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(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

Yoshinori Seko, MD; Osamu Sato, MD; Atsuhiko Takagi, MD; Yusuke Tada, MD; Hiroshi Matsuo, MD; Hideo Yagita, PhD; Ko Okumura, PhD; Yoshio Yazaki, MD

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

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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

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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

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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

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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.

View larger version (43K): [in this window] [in a new window]   Figure 1. Southern blot analysis of the expression of T-cell receptor (TCR) V and Vß gene segments in infiltrating cells in the aortic tissue from patients with atherosclerotic aortic aneurysm (cases 1 to 3) and Takayasu's arteritis (cases 4 to 7). In all samples, polymerase chain reaction was performed using each of the 18 TCR V family-specific as well as a C-specific 5'-primer and a common C 3'-primer, or each of the 22 TCR Vß family-specific as well as a Cß-specific 5'-primer and a common Cß 3'-primer. Amplified DNAs were hybridized with a 32P-labeled 5'-C or 5'-Cß primer.

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.

View this table: [in this window] [in a new window]   Table 1. Expression of Cytokine mRNA in the Aortic Tissue of Patients With Atherosclerotic Aortic Aneurysm and Takayasu's Arteritis

	Discussion

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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

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