Analysis of T cell receptor V repertoires of annular erythema associated with Sjögren’s syndrome

ARTICLE

Sjögren's syndrome (SjS) is characterized by lymphocytic infiltration into the lacrimal and salivary glands leading to symptomatic dry eyes and mouth [1]. SjS frequently accompanies various kinds of cutaneous manifestations other than extraglandular manifestations, including hypergammaglobulinemic purpura, urticarial vasculitis, autoimmune anhidrosis, and annular erythema [2-4]. Immunohistochemical studies have shown that major dermal infiltrates in the lesional skin of annular erythema associated with SjS (AESjS) consist primarily of CD4⁺ T lymphocytes [3]. As facial infiltrative erythema can be seen associated with both lupus erythematosus and SjS, we have analyzed the T cell receptor (TCR) Vß repertoire of infiltrating cells in the lesional skin of AESjS using the reverse transcriptase polymerase chain reaction (RT-PCR) to study the mechanism of induction of AESjS and the difference between AESjS and the butterfly-like erythema associated with systemic lupus erythematosus (SLE).

Materials and methods

Patients

Seven patients with AESjS were enrolled in this study (6 females and 1 male, aged 37-69 years), the details of whom are summarized in Table I. Japanese diagnostic criteria for SjS [5] were used. All cases had primary SjS (5 definite and 2 probable) and none of them had a history of having taken any photosensitizing agents. They had not been treated with systemic immunosuppressive drugs when the AESjS had developed. Antinuclear antibodies (ANA) were detected by indirect immunofluorescence using Hep-2 cells. Anti-SS-A and anti-SS-B antibody titers were determined by micro-immunodiffusion using pig spleen for SS-A and rabbit thymus for SS-B as antigens (MBL Co. Ltd., Tokyo, Japan). The biopsied skin tissues from the annular erythema were divided up and one half was snap-frozen in OCT compound (Miles, Elkhart, IN) in liquid nitrogen and immediately stored at ­ 80° C and the other was fixed in 10% formalin solution for routine pathological examination. Histopathological examination showed deep perivascular and periappendageal infiltration by lymphocytes in all cases. As a control, three patients with typical SLE (aged 16-40 years old; 2 females and 1 male) who were diagnosed from clinical, histological and laboratory findings were also examined.

RT-PCR analysis of annular erythema

Peripheral blood mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation (Pharmacia. Uppsala, Sweden). After adherence steps, peripheral blood lymphocytes (PBL) were resuspended in RPMI 1640 supplemented with 7% fetal calf serum (FCS). Total RNA was isolated from fifty, 5-µm frozen biopsied tissue sections and PBL (1 x 10⁶) using RNA zol (Chinna/Biotex, Houston, TX, USA). Then, 100 ng of total RNA was reverse transcribed to cDNA by RAV-2 reverse transcriptase (Takara, Tokyo, Japan). The PCR was performed with 30 µl of a mixture containing 1.5 U Taq polymerase (Perkin Elmer Cetus, CT, USA), 200 M dNTP, 1.5 mM MgCl₂, 50 mM KCl, 10 mM Tris-HCl buffer, pH 8.3, and 20 pmol of Vß family-specific oligonucleotide primers as published by Dunn et al. [6], which were synthesized by a DNA synthesizer (Applied Biosystems model 3919). As internal controls, a pair of 5' sense C alpha-specific primers and 3' antisense C alpha-specific primers were also amplified, as described previously by Choi et al. [7]. The amplification consisted of 30 cycles of 94° C for 1 min, 57° C for 1 min and 72° C for 1 min and 5 sec followed by a final extension for 7 min according to the method of Dunn et al. [6]. PCR products were electrophoresed in 1.7% agarose gel. The gel was stained with 1% ethidium bromide and visualized under ultraviolet light. In preliminary experiments, PCR amplification was performed for 27, 30 and 35 cycles, to ensure that the amplification occurred in a linear range as quantified by a densitometer (EPA-3000, Chemiway, Tokyo). Each intensity was assessed as the ratio with C alpha which was expressed to a similar degree in all experiments, and the relative amount of each TCR Vß gene expression was calculated as a ratio of the total Vß band intensity. Reproducibility of the method was confirmed in preliminary experiments, showing that two separate assays of the same cDNA from a healthy donor resulted in almost identical data. As a control, 3 normal skin biopsies obtained from the face, during plastic surgery procedures were also assayed.

Results

Results of TCR Vß repertoire expression showed diversity, however, TCR Vß 2 was commonly demonstrated in AESjS in 6 out of 7 patients. The TCR Vß expression was also estimated on paired PBL from 4 AESjS patients (Patient 4 (Exp. 1), Patient 5 (Exp. 2), Patient 6 (Exp. 3), Patient 7 (Exp. 4)). Densitometric analysis showed predominant expression of TCR Vß 2 in AESjS as compared to paired PBL (Fig. 1). In one patient (Exp. 4), biopsies were taken from annular erythema occurring on the face and chest. Vß 2, 6, 18 and 19 were strongly detected in both lesional skin samples as compared to PBL (Fig. 1). In a comparative study, the Vß 6, 13-2 and 14 were strongly expressed in the butterfly rash in SLE, as compared to paired PBL. Otherwise, TCR Vß 7 and 20 were demonstrated in 2 SLE patients, and Vß 4, 12, 15, 17 and 18 were also detected. TCR Vß 6 was demonstrated in both SjS and SLE. In normal skin, diverse usage of TCR Vß was noted, and TCR Vß 6, 12, 13-2, 14 and 17 were commonly expressed.

Discussion

AESjS mimics Sweet's syndrome clinically and shows distinct histopathological features resembling Darier's annular erythema. It can be distinguished from subacute cutaneous lupus erythematosus (SCLE) [2-4]. Facial infiltrative erythema is occasionally present in association with both LE and SjS. AESjS has been recently recognized as one of the specific, cutaneous manifestations associated with SjS, however, the mechanism is still unknown. Although the self-antigens in SjS have not yet been identified, retroviral particle alone, or virus-induced products, might be candidates as these self-antigens in the progression of SjS [8-11]. In addition, there is the possibility that the reactive T cells are antigen-driven and/or superantigen-driven. SjS patients occasionally develop annular erythema following upper respiratory infection [12], and it has also been reported that lymphocytes from patients with AESjS showed a strong response to staphylococcal enterotoxin B [13]. In AESjS, cell adhesion molecules on vascular endothelial cells, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), are up-regulated [14-15]. Therefore these bacterial superantigens may contribute to lymphocyte infiltration into the skin through the expression of cell adhesion molecules.

Recently, Sumida et al. [16] has reported the predominant expression of Vß 2 or Vß 13 on infiltrating T cells in the lips of SjS patients, which may be responsible for triggering the autoimmunity in this disease. They suggest the possibility that previous bacterial infection in the lips of SjS patients led to stimulation and expansion of T cells expressing these two genes. Murata et al. [17] have demonstrated that the TCR Vß 2 gene was predominantly expressed in interstitial nephritis in SjS. They showed that the TCR Vß gene on infiltrating T cells from the kidneys of SjS patients was more restricted than those on infiltrating T cells from labial salivary glands and PBL. On the other hand, diverse usage of TCR Vß was observed in the lacrimal glands in SjS [18]. In this study, the TCR Vß repertoire of infiltrating T cells in the lesional skin of AESjS was not strictly restricted, however, Vß 2, which was preferentially detected in lips or kidneys associated with SjS, was predominantly expressed in 3 cases as compared to paired PBL. Furthermore, it is of note that Vß 2, 6, 18 and 19 genes were strongly detected in AESjS lesional skin from different sites compared to PBL in one patient. In particular, Vß 2 was commonly detected in infiltrating lymphocytes and PBL, and was not commonly detected in lesional skins, suggesting that TCR Vß 2 may contribute to the development of AESjS.

In our study, anti-SS-A antibody was detected in 4 cases (patient 1, 4, 5, 7), Vß 6 was commonly detected and Vß 2 was noted in 3 of these cases. However, Vß 2 and 6 were also detected in cases with negative anti-SS-A antibody.

CONCLUSION

REFERENCES

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