonal T-cell populations. In contrast, the rearrangement of one functional β-chain gene is generally sufficient to exclude rearrangement of the second chromosome; thus, there is only one TCR β-chain transcript per cell. In addition, somatic mutations have been reported for TCRAV genes but not BV. Because of that, the β-chain is a more informative lineage marker for the definition of repertoires in PCR-based studies.

Current theories on the origin of atherosclerosis emphasize the primary role of lipids [39], oxidative stress [40]and endothelial cell dysfunction [8]in the development of the disease. In addition, immunohistochemical studies have shown that the atherosclerotic plaque contains high numbers of activated T-lymphocytes with a memory phenotype [6, 7]. These, as well as other studies showing an aberrant expression of MHC class II molecules by vascular smooth muscle cells in the plaque [3, 4], raise the possibility that the immune response may also play a disease-specific primary role in atherogenesis. In support of this possibility the immunosuppressive drug, cyclosporin A (CyA), has been shown to inhibit the development of intimal thickening during vascular response to injury [41], and reduce early atherosclerosis in cholesterol-fed rabbits [42]. In contrast, in different studies, suppression of cell-mediated immunity by CyA enhanced the development of atherosclerosis in cholesterol-fed rabbits [43]and hyperlipidemic C57BL/6 mice [44]. These results suggest that T-cells may have a protective rather than a pathogenic role in atherogenesis. In agreement with this possibility, Hansson and colleagues demonstrated that in rats depleted of T-lymphocytes by antibody treatment, significantly larger arterial lesions developed after balloon-catheter injury [45]. Furthermore, larger lesions also developed in athymic rnu/rnu rats that lack T-lymphocytes, compared to the rnu/+ littermates with normal T-cell counts [45]. Finally, cholesterol-fed C57BL/6 mice deficient in MHC class I have recently been shown to develop more severe atherosclerosis than do control immunocompetent mice [46]. One proposed mechanism to explain the regulatory role of T-cells in atherogenesis is through secretion of IFN-{gamma} which has been shown to inhibit vascular smooth muscle cell proliferation [47]. Thus, T-cells may down-modulate the repair process of the vasculature and thus have a protective function in atherogenesis. This type of function would not require a limited T-cell population, restricted by their {alpha}/β heterodimer antigen receptor. Indeed it could simply be achieved by a non-specific infiltration of T-cells responding to the up-regulation of lymphocyte homing receptors on dysfunctional endothelial cells and to the secretion of chemotactic mediators by the developing lesions [48].

The availability of genetically modified mouse models of atherosclerosis [49], combined with the possibility of further genetic manipulation, may allow a systematic dissection of the role of T-lymphocytes in this disease. For example, human apolipoprotein B transgenic mice [50]can be genetically altered to delete all or specific T-cell subsets. In addition, gene knockouts and anti-cytokine treatments may be used to polarize helper T-cells into Th1 or Th2 phenotypes. These models will provide a powerful tool to understand the role of T-cells in vascular damage, including their effect on the regulation of macrophage activation. Furthermore, such models could serve to investigate the role played by T-cell-derived factors, such as cytokines and metalloproteinases, in the development of the atherosclerotic plaque. The precise characterization of the T-cell response during atherogenesis may have important therapeutic implications. Just as oncologists use various modalities of therapy to treat cancer, modulation of the inflammatory T-cell response in the blood vessel might be employed to treat atherosclerosis in combination with other treatments and drugs.

Time for primary review 41 days.


   Acknowledgements
 
This work was supported by a Grant-in-aid of the American Hearth Association, California Affiliate. G.T.S. was supported by a Junior Research Fellowship from the British Heart Foundation. The authors are grateful to Nigel Heaton and his team from the Liver Transplant Unit of King's College Hospital for supplying the aortic specimens. The authors gratefully acknowledge the excellent technical assistance of Jane Codd.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Jonasson L., Holm J., Skalli O., Bondjers G., Hansson K. Regional accumulation of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis (1986) 6:131–138.[Abstract/Free Full Text]
  2. Hansson G.K. Immune and inflammatory mechanisms in the development of atherosclerosis. Br Heart J (1993) 69(suppl):S38–S41.[Free Full Text]
  3. Xu Q., Oberhuber G., Gruschwitz M., Wick G. Immunology of atherosclerosis: Cellular composition and MHC class II antigen expression in aortic intima, fatty streaks, and atherosclerotic plaques in young and aged human specimens. Clin Immunol Immunopathol (1990) 56:344–359.[CrossRef][ISI][Medline]
  4. Hansson G.K., Jonasson L., Holm J., Claesson-Welsh L. Class II MHC antigen expression in atherosclerotic plaque: smooth muscle cells express HLA-DR, HLA-DQ and the invariant gamma chain. Clin Exp Immunol (1986) 64:261–268.[ISI][Medline]
  5. Hansson G.K., Jonasson L., Seifert P.S., Stemme S. Immune mechanisms in atherosclerosis. Arteriosclerosis (1989) 9:567–578.[Abstract/Free Full Text]
  6. Hansson G.K., Holm J., Jonasson L. Detection of activated T lymphocytes in the human atherosclerotic plaque. Am J Pathol (1989) 135:169–175.[Abstract]
  7. Stemme S., Holm J., Hansson G.K. T lymphocytes in human atherosclerotic plaques are memory cells expressing CD45RO and the integrin VLA-1. Arterioscler Thromb (1992) 12:206–211.[Abstract/Free Full Text]
  8. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature (1993) 362:801–809.[CrossRef][Medline]
  9. Wick G., Schett G., Amberger A., Kleindienst R., Xu Q. Is atherosclerosis an immunologically mediated disease? Immunol Today (1995) 16:27–33.[CrossRef][ISI][Medline]
  10. Xu Q., Willeit J., Marosi M., et al. Association of serum antibodies to heat-shock protein 65 with carotid atherosclerosis. Lancet (1993) 341:255–259.[CrossRef][ISI][Medline]
  11. Stemme S., Faber B., Holm J., Wiklund O., Witztum J.L., Hansson G.K. T lymphocytes from human atherosclerotic plaques recognize oxidized low density protein. Proc Natl Acad Sci USA (1995) 92:3893–3897.[Abstract/Free Full Text]
  12. Mattila K.J., Valle M.S., Nieminen M.S., Valtonen V.V., Hietaniemi K.L. Dental infections and coronary atherosclerosis. Atherosclerosis (1993) 103:205–211.[CrossRef][ISI][Medline]
  13. Thom D., Grayston T., Siscovick D.S., Wang S.P., Weiss N.S., Daling J.R. Association of prior infection with Chlamydia pneumoniae and angiographically demonstrated coronary artery disease. J Am Med Assoc (1992) 268:68–72.[Abstract]
  14. Bruggeman C.A., Dam-Mieras V. The possible role of cytomegalovirus in atherogenesis. Prog Med Virol (1991) 38:1–26.[ISI][Medline]
  15. Yamashiroya H.M., Ghosh L., Yang R., et al. Herpesviridae in coronary vessels and aorta of young trauma victims. Am J Pathol (1988) 130:71–79.[Abstract]
  16. Oksenberg J.R., Stuart S., Begovich A.B., et al. Limited heterogeneity of rearranged T-cell receptor V alpha transcripts in brains of multiple sclerosis patients. Nature (1990) 345:344–346.[CrossRef][Medline]
  17. Oksenberg JR, Panzara MA, Steinman L. The polymerase chain reaction and the analysis of the T cell receptor repertoire. Austin, TX: RG Landes Company, 1992.
  18. Marguerie C., Lunardi C., So A. PCR-based analysis of the TCR repertoire in human autoimmune diseases. Immunol Today (1992) 13:336–338.[CrossRef][ISI][Medline]
  19. Karin N., Szafer F., Mitchell D., Gold D.P., Steinman L. Selective and non selective stages in homing of T lymphocytes to the central nervous system during experimental allergic encephalomyelitis. J Immunol (1993) 150:4116–4124.[Abstract]
  20. Jorgensen J.L., Esser U., Fazekas de St. Groth B., Reay P., Davis M.M. Mapping TCR-peptide contacts by variant peptide immunization of single-chain transgenics. Nature (1992) 355:224–230.[CrossRef][Medline]
  21. Katayama C.D., Eidelman F.J., Duncan A., Hooshmand F., Hedrick S.M. Predicted complementarity determining regions of the T cell antigen receptor determine antigen specificity. EMBO J (1995) 14:927–938.[ISI][Medline]
  22. Garboczi D.N., Ghosh P., Utz U., Fan Q.R., Biddison W.E., Wiley D.C. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature (1996) 384:134–141.[CrossRef][Medline]
  23. Oksenberg J.R., Panzara M.A., Begovich A.B., et al. Selection for T-cell receptor Vβ-Dβ-Jβ gene rearrangements with specificity for a myelin basic protein peptide in brain lesions of multiple sclerosis. Nature (1993) 362:68–70.[CrossRef][Medline]
  24. Stemme S., Rymo L., Hansson G.K. Polyclonal origin of T lymphocytes in human atherosclerotic plaques. Lab Invest (1991) 65:654–660.[ISI][Medline]
  25. Swanson S.J., Rosenzweig A., Seidman J.G., Libby P. Diversity of T cell antigen receptor V beta gene utilization in advanced human atheroma. Arterioscler Thromb (1994) 14:1210–1214.[Abstract/Free Full Text]
  26. Stary H.C., Chandler A.B., Glagov S. A definition of initial, fatty streak, and intermediate lesions of atherosclerosis. Arterioscler Thromb (1994) 14:840–856.[Abstract/Free Full Text]
  27. Stary H.C., Chandler A.B., Dinsmore R.E. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. Circulation (1995) 92:1355–1374.[Abstract/Free Full Text]
  28. D'Aquila R.T., Bechtel L.J., Videler J.A., Eron J.J., Gorczyca P., Kaplan J.C. Maximizing sensitivity and specificity of PCR by preamplification heating. Nucleic Acid Res (1991) 19:3749.[Free Full Text]
  29. Oksenberg JR, Steinman L. Detection an analysis of T cell receptor gene products by RT-PCR. In: Siebert PD, Larrick JW, editors. Reverse transcriptase PCR. London: Ellis Horwood, 1995:61–85.
  30. Lazaro A.M., Fernandez-Vina M.A., Liu Z., Stastny P. Enzyme-linked DNA oligotyping. A practical method for clinical HLA-DNA typing. Hum Immunol (1993) 36:243–248.[CrossRef][ISI][Medline]
  31. Holodniy M., Winters M.A., Merigan T.C. Detection and quantitation of gene amplification products by a nonisotopic automated system. BioTechniques (1992) 12:36–39.[ISI][Medline]
  32. Gorski J., Yassai M., Kissella B., Keever C., Flomenberg N. Circulating T cell repertoire complexity in normal individuals and bone marrow recipients analyzed by CDR3 size spectratyping. J Immunol (1994) 152:5109–5119.[Abstract]
  33. Lehman EL, D'Abrera HJM. Non parametrics statistical methods based on ranks. New York: McGraw Hill, 1975.
  34. Rosenberg W.M.C., Moss P.A.H., Bell J.I. Variation in human T cell receptor V beta and J beta repertoire: analysis using anchor polymerase chain reaction. Eur J Immunol (1992) 22:541–549.[ISI][Medline]
  35. Akolkar P.N., Gulwani-Akolkar B., Pergolizzi R., Bigler R.D., Silver J. Influence of HLA genes on T cell receptor V segment frequencies and expression levels in peripheral blood lymphocytes. J Immunol (1993) 150:2761–2773.[Abstract]
  36. Ramakrishnan N.S., Grunewald J., Janson C.H., Wigzell H. Nearly identical T-cell receptor V-gene usage at birth in two cohorts of distinctly different ethnic origin: influence of environment in the final maturation in the adult. Scand J Immunol (1992) 36:71–78.[CrossRef][ISI][Medline]
  37. Hall M.A., Lanchbury J.S. Healthy human T-cell receptor beta chain repertoire. Hum Immunol (1995) 43:207–218.[CrossRef][ISI][Medline]
  38. Shanmugam A., Copie-Bergman C., Falissard B. TCR alpha beta gene usage for myelin basic protein recognition in healthy monozygous twins. J Immunol (1996) 156:3747–3754.[Abstract]
  39. Steinberg D. Modified forms of low-density lipoprotein and atherosclerosis. J Intern Med (1993) 233:227–232.[ISI][Medline]
  40. Haliwell B. The role of oxygen radicals in human disease, with particular reference to the vascular system. Haemostasis (1993) 231:118–126.
  41. Jonasson L., Holm J., Hansson G.K. Cyclosporin A inhibits smooth muscle proliferation in the vascular response to injury. Proc Natl Acad Sci USA (1988) 85:2303–2306.[Abstract/Free Full Text]
  42. Drew A.F., Tipping P.G. Cyclosporin treatment reduces early atherosclerosis in the cholesterol-fed rabbit. Atherosclerosis (1995) 116:181–189.[CrossRef][ISI][Medline]
  43. Roselaar S.E., Schonfeld G., Daugherty A. Enhanced development of atherosclerosis in cholesterol-fed rabbits by suppression of cell mediated immunity. J Clin Invest (1995) 96:1389–1394.[ISI][Medline]
  44. Emeson E.E., Shen M.-L. Accelerated atherosclerosis in hyperlipidemic C57BL/6 mice treated with cyclosporin A. Am J Pathol (1993) 142:1906–1915.[Abstract]
  45. Hansson G.K., Holm J., Holm S., Fotev Z., Hedrich H., Fingerle J. T lymphocytes inhibit the vascular response to injury. Proc Natl Acad Sci USA (1991) 88:10530–10534.[Abstract/Free Full Text]
  46. Fyfe A.I., Qiao J.-H., Lusis A.J. Immune-deficient mice develop typical atherosclerotic fatty streaks when fed an atherogenic diet. J Clin Invest (1994) 94:2516–2520.[ISI][Medline]
  47. Thyberg J., Hansson G.K. Cyclosporin A inhibits induction of DNA synthesis by PDGF and other peptide mitogens in cultured rat aortic smooth muscle cells and dermal fibroblasts. Growth Factors (1991) 4:209–219.[Medline]
  48. McMurray H.F., Parthasarathy D., Steinberg D. Oxidatively modified low density lipoprotein is a chemoattractant for human T-lymphocytes. J Clin Invest (1993) 92:1004–1008.[ISI][Medline]
  49. Breslow J.L. Mouse models of atherosclerosis. Science (1996) 272:685–688.[Abstract]
  50. Callow M.J., Verstuyft J., Tanirala R., Palinski W., Rubin E.M. Atherogenesis in transgenic mice with human apolipoprotein B and lipoprotein(a). J Clin Invest (1995) 96:1639–1646.[ISI][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Oksenberg, J. R
Right arrow Articles by Erusalimsky, J. D
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oksenberg, J. R
Right arrow Articles by Erusalimsky, J. D
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?