|Click here for information on how to order reprints of this article.|
Emiko Isogai, DVMa
Hiroshi Isogai, DVMb
Satoshi Kotake, MDc
Shigeaki Ohno, MDc
Mami Ishihara, MDd
Koki Aoki, MDe
Michiko Tojo, MDf
Fumio Kaneko, MDf
Keiji Oguma, MDg
aDepartment of Preventive Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan
bDivision of Experimental Animals,
cDepartment of Ophthalmology, Hokkaido University School
dDepartment of Ophthalmol, Yokohama City Univ., School of Medicine, 3-9 Fukuura, Yokohama, Japan
eAoki Eye Clinic, Kita 2-1, Hondori 6,
fDepartment of Dermatology,
gDepartment of Microbiology,
KEY WORDS: Behçet's disease, heat shock protein 60, retinal ganglion cell protein, Streptococcus sanguis
Background: Behçet's disease (BD) is a multisystemic disease of unknown origin. Streptococcus sanguis has been implicated in the etiology of BD.
Design: We evaluated the antibody response against cross-reactive epitopes on heat shock protein (hsp) 60 and retinal ganglion cell protein homologue to S. sanguis in patients with BD.
Materials and Methods: Patients with active BD (n = 32) and control subjects (sarocoidosis, n = 10; allergic conjunctivitis, n = 10; adenovirus conjunctivitis, n = 10; Lyme disease, n = 10; healthy controls, n = 41) were included. Serum IgA, IgG, and IgM against 5 synthetic peptides were tested in enzyme-linked immunosorbent assay (ELISA).
Results: We determined the antibody response against human hsp336-351 and retinal ganglion cell peptides. The number of IgG- or IgA-positive patients was significantly higher among patients with BD than that in healthy controls. The mean level of these antibodies in sera from BD patients was significantly higher than in controls. The levels of antibodies against Brn3b11-25/177-189 and Bes1229-243 were significantly higher in patients with BD than that in healthy controls. The antibody levels against 5 peptides significantly correlated with each other. Some patients with other "control" diseases such as sarcoidosis, Lyme disease, and allergic conjunctivitis also showed positive IgG or IgM antibody levels against hsp336-351.
Conclusions: Antibody cross-reactivity from the sera of patients with BD with synthetic peptides have homologies with proteins from S. sanguis.
Behçet's disease (BD) is multisystemic disorder presenting with recurrent oral and genital ulceration as well as uveitis often leading to blindness.1,2 We present evidence for an HLA association with BD. HLA-B51, one of the split antigens of HLA-B5, was found to be the most strongly associated genetic marker.3,4 Genetic predisposition such HLA-B51 and certain microorganisms are believed to play important roles. We found that the proportion of Streptococcus sanguis in the oral flora of patients with BD was significantly increased compared with controls.5,6 Patients with BD showed hypersensitivity in skin tests with the streptococcal antigens, and sometimes symptoms typical of BD can be provoked by an injection of the antigen.7 Herpes simplex virus and serum antibodies against the virus have been found in a higher proportion of patients with BD than in controls.1,8 Other viruses such as hepatitis c virus and parvovirus B19, may also have some role.1 However, none of these viral agents have been proved to cause BD.
Previous investigations have suggested that immunologic cross-reactivity between peptides within streptococcal heat shock protein (hsp) and human peptides might be involved in the pathogenesis of BD.9-14 Upregulation of hsp expression was found at lesional skin sites in BD.15 Tanaka et al.13 reported that antistreptococcal hsp60 antibody and antiretinal hsp60 antibody titers were both significantly higher in BD patients than in healthy donors and that significant differences exist in the antigenicity of various hsp60s. The concept of overexpression of self-hsp, either on the cell surface or as peptides presented by MHC products, has been central to the hypothesis that hsp-specific antibodies and T cells play a role in the pathogenesis of human autoimmune disease.16 T cell response to hsp60 have been found in patients with BD.9,11,14,17 Although the level of hsp-specific antibodies in serum have increased in BD,11,13,14,17 these immune responses to hsp have been also found in a number of human autoimmune diseases.16 Thus, it is difficult to explain the pathogenesis of BD by hsp alone.
We recently succeeded in isolating and determining the sequence of bes-1 encoding a streptococcal antigen correlated with BD.18 The residues in a portion of the amino acid sequence showed a 60% correspondence to those of the human intraocular peptide Brn-3b. Brn-3b is a POU (Pit-Oct Unc) domain expressed in a subset of retinal ganglion cells.19 In analysis of the gene product of an immunopositive clone by Western blot, patients with BD showed a positive reaction but healthy controls did not.18 Thus, cross-reactivity between S. sanguis peptide and human intraocular peptide has been determined. In this study, we showed significant antibody responses against molecular mimic peptides, hsp, and retinal ganglion cell-associated protein in BD.
Patients and Methods
Serum specimens from 32 patients with BD (22 men and 10 women, 36 to 51 years old) were examined. The diagnosis of BD was made under the criteria of the BD Research Committee of the Ministry of Health and Welfare of Japan, with no evidence of any concomitant disorders that might cause similar manifestations. As "disease controls," 20 patients with uveitis (10 with sarcoidosis20 and 10 with Lyme disease21), 20 patients with conjunctivitis (10 with allergic conjunctivitis, 5 men and 5 women, 16 to 48 years old and 10 with adenovirus conjunctivitis showing adenovirus types 3, 4, 8, 19, and 37; 5 men and 5 women, 21 to 54 years old) were examined. Forty-one unrelated healthy Japanese individuals with no history of BD served as healthy controls (34 men and 7 women; 19 to 59 years old).
A peptide-derived sequence of human hsp336-351(QPHDLGKVGEVIVTKDD)
that had been reported to stimulate T lymphocytes in patients22,23 was produced by the American Peptide Company,
Enzyme-linked immunosorbent assay (ELISA) was performed as described previously24 to detect IgA, IgG, and IgM antibodies in 100 mL of serum from the patients with BD, the disease controls, and healthy controls. The cut-off value used in the present study was determined at mean plus or minus 2 SD.
The significance of the positive level of anti-synthetic peptide antibodies among the patients, the disease controls, and healthy controls was tested by chi-square analysis with Yates' correction. The results were expressed as mean plus or minus standard error (SE). Differences between the patients and control groups were determined using the Mann-Whitney test. Spearman's rank correlation was used for antibody titers against synthetic peptides. P < 0.05 was taken as the level of significance.
We determined the antibody response against human hsp336-351, Brn-3b11-25, Brn-3b177-189, Bes-1229-243, and Bes-1373-385. The positive percent in ELISA is shown in Table 1. The number of IgG-positive patients with BD was significantly higher than the number IgG-positive healthy controls in the IgG responses against hsp336-351, Brn-3b11-25, Bes-1229-243 homologue to Brn-3b11-25, and Brn-3b177-189 (P < 0.01). The positive percentages in the responses were 46.9%, 46.9%, 56.3%, and 43.8%, respectively. In sarcoidosis, Lyme disease, and allergic conjunctivitis, IgG responses against hsp336-351 were observed in 30%, 80%, and 30% of patients; not response was seen in patients with adenovirus conjunctivitis. The percentage of IgM-positive patients with various ocular manifestations was similar to that of healthy controls, whereas 70% of patients with Lyme disease showed a positive reaction. Positive IgA antibody response against hsp336-351 was observed in half of the sera from BD patients. There were no positive IgA responses against the peptide in disease and healthy controls, with one exception. A small number of disease and healthy controls showed a positive reaction against Brn-3b11-25, Brn-3b177-189, Bes-1229-243, and Bes-1373-385 in ELISA.
As shown in Table 2, IgG antibody titers to various peptides increased significantly in BD, compared with disease controls and healthy controls. IgG and IgA antibody titers to hsp336-351 were significantly higher than in healthy controls. In sarcoidosis, Lyme disease, and allergic conjunctivitis, levels of IgG antibody against hsp336-351 were also high (P < 0.05 vs healthy controls). There were no significances in IgM responses against various peptides except in patients with Lyme disease.
A distribution pattern with a double peak was observed in BD (Fig. 1). Healthy controls showed single a peak in distribution. Similarly, a single peak was observed, with peak-shift from left to right, in the other disease controls.
A correlation was recognized among the antibody responses against hsp, Brn-3b, and Bes-1 (Table 3; P < 0.01 or P < 0.05). The correlation coefficients in the antibody response ranged from 0.101 to 0.964.
It is known that an IgA immune response to hsp336-351 is significantly induced in patients with BD.16 Our data also indicate that the mean antibody titer in patients with BD was high compared with that in controls. These findings indicate that the immune response against a specific epitope such as hsp336-351 may be characterized in BD. However, interestingly, IgG reactivity with hsp336-351 was divided into 2 groups. As shown in Figure 1, the first peak was similar to that in healthy controls, and second peak was only recognized in patients with BD. It is possible that there are several processes of the humoral immune responses against the cross-reactive specific epitopes on the bacterial hsp60, at least mucosal and systemic immune responses.
Human hsp60 is thought to be a target molecule for the induction of local inflammation caused by stimulation of bacterial hsp60.16,25 Indeed, the uncommon serotypes of S. sanguis found in BD cross-react with the 65-kD hsp that also shares antigenicity with an oral mucosal antigen.12
Antibodies against peptides of human ocular ganglion cell protein and S. sanguis were examined. Peptides such as Brn-3b11-25, and Brn-3b177-189, yielded significant antibody levels in BD. The percentages of the patients who showed positive reactions against peptides Brn-3b11-25, and Brn-3b177-189, were 46.9 and 56.3, respectively. The percentage was similar to that for anti-hsp336-351. A significant correlation between the antibody response against hsp336-351 and that of the other peptides support the suggestion that S. sanguis infection can be associated with the pathogenesis of BD.
The majority of patients with BD experience oral ulceration as an initial symptom.1 However, because recurrent oral ulceration is a common manifestation of many local or general disorders, a variety of differential diagnosis must be considered. The antibody reaction to peptides can be useful to know whether recurrent oral ulceration may progress to BD.
Although we currently cannot distinguish whether the development of BD is related to direct infection by S. sanguis or whether it is simply the result of an immune reaction, the possibility remains that S. sanguis infection might be a trigger for the development of both mucosal and ocular inflammation. There can be a cross-reactive response against S. sanguis and molecular mimic host cells. Self-epitope with S. sanguis and presence of cross-reactive antibodies in the sera of patients with BD suggest that this bacterium may be associated with the initiation and progression of some cases of BD.
The most difficult step is to definitively prove the relevance of molecular mimicry to naturally occurring human diseases. Various correlations ranging from the reasonably convincing to less convincing have been well known. Genetic factors may be important to developing an abnormal host response to the cross-reactive antigens.
1. Kaklamani VG, Variopoulos G, Kaklamanis PG: Behçet's disease. Semin Arthritis Rheum 27:197-217, 1998.
2. Sakane T, Takeno M, Suzuki N, et al: Behçet's Disease. N Engl J Med 341:1284-1291, 1999.
3. Mizuki N, Inoko H, Mizuki N et al: Human leukocyte antigen serologic and DNA typing of Behçet's disease and its primary association with B51. Invest Ophthalmol Vis Sci 33:3332-3340, 1992.
4. Ohno S, Ohguchi M, Hirose S, et al: Close association of HLA-Bw51 with Behçet's disease. Arch Ophthalmol 100:1455-1458, 1982.
5. 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.
6. Isogai E, Ohno S, Takeshi K, et al: Close association of Streptococcus sanguis uncommon serotypes with Behçet's disease. Bifidobacteria Microflora 9:27-41, 1990.
7. Mizushima Y (The Behçet's Disease Research Committee of Japan): Skin hypersensitivity to Streptococcus sanguis and the induction of symptoms by the antigens in Behçet's disease: A multicenter study. J Rheumatol 16:506-511, 1989.
8. Eglin RP, Lehner T, Subak-Sharpe JH: Detection of RNA complementary to herpes simplex virus in mononuclear cells from patients with Behçet's syndrome and recurrent oral ulcers. Lancet 2:1356-1361, 1982.
9. Direskeneli H, Hasan A, Shinnick T, et al: Recognition of B-cell epitopes of the 65 kDa HSP in Behçet's disease. Scand J Immunol 43:464-471, 1996.
10. Hasan A, Fortune F, Wilson A, et al: Role of dT cells in the pathogenesis and diagnosis of Behçet's disease. Lancet 347:789-794.
11. Lehner T: The role of heat shock protein, microbial and autoimmune agents in the aetiology of Behçet's disease. Intern Rev Immunol 14:21-32, 1997.
12. Lehner T, Lavery E, Smith R, et al: Association between the 65-kilodalton heat shock protein, Streptococcus sanguis, and the corresponding antibodies in Behçet's disease. Infect Immun 59:1434-1441, 1991.
13. Tanaka T, Yamakawa N, Koike N, et al: Behcet's disease and antibody titers to various heat shock protein 60s. Ocular Immunol Inflamm 7:69-74, 1999.
14. De Smet MD, Ramadan A: Circulating antibodies to inducible heat shock protein 70 in patients with uveitis. Ocular Immunol Inflamm 9:85-92, 2001.
15. Ergun T, Ínce Ü, Eksioglu-Demiralp E, et al: HSP 60 expression in mucocutaneous lesions of Bechet's disease. J Am Acad Dermatol 45:904-909, 2001.
16. Zügel U, Kaufmann HE: Role of heat shock proteins in protection from and pathogenesis of infectious diseases. Clin Microbiol Rev 12:19-39, 1999.
17. Tanaka T, Yamakawa N, Yamaguchi H, et al: Common antigenicity between Yersinia enterocolitica-derived heat-shock protein and the retina, and its role in uveitis. Ophthalmic Res 28:284-288, 1996.
18. Yoshikawa K, Kotake S, Kubota T, et al: 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.
19. Xiang M, Zhou L, Peng Y, et al: Brn-3b: A POU domain gene expressed in a subset of retinal ganglion cells. Neuron 11:689-701, 1993.
20. Ishihara M, Ohno S, Ono H, et al: Seroprevalence of anti-Borrelia antibodies among patients with confirmed sarcoidosis in a region of Japan where Lyme borreliosis is endemic. Grafe's Arch Clin Exp Ophthlamol 236:280-284, 1998.
21. Isogai E, Isogai H, Kotake S, et al: Detection of antibodies against Borrelia burgdorferi in patients with uveitis. Am J Ophthalmol 112:23-30, 1991.
22. Pervin K, Chiderstone A, Shinnick T et al: T cell eptitope expression of mycobacterium and homologous human 65-kilodalton heat shock protein peptides in short term cell lines from patients with Behçet's disease. J Immunol 151:2273-2282, 1993.
23. Kaneko S, Suzuki N, Yamashita N, et al: Characterization of T cells specific for an epitope of human 60-kD heat shock protein (hsp) in patients with Behçet's disease. Clin Exp Immunol 108:204-212, 1997.
24. Ikushima M, Kawahashi S, Ohzeki Y, et al: A new specific serodiagnosis system for Lyme disease: Use of synthetic peptides derived from outer surface protein C of Borrelia burgdorferi. Opportunistic Pathogens 9:21-25, 1997.
25. Kaufman, SHE, Schoel B, Wand-Wurttenburger A, et al: T-cells, stress proteins, and pathogenesis of mycobacterial infections. Curr Trop Microbiol Immunol 155:125-141, 1990.
Table 1. Antibodies Against Synthetic Peptides in Patients with Behçet's Disease, Disease Controls and Healthy Controls
Group ELISA-positive individuals, n (%)
IgG IgM IgA
hsp Brn-3b11-25 Bes-1229-243 Brn-3b177-189 Bes-1373-385 hsp Brn-3b11-25 Bes-1229-243 Brn-3b177-189 Bes-1373-385 hsp Brn-3b11-25 Bes-1229-243 Brn-3b177-189 Bes-1373-385
Behçet's 15 15 18 14 1 3 2 2 2 2 16 1 3 0 0
disease (46.9) (46.9) (56.3) (43.8) (3.1) (9.4) (6.3) (6.3) (6.3) (6.3) (50.0) (3.2) (3.2) (0) (0)
Sarcoidosis 2 0 0 0 0 1 0 0 0 0 0 0 0 0 0
(10 patients) (20.0) (0) (0) (0) (0) (10.0) (0) (0) (0) (0) (0) (0) (0) (0) (0)
Lyme 8 0 0 0 0 7 0 0 0 0 0 0 0 0 0
disease (80.0) (0) (0) (0) (0) (70.0) (0) (0) (0) (0) (0) (0) (0) (0) (0)
Allergic 3 0 1 1 0 1 0 0 0 0 1 0 0 0 0
conjunctivitis (30.0) (0) (10.0) (10.0) (0) (10.0) (0) (0) (0) (0) (10.0) (0) (0) (0) (0)
Adenvirus 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
conjunctivitis (0) (0) (0) (0) (0) (10.0) (0) (0) (0) (0) (0) (0) (0) (0) (0)
Healthy 1 2 2 3 1 0 2 2 1 1 0 0 0 0 0
controls (2.4) (4.9) (4.9) (7.3) (2.4) (0) (4.9) (4.9) (2.4) (2.4) (0) (0) (0) (0) (0)
Hsp, human hsp336-351 (QPHDLGKVGEVIVTKDD), No.1: Brn-3b11-25 (AFSMPHGGSLHVEPK), No.2: Bes-1229-243 (AFIVPHGGHFHYIPK), No.3: Brn-3b177-189 (HHHHHHHQPHQAL), No.4: Bes-1373-385 (HGDHHHFIPYDKL).
Table 2. Level of Antibodies Against Synthetic Peptides in Patients with Behçet's Disease, Disease Controls, and Healthy Controls
IgG IgM IgA
hsp Brn-3b11-25 Bes-1229-243 Brn-3b177-189 Bes-1373-385 hsp Brn-3b11-25 Bes-1229-243 Brn-3b177-189 Bes-1373-385 hsp Brn-3b11-25 Bes-1229-243 Brn-3b177-189 Bes-1373-385
Behçet's 0.598 1.285 1.715 1.072 0.372 0.386 0.377 0.499 0.346 0.350 0.220 0.192 0.190 0.209 0.313
disease ± 0.241 ± 0.315 ± 0.101 ± 0.108 ± 0.037 ± 0.031 ± 0.043 ± 0.054 ± 0.049 ± 0.041 ± 0.011 ± 0.010 ± 0.015 ± 0.008 ± 0.008
Sarcoidosis 0.502 0.713 0.966 0.598 0.375 0.395 0.385 0.501 0.372 0.351 0.152 0.238 0.182 0.236 0.309
± 0.115 ± 0.101 ± 0.123 ± 0.104 ± 0.065 ± 0.063 ± 0.042 ± 0.055 ± 0.041 ± 0.043 ± 0.017 ± 0.035 ± 0.053 ± 0.035 ± 0.041
Lyme 0.681 0.732 0.956 0.535 0.418 0.529 0.412 0.461 0.366 0.347 0.150 0.185 0.154 0.222 0.312
disease ± 0.052 ± 0.112 ± 0.138 ± 0.056 ± 0.047 ± 0.036 ± 0.085 ± 0.056 ± 0.040 ± 0.053 ± 0.022 ± 0.016 ± 0.019 ± 0.056 ± 0.033
Allergic .514 0.574 0.885 0.575 0.469 0.416 0.560 0.570 0.384 0.410 0.152 0.207 0.150 0.204 0.271
conjunctivitis ± 0.047 ± 0.091 ± 0.139 ± 0.099 ± 0.092 ± 0.038 ± 0.078 ± 0.072 ± 0.057 ± 0.058 ± 0.020 ± 0.019 ± 0.024 ± 0.024 ± 0.020
Adenvirus 0.333 0.435 0.650 0.672 0.295 0.259 0.373 0.363 0.237 0.239 0.134 0.200 0.153 0.198 0.278
Healthy 0.295 0.655 0.895 0.508 0.327 0.340 0.390 0.523 0.379 0.384 0.157 0.264 0.167 0.245 0.333
controls ± 0.017 ± 0.050 ± 0.064 ± 0.045 ± 0.035 ± 0.025 ± 0.033 ± 0.045 ± 0.038 ± 0.035 ± 0.034 ± 0.057 ± 0.056 ± 0.077 ± 0.079
* Significantly higher than healthy controls (P<0.05)
Table 3. Correlation Among the Antibody Responses to Cross-Reactive Epitopes on the hsp and Retinal Ganglion Cell Protein Homologous of S. Sanguis
hsp336-351 Brn-3b11-25 Bes-1229-243 Brn-3b177-189 Bes-1373-385
Brn-3b 11-25 0.570* 1.000
Bes-1229-243 0.704* 0.652* 1.000
Brn-3b177-189 0.581** 0.680* 0.724* 1.000
Bes-1373-385 0.706* 0.586** 0.622* 0.861* 1.000
Brn-3b11-25 0.275 1.000
Bes-1229-243 0.808* 0.346 1.000
Brn-3b177-189 0.802* 0.249 0.947* 1.000
Bes-1373-385 0.831* 0.276 0.964* 0.947* 1.000
Brn-3b11-25 0.447** 1.000
Bes-1229-243 0.104 0.283 1.000
Brn-3b177-189 0.421** 0.458** 0.120 1.000
Bes-1373-385 0.341 0.202 0.480** 0.248 1.000
* P < 0.01.
** P < 0.05.
Figure 1. Distribution of antibody-positive subjects in the IgG response against hsp336-351. Gray bar: healthy controls; black bar: patients with BD.
©2000-2013. All Rights Reserved. Veterinary Solutions LLC