(Circulation. 1996;93:1788-1790.)
© 1996 American Heart Association, Inc.
Articles |
Restricted Usage of T-Cell Receptor V
-Vß Genes in Infiltrating Cells in Aortic Tissue of Patients With Takayasu's Arteritis
From the Third Department of Internal Medicine (Y.S., Y.Y.), Faculty of Medicine, University of Tokyo (Japan); the Department of Immunology (Y.S., H.Y., K.O.), School of Medicine, Juntendo University, Tokyo, Japan; the Institute for Adult Diseases (Y.S.), Asahi Life Foundation, Tokyo, Japan; the Second Department of Surgery (O.S., A.T.), Faculty of Medicine, University of Tokyo (Japan); the Second Department of Surgery (Y.T.), Yamanashi (Japan) Medical College; and the Division of Angiology (H.M.), Department of Internal Medicine, National Cardiovascular Center, Osaka, Japan.
Correspondence to Yoshinori Seko, MD, Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background Infiltration by perforin-secreting killer lymphocytes, such as T cells and natural killer cells, has been shown to be involved in the pathogenesis of vascular cell damage in Takayasu's arteritis.
Methods and Results To investigate the immunological
mechanisms involved, especially the nature of T-cell infiltration in
Takayasu's arteritis as well as atherosclerosis, we
analyzed the expression of T-cell receptor (TCR) V
and Vß
genes in infiltrating cells in the aortic tissue of patients with
Takayasu's arteritis and atherosclerotic aortic aneurysm by
polymerase chain reaction (PCR). We also analyzed the
expression of cytokine genes by PCR. We found that the
repertoires of TCR V as well as Vß gene transcripts in Takayasu's
arteritis were restricted. The infiltrating cells expressing V2,
V16, V17, Vß7, and Vß13.1 were found in 3 of 4 patients. In
contrast, TCR V-Vß repertoires in atherosclerotic aortic
aneurysm were polyclonal. There was no significant difference
in the pattern of cytokine gene expression between the two
diseases.
Conclusions The restricted usage of TCR V as well as
Vß genes by infiltrating T cells in Takayasu's arteritis may
indicate that a specific antigen in the aortic tissue was targeted. Our
findings provide the evidence that distinct immunological mechanisms
are involved in the pathogenesis of Takayasu's arteritis and
atherosclerotic aortic aneurysm.
Key Words: Takayasu's arteritis • immunology • polymerase chain reaction • atherosclerosis
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Takayasu's arteritis is a chronic vasculitis of unknown pathogenesis involving the aorta, its main branches, and the pulmonary arteries. In the acute phase, extensive inflammation with massive cell infiltration and destruction of vascular tissue occurs, whereas in the chronic phase, local inflammation causing persistent vascular cell damage may develop lesions with pathological features resembling the atherosclerotic aortic aneurysm. On the other hand, histological findings of inflammatory cell infiltration and destruction of the vessel wall of the atherosclerotic aortic aneurysm suggest that cell-mediated autoimmune mechanisms play an important role in the pathogenesis involved.1 2 3 We previously reported that perforin-secreting killer lymphocytes such as T cells and natural killer (NK) cells infiltrate and a 65-kD heat-shock protein is strongly expressed in the aortic tissue of patients with Takayasu's arteritis.4 This strongly suggests that cell-mediated autoimmunity plays an important role in the vascular cell damage involved in Takayasu's arteritis as well as in atherosclerosis. However, the precise mechanisms involved in the pathogenesis of both vascular diseases are still unknown.
The purpose of the present study was to clarify in more detail the
immunological mechanisms involved in these two diseases and to examine
the differences in them. For this purpose, to examine the antigen
specificity of the infiltrating T cells, we analyzed the
expression of T-cell receptor (TCR) V and Vß genes by polymerase
chain reaction (PCR). We also analyzed the expression of
cytokine genes by a PCR method.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Patients
Aortic tissue samples were obtained at aortic bypass surgery from four patients with Takayasu's arteritis (two men and two women; average age, 56.3 years) and four patients with atherosclerotic aortic aneurysm (three men and one woman; average age, 69.3 years) in whom clinical diagnoses of Takayasu's arteritis and atherosclerotic aortic aneurysm had been determined previously by history, physical examination, blood analyses (such as C-reactive protein and erythrocyte sedimentation rate), and angiography.
Preparation of RNA and cDNA Synthesis
Total cytoplasmic RNA was prepared from freshly dissected aortic
tissue samples by a method using RNA zol (CINNA/BIOTECX Laboratories
International, Inc, according to the manufacturer's instructions); 10
to 20 µg of total RNA was used for the synthesis of
single-stranded cDNA with reverse transcriptase. Briefly, in a
volume of 40 µL 1x RTase buffer (50 mmol/L Tris-HCl, pH 8.3, 75
mmol/L KCl, 3 mmol/L MgCl2), 10 mmol/L dithiothreitol, 0.5
mmol/L deoxyribonucleotide triphosphates, 25 mg/L oligo
d(T),12-18 and 400 units of Moloney murine leukemia virus
(M-MLV H RT) reverse transcriptase (GIBCO BRL) were incubated with RNA
(10 to 20 µg) for 50 minutes at 42°C. The reaction mixture was
denatured at 90°C for 5 minutes, then treated with RNase H (GIBCO
BRL) for 20 minutes at 37°C.
Amplification of cDNA by PCR
For the analyses of TCR genes, single-stranded cDNA
from aortic tissues was amplified using a 5'-V– or
5'-Vß–specific primer and a 3'-C or 3'-Cß primer at a final
concentration of 0.5 µmol/L each in the reaction mixture. We
synthesized 18 different V-specific
oligonucleotides and a C-specific
oligonucleotide as 5'-sense primers and another
C-specific oligonucleotide as a 3'-antisense
primer. We also synthesized 22 different Vß-specific
oligonucleotides and a Cß-specific
oligonucleotide as 5'-sense primers and another
Cß-specific oligonucleotide as a 3'-antisense
primer. The sequences for primers were the same as previously
reported.5 Amplification was performed with 0.4 unit of
AmpliTaq DNA polymerase (Perkin-Elmer Cetus) in a DNA
thermal cycler (Perkin-Elmer Cetus). The PCR was performed with 40
cycles of denaturation at 94°C for 1 minute, primer annealing at
55°C for 2 minutes, and primer extension at 72°C for 3 minutes.
For the analyses of cytokine genes, single-stranded
cDNA from aortic tissues was amplified with 1 unit of
AmpliTaq DNA polymerase and 1 unit of Perfect Match
polymerase enhancer (Stratagene) using 5'- and 3'-primers specific for
interleukin (IL)-1, IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, interferon (IFN)-
, tumor necrosis factor (TNF)-, TNF-ß,
granulocyte/macrophage colony stimulating factor (GM-CSF),
tumor growth factor (TGF)-ß, and ß-actin (CLONTECH
Laboratories), respectively. The PCR was performed with 30 cycles of
denaturation at 94°C for 1 minute, primer annealing at 60°C for 2
minutes, and primer extension at 72°C for 3 minutes. Expression of
these cytokines was examined using ethidium
bromide–stained agarose gel electrophoresis.
Southern Blot Analysis
Ten microliters of V-C or Vß-Cß amplified products
were subjected to electrophoresis on 2% agarose gel and transferred to
a nylon membrane. We used 5'-C or 5'-Cß primer as probes for the
detection of V-C or Vß-Cß amplified products,
respectively. 5'-C and 5'-Cß primers were labeled by terminal
deoxynucleotidyl transferase at the 3' end with
32P–-dCTP. Filters were prehybridized for 2 hours
at 55°C in (6x SSPE, 2x Denhardt's solution, 0.5% SDS, and 100
mg/L salmon sperm DNA) and hybridized overnight at 55°C in the same
solution with a 32P-labeled 5'-C or 5'-Cß primer. The
filters were subsequently washed in 0.1x SSPE and 0.1% SDS for 1 hour
at 55°C and then were autoradiographed.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Histological examination showed marked thickening of the intima and adventitia, irregular disruption of the medial elastic fibers, and inflammatory cell infiltration involving the vasa vasorum of the media and the adventitia. Along with clinical features, angiography, and blood analyses, a diagnosis of Takayasu's arteritis was established in all four patients.
Polyclonal Expression of TCR V and Vß Genes in Infiltrating
Cells in the Aortic Tissue With Atherosclerotic Aortic
Aneurysm
The Figure
(cases 1 to 3) shows the results of
Southern blot analysis. Almost all TCR V as well as Vß
genes were expressed in infiltrating cells in the aortic tissue with
atherosclerotic aortic aneurysm. These results also indicate
that the PCR using the V and C as well as Vß and Cß primers
could amplify the individual V and Vß gene transcripts.
|
Restricted Expression of TCR V and Vß Genes in Infiltrating
Cells in the Aortic Tissue With Takayasu's Arteritis
The Figure
(cases 4 to 7) also shows the results of Southern blot
analysis of the PCR-amplified products obtained from
infiltrating cells in the aortic tissue with Takayasu's arteritis. As
shown in the Figure, in contrast to the expression in that with
atherosclerotic aortic aneurysm, only a few or limited numbers
of V as well as Vß genes were preferentially rearranged and
transcribed in infiltrating cells in the aortic tissue of all patients
with Takayasu's arteritis. Although there were no specific patterns in
the expression of V and Vß genes among these patients, V2,
V16, V17, Vß7, and Vß13.1 were rearranged in three of four
patients.
Expression of Cytokine Genes in the Aortic Tissue of
Patients With Atherosclerotic Aortic Aneurysm and
Takayasu's Arteritis
Amplification of cDNAs from the aortic tissue for cytokine
transcripts resulted in strong bands for IL-6, weak to moderate bands
for IL-1ß, and weak bands for IL-3, IL-4, IL-5, IL-7, IFN-,
TNF-, and TNF-ß (Table). There was no significant
difference in the pattern of cytokine gene expression between
atherosclerotic aortic aneurysm and Takayasu's arteritis.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In this study, we demonstrated that TCR V
as well as Vß gene
usage by infiltrating cells in the aortic tissue of patients with
Takayasu's arteritis was restricted. This contrasts sharply with the
polyclonal usage of TCR V and Vß genes by those of patients with
atherosclerotic aortic aneurysm, indicating that distinct
immunological mechanisms are involved in the pathogenesis of these two
diseases. However, the pattern of cytokine expression in these
diseases was similar.
We previously reported that 
T lymphocytes, cytotoxic T
lymphocytes (CTLs), T-helper (Th) cells, NK cells, and
macrophages infiltrate the aortic tissue in Takayasu's
arteritis.4 We also showed that macrophages, NK
cells, CTLs, and Th cells infiltrate the aortic tissue in
atherosclerotic aortic aneurysm.4 If the
T-cell–mediated autoimmune process plays a major role in the
vascular cell damage involved, the analysis of T-cell
repertoire at the site of inflammation is of great importance. In
particular, characterization of TCR gene usage in the aortic tissue
could be a direct way to examine the importance of T cells in the
pathogenesis of these diseases. The restricted usage of TCR V as
well as Vß genes by infiltrating cells in Takayasu's arteritis, as
revealed in the present study, indicates that a specific antigen,
presented at the groove of major histocompatibility complex
molecules, in the aortic tissue was targeted. We also examined the
human leukocyte antigen (HLA) class I alleles of the patients with
Takayasu's arteritis (data not shown). However, because the number of
patients studied was rather small, we could not conclude whether there
is a correlation between the patterns of TCR usage and the HLA class I
alleles. The polyclonal TCR V-Vß repertoires in
atherosclerotic aortic aneurysm may indicate that nonspecific
T-cell recruitment by inflammatory cytokines occurred. Swanson
et al6 also reported polyclonal expression of TCR Vß
genes in human atheroma. However, we could not exclude the
possibility that antigen-specific T cells infiltrated in a small
population. Recently, we have found that infiltrating NK cells and CTLs
secrete perforin directly onto the aortic vascular cells in
atherosclerotic aortic aneurysm, as in Takayasu's arteritis
(personal observations; Y. Seko, March 1995). It is unclear why
infiltrating T cells recognize and damage the aortic vascular cells in
atherosclerotic aortic aneurysm and whether the antigen
recognized by infiltrating T lymphocytes is the same as that
recognized by infiltrating ß T lymphocytes in Takayasu's
arteritis. To investigate the antigen specificity of the infiltrating
T lymphocytes in Takayasu's arteritis, we are currently
analyzing the TCR repertoire of and chains by the PCR
method.
|
Acknowledgments |
|---|
This work was supported by a grant for cardiomyopathy and a grant for intractable vasculitis from the Ministry of Health and Welfare, Japan, a grant for scientific research from the Ministry of Education, Science, and Culture, Japan, and grants from Kowa Life Science Foundation, Ichiro Kanehara Foundation, Kanae Foundation of Research for New Medicine, and Japan Foundation of Cardiovascular Research. We thank Mrs K. Takahashi for excellent technical assistance.
Received January 22, 1996; revision received March 4, 1996; accepted March 4, 1996.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK. Regional accumulation of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis. 1986;6:131-138.
2. Emeson EE, Robertson AL Jr. T lymphocytes in aortic and coronary intimas: their potential role in atherogenesis. Am J Pathol. 1988;130:369-376. [Abstract]
3.
Hansson GK, Jonasson L, Seifert PS, Stemme S.
Immune mechanisms in atherosclerosis.
Arteriosclerosis. 1989;9:567-578.
4. Seko Y, Minota S, Kawasaki A, Shinkai Y, Maeda K, Yagita H, Okumura K, Sato O, Takagi A, Tada Y, Yazaki Y. Perforin-secreting killer cell infiltration and expression of a 65-kD heat-shock protein in aortic tissue of patients with Takayasu's arteritis. J Clin Invest. 1994;93:750-758.
5.
Seko Y, Ishiyama S, Nishikawa T, Kasajima T, Hiroe M,
Kagawa N, Osada K, Suzuki S, Yagita H, Okumura K, Yazaki Y.
Restricted usage of T cell receptor V-Vß genes in
infiltrating cells in the hearts of patients with acute myocarditis and
dilated cardiomyopathy. J
Clin Invest. 1995;96:1035-1041.
6.
Swanson SJ, Rosenzweig A, Seidman JG, Libby P.
Diversity of T-cell antigen receptor Vß gene utilization in
advanced human atheroma.
Arterioscler Thromb. 1994;14:1210-1214.
This article has been cited by other articles:
 |
|
 |
|
  R. He, D.-C. Guo, W. Sun, C. L. Papke, S. Duraisamy, A. L. Estrera, H. J. Safi, C. Ahn, L. Maximilian Buja, F. C. Arnett, et al. Characterization of the inflammatory cells in ascending thoracic aortic aneurysms in patients with Marfan syndrome, familial thoracic aortic aneurysms, and sporadic aneurysms. J. Thorac. Cardiovasc. Surg., October 1, 2008; 136(4): 922 - 929. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kuivaniemi, C. D. Platsoucas, and M. D. Tilson III Aortic Aneurysms: An Immune Disease With a Strong Genetic Component Circulation, January 15, 2008; 117(2): 242 - 252. [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Park, S. W. Lee, Y. B. Park, and S. K. Lee Serum cytokine profiles and their correlations with disease activity in Takayasu's arteritis Rheumatology, May 1, 2006; 45(5): 545 - 548. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Weyand and J. J. Goronzy Medium- and Large-Vessel Vasculitis N. Engl. J. Med., July 10, 2003; 349(2): 160 - 169. [Full Text] [PDF]
|
|
|
|
 |
|
 G. K. Hansson, P. Libby, U. Schonbeck, and Z.-Q. Yan Innate and Adaptive Immunity in the Pathogenesis of Atherosclerosis Circ. Res., August 23, 2002; 91(4): 281 - 291. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. K. Schiller, W. A. Boisvert, and L. K. Curtiss Inflammation in Atherosclerosis: Lesion Formation in LDL Receptor-Deficient Mice With Perforin and Lystbeige Mutations Arterioscler Thromb Vasc Biol, August 1, 2002; 22(8): 1341 - 1346. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Noris, E. Daina, S. Gamba, S. Bonazzola, and G. Remuzzi Interleukin-6 and RANTES in Takayasu Arteritis : A Guide for Therapeutic Decisions? Circulation, July 6, 1999; 100(1): 55 - 60. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||