Bes-1 DNA Fragment Encoding Streptococcal Antigen in Skin Lesions From Patients With Behçet’s Disease

Michiko Tojo, MD*

Hirokatsu Yanagihori, MD*

Xueyi Zheng, MD*

Noritaka Oyama, MD*

Emiko Isogai. DVM†

Koichi Kimura, MD‡

Koichiro Nakamura, MD*

Fumio Kaneko, MD*

*Department of Dermatology, Fukushima Medical University School of Medicine, Fukushima, Japan

†Department of Preventive Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan

‡Department of Biomedical Engineering, Hokkaido Institute of Technology, Sapporo, Japan

KEY WORDS: Behçet’s disease, Streptococcus sanguis, Bes-1 DNA, polymerase chain reaction

Abstract

Although the etiology and pathogenesis of Behçet’s disease (BD) is poorly understood, streptococcal infectious allergy has long been postulated as one of the triggers for the onset of the disease. Bes-1 gene encoding a streptococcal antigen, which we recently cloned, seems to be homologous to the human intraocular peptide Brn-3b. To investigate the relationship between Bes-1 and BD, polymerase chain reaction (PCR) analyses were performed to detect Bes-1 DNA in samples of lesional tissues from patients with BD and patients with other inflammatory disorders who served as control subjects. Three (one each of clinically complete type, incomplete type, and suspected type) of 11 BD cases and one phlegmone case resulting from streptococcal infection were positive for Bes-1 DNA. PCR in situ hybridization was performed to determine the distribution of Bes-1 DNA in the BD lesional skin samples, and amplification of the Bes-1 sequence was detected in the nuclei of cells in the dermal vessel walls and in infiltrating mononuclear cells in lesions taken from patients with BD. These findings indicate that Bes-1 DNA might be involved in the pathogenesis of BD.

Introduction

Behçet’s disease (BD) is a multisystem inflammatory disease characterized by oral and genital ulceration and various cutaneous, arthritic, ocular, vascular, and neurologic manifestations.1,2 The etiology and pathogenesis of BD is not known, although several hypothesis such as viral or bacterial infection or autoimmunity based on the genetic background have been suspected as causative factors in the establishment of BD. Patients with BD generally have a high incidence of chronic streptococcal infections such as tonsillitis and dental caries in the oral cavity, and the hyperreactivity to streptococcal antigens might be related to these chronic infection foci.3 Lymphocyte function is also abnormal in patients with BD.4 Lymphocytes specific for selected self-peptides derived from heat shock protein (HSP) 60, which are highly homologous with bacterial HSP 65, have been found in patients with BD and the expression of HSP 60 is aberrant in the oral mucous membrane.5 Although it is difficult to explain the pathogenesis of BD by streptococcal infection and/or HSP alone, it might play a pathogenic role in a subset of patients only. Genetic factors might be important for developing an abnormal host response to these crossreactive antigens, and our interest has been focused on streptococcal infection as one of the extrinsic factors. We and others demonstrated previously that patients with BD had an intense delayed-type hypersensitivity to streptococci as demonstrated by the cutaneous reaction6 using streptococcus antigens and overproduction of inflammatory cytokines by peripheral blood mononuclear cells (PBMCs).7,8 In our previous study, we found that Streptococcus sanguis, which is serologically different from the standard strains, was dominant in the oral flora of patients with BD.9,10 These patients showed significantly higher titers of antibody against several 80 to 150 kDa membrane proteins from the isolated strain as well as greater hypersensitivity to streptococcal antigens than did normal controls.11,12 Furthermore, the strain adhered avidly to the epithelial cells of the lesions from patients with BD.10,13 Recently, we cloned and determined the sequence of the Bes-1 gene encoding the immunogenic antigen of S. sanguis KTH-1 (uncommon serotype 1, strain 113-20) isolated from the patients with BD.14 A portion of the amino acid sequence showed 60% homology with the human intraocular peptide Brn-3b, which is a POU domain expressed in a subset of retinal ganglion cells.15 Western blot analysis of the gene product of an immunopositive clone showed that patients with BD, but not healthy control subjects, had a positive reaction. Thus, there is a crossreactivity between S. sanguis peptide and human intraocular peptide.16,17

In this study, we performed polymerase chain reaction (PCR) and PCR in situ hybridization (PCR–ISH) analyses to detect Bes-1 DNA in the mucocutaneous lesional samples from patients with BD, including those with recurrent oral ulcer, genital ulcer, folliculitis, erythema nodosum (EN)-like eruptions, and compared the results with those from samples of lesions from other related inflammatory diseases, including non-BD-EN, Sweet’s disease, and phlegmone resulting from streptococcal infection.

Patients

Eleven patients with BD (3 men and 8 women) who were diagnosed using the international diagnostic criteria of BD18 were studied. Seven patients (1 man and 6 women) with other inflammatory disorders, including 3 with Sweet’s disease, 3 with non-BD-EN, and a case of phlegmone resulting from streptococcal infections, served as control subjects. The 3 clinical types of BD were defined according to the revised Japanese criteria19: (1) clinically complete type with 4 major symptoms (n = 2); (2) incomplete type with 3 major symptoms, major symptoms other than ocular symptoms, or 2 minor features (n = 8); and (3) suspected type with 2 major symptoms (n = 1). Patients with non-BD inflammatory diseases who had no history of recurrent herpetic infection were examined as control subjects. The mean age in the BD group was 35 years (range, 23–50 y) and 38 years (range, 23–70 y) in the control group. The mean duration of the disease was 5.5 years (1 mo to 24 y) in the BD group and 4.8 weeks (1 wk to 2.5 mo) in the control group. The clinical condition of each patient is shown in Table 1.

Preparation of DNA Samples

The samples of mucocutaneous lesions, including recurrent oral ulcers (n = 2), genital ulcers (n = 3), folliculitis (n = 3), and EN-like eruptions (n = 7), were obtained from patients with BD; and as controls, skin samples of inflammatory diseases, including non-BD-EN (n = 3), Sweet’s disease (n = 3), and phlegmone resulting from streptococcal infection (n = 1), and of healthy control (n = 2) were examined (Table 1). DNA was extracted from each skin sample as described previously.20 Briefly, the skin sample was treated with lysis buffer composed of 10mM Tris-HCl (pH 7.4), 10 mM EDTA, 150 mM NaCl, 0.4% SDS, and 100 mg/mL proteinase K, and extracted with phenol and chloroform. The DNA was precipitated with ethanol, dried, resuspended in distilled water, and stored at -20˚C. Every DNA sample was extracted at least twice and distilled water was extracted alongside the specimen as an extraction control.

Polymerase Chain Reaction

Nested PCR was carried out for amplification of Bes-1 DNA using 2 primer sets. The oligonucleotide primers (Bes1-1 and 2; 21 bp), which flank and amplify the 999-bp region of the S. sanguis genome coding for Bes-1, including the Brn-3b homologous lesion and double-restriction site for Pst-1, were synthesized. The oligonucleotide primers (Bes1–3 and 4; 18 bp), which flank and amplify the 276-bp region in the middle of the sequence of the previous primers, were also synthesized. The primer sequences were (forward/reverse): Bes1–1F 5’-TAATAACCCTGACCAAGCCTA-3’/ Bes1-2R 5’-CCCTTTCAAAAGTCATAAATC-3’ (999 bp) and Bes1–3F 5’-AGCTATGGACTGAAGAAA-3’/ Bes1–4-R 5’-AAGCTGCTGGAGATTGGT-3’ (276 bp). The PCR mixture in a final volume of 25 mLcontained 1 mg of DNA, 1 mM each of both primers, 50 mM Tris-HCl (pH 9.0), 20 mM ammonium sulfate, 1.5 mM MgCl2, 200 mM each of deoxynucleoside triphosphates (dNTPs), and 5 units of amplitaq DNA polymerase (Takara Shuzo Co., Osaka, Japan). The amplification conditions were 3 minutes at 94˚, 30 cycles of 1 minute at 94˚, 1 minute at 50˚, 1 minute at 72˚, and a final extension for 5 minutes at 72˚. All DNA samples were confirmed to be amplifiable using PCR primers specific for a conserved region of the human b-globin gene. Every sample was tested at least twice. Positive and negative controls were used in each case. As a positive control, the template DNA was Bes-1 DNA, and distilled water and an extraction control were used as a negative control. After amplification, the PCR products were analyzed by 1.5% agarose gel electrophoresis.

PCR-RFLP

Restriction digestion of amplified DNA was performed by adding 10 units of Pst-I to a 10-mL sample of PCR product and incubating for 1 hour at 37˚. The digested DNA was analyzed using the same 1.5% agarose gel electrophoresis.

In situ PCR

In situ nested PCR was conducted on biopsy materials, which were fixed in 10% formalin and embedded in paraffin. Briefly, 10-mm thick sections were placed on slides pretreated with aminopyltriethoxilane, dewaxed, and rehydrated according to a standard protocol. The sections were treated with 0.1 M HCl and 10 mg/mL proteinase K each for 20 minutes at room temperature. Amplification was carried out in a 25-mL chamber (TaKaRa, Japan). Each 25-mL reaction for Bes-1 DNA amplification contained 10 mM Tris/HCl, 50 mM KCl, 3.5 mM MgCl₂, 200 mM dNTP, 100 mM digoxigenin-labeled dUTP, 1U Taq polymerase, and 25 pmol primers. Amplification conditions were denaturation for 10 minutes at 80˚ and 20 cycles of incubation at 95˚ for 1 minute, 55˚ for 3 minutes, and 72˚ for 3 minutes. This was followed by a 10-minute extension. The sequences of primers were as previously described (exterior: Bes1–1F and Bes1–2R, interior: Bes1–3F and Bes1–4R). Signals were detected and visualized using a digoxigenin nucleic acid detection kit (Roche Diagnostics, GmbH, Manheim, Germany)

Results

The results are summarized in Table 2. The amplified PCR product contained the 999-bp band as seen by ultraviolet fluorescence after ethidium bromide staining. Three of the 11 BD cases were positive for Bes-1 (Fig. 1, no. 4, 7, 8, and 22) and one of the control samples was positive (Fig. 1, no. 9). To exclude the possibility of Bes-1 DNA contamination at the surface of the epidermis in the skin samples, the epidermis was removed from the positive samples by microscopic dissection and they were analyzed by PCR again. Because the same products were formed, it is likely that the Bes-1 DNA came from dermal components (Fig. 2, no.4, 4’ and 7, 7’). To establish the specificity of the first PCR product with primers Bes1-1 and 2, all the 999-bp bands were analyzed by restriction endonuclease digestion. They yielded the expected 160-, 389-, and 450-bp fragments when digested with Pst-I (Fig. 3, no.7). Three lesions from patients with BD (no.4, 7, and 8) and the lesion from a patient with phlegmone (no. 9) showed positive hybridization signals for Bes-1 DNA by PCR–ISH (Fig. 4; Table 2). The reaction product was detected on the nuclei of cells adhering to the dermal vessel walls and in some of the infiltrating cells around the vessels in severely inflamed sites of BD lesions (Fig. 4). The negative controls (without Taq DNA polymerase) did not show amplification of Bes-1 DNA (data not shown).

Discussion

A streptococcal etiology of BD has long been postulated.3,5–8 In this study, the 999-bp fragment of Bes-1 DNA was detected by PCR in the mucocutaneous lesions from patients with BD. The product yielded the expected fragments when digested with Pst-1 restriction endonuclease. Among the patients with BD in this study, 37% were positive for Bes-1 DNA and this was confirmed using skin samples without epidermis. We also demonstrated by PCR the presence of Bes-1 DNA in samples of a genital ulcer (no. 4, incomplete type) and an EN-like eruption (no. 22, complete type) in patients with BD, and Bes-1 DNA was found in the skin sample of an EN-like eruption (no. 7) and an recurrent oral ulcer (no. 8) in one patient with BD (suspected type). Bes-1 DNA was also detected by PCR–ISH in the mucocutaneous lesions, which were positive by PCR. The reaction product was deposited on the nuclei of cells adhering to the dermal vessel walls and some of the infiltrates around the vessels in the main inflammatory lesions. The PCR–ISH-positive cells might be correlated with Bes-1 DNA in the lesions in patients with BD.

One of the control cases with non-BD (no. 9) who had a phlegmone lesion infected with bacteria, was positive for Bes-1 DNA by PCR and PCR–ISH. Although the bacterial culture from this patient was inconclusive with respect to the species of bacteria, the results suggest that the phlegmone lesion might have been affected by S. sanguis and/or the correlated organisms infection.

Previously, we also found HSV-1 and/or HSV-2 DNAs in one (incomplete type) of 11 BD cases, 2 of 3 cases of Sweet’s disease, and 1 of 3 non-BD-EN cases.21 HSV-1/2 DNA was found in a lower percentage of BD cases (1 of 11 cases), and Bes-1 DNA was found more frequently (3 of 11 cases) than HSV–DNA. Moreover, high titers of HSV-1/2 antibodies were closely correlated with positivity for HSV-1/2 DNA in the skin lesions, even in patients with both BD and non-BD. Thus, the presence of Bes-1 DNA seems to be more closely related to the pathogenesis of BD than the presence of HSV1/2.

In conclusion, the present results suggest a causative role for Bes-1 in the pathogenesis of BD in the correlation with chronic focal infection in the oral cavity. Consistent findings from PCR–ISH is crucial to demonstrate a causal association between an infectious agent, Bes-1, and the skin lesions in patients with BD. Therefore, further studies, probably using double staining for the characterization of PCR–ISH-positive cells, might elucidate the role of the streptococcal antigen, Bes-1, in the BD lesions.

References

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4. Mochizuki M, Suzuki N, Takeno M: Fine antigen specificity of human gamma delta T cell lines (V gamma 9+) established by repetitive stimulation with a serotype (KTH-1) of a gram-positive bacterium, Streptococcus sanguis. Eur J Immunol 24:1536–1543, 1994.

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7. Hirohata S, Oka H, Mizushima Y: Streptococcal-related antigens stimulate production of IL-6 and interferon-g by T cells from patients with Behçet’s disease. Cell Immunol 140:410–419, 1992.

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9. Isogai E, Ohno S, Takeshi K, et al: Close association of Streptococcus sanguis uncommon serotype with Behçet’s disease. Bifidobact Microflora 9:27–41, 1990.

10. Isogai E, Ohno S, Kotake S, et al: Chemiluminescence of neutrophils from patients with Behçet’s disease and its correlation with an increased proportion of uncommon serotypes of Streptococcus sanguis in the oral flora. Arch Oral Biol 35:43–48, 1990.

11. Yokota K, Hayashi S, Fujii N, et al: Antibody response to oral streptococci in Behçet’s disease. Microbiol Immunol 36:815–822, 1992.

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13. Isogai E, Isogai H, Fujii N, et al: Adhesive properties of Streptococcus sanguis isolated from patients with Behçet’s disease. Microbial Ecology in Health and Disease 3:321–328, 1990.

14. Yoshikawa K, Kotake S, Kubota T, Kimura K, Isogai E, Fujii N: Cloning and sequencing of BES-1 gene encoding the immunogenic antigen of Streptococcus sanguis KTH-1 isolated from the patients with Behçet’s disease. Zentbl Bakteriol 287:449–460, 1998.

15. Xiang M, Zhou L, Peng Y, Eddy RL, Shows TB, Nathans J: Brn-3b: a POU domain gene expressed in a subset of retinal ganglion cells. Neuron 11:689–701, 1993.

16. Isogai E, Isogai H, Kotake S, et al: Antibody cross reactivity from sera of patients with Behçet’s disease with synthetic peptides that have homologies with proteins from Streptococcus sanguis. J Appl Res 2:1–7, 2002.

17. Isogai E, Isogai H, Kotake S, Ohno S, Kimura K, Oguma K: Role of Streptococcus sanguis and traumatic factors in Behçet’s disease. J Appl Res 3:1–7, 2003.

18. International Study Group for Behçet’s Disease (ISGBD): Criteria for diagnosis of Behçet’s disease. Lancet 335:1078–1080, 1990.

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21. Tojo M, Zheng X, Yanagihori H, et al: Detection of herpes virus genomes in skin lesions from patients with Behçet’s disease and other related inflammatory diseases. Acta Dermatol Venereol 83:124–127, 2003.

Table 2. Results in Patients Positive for Bes-1 DNA

No. of samples PCR PCR–ISH

1 — —

2 — —

3 — Yes

4 Yes Yes

5 — —

6 — —

7 Yes Yes

8 Yes Yes

9 Yes Yes

10 — —

11 — —

12 — —

13 — —

14 — ND

15 — ND

16 — ND

17 — ND

18 — ND

19 — ND

20 — ND

21 — ND

22 Yes ND

23 — ND

24 — ND

ND, not done.

M 4 7 8 9 22 Bes1

Figure 1. Electrophoresis on 1.5% agarose gel of amplified DNA showing the 999-bp DNA for Bes-1. The gel was stained with ethidium bromide and viewed under ultraviolet light (M, size marker; Bes1, positive control). The positive bands are present in lanes 4, 7, 8, 9, and 22.

M 4 4’ 7 7’

Figure 2. The epidermis was removed from Bes-1-positive samples by microdissection. Representative data are shown (no. 4 and 7). Lanes 4 and 7 include epidermis and lanes 4’ and 7’ are without epidermis. Positive bands were detected in all lanes.

M Bes1 M No. 7

Figure 3. Electrophoresis of restriction endonuclease digested DNA. Representative data are shown (no. 7). Positive control, Bes-1, and no. 7 showed the expected fragment pattern; uncut 999-bp sample and sample cut with Pst-1 showing 160-, 389-, and 450-bp fragments.

Figure 4. PCR–ISH analysis of EN-like eruption in one BD case (no. 7) shows the positive signals (arrows) for Bes-1 DNA in cells adhering to the superficial dermal vessel wall (A) and deep dermal vessel walls (B).