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Increased proportion of CD3+CD4−CD8− double-negative T cells in peripheral blood of children with Behcet's disease
Autoimmunity Reviews 6 (2007) 237 – 240 www.elsevier.com/locate/autrev
A meeting review
Increased proportion of CD3+CD4−CD8− double-negative T cells in peripheral blood of children with Behcet's disease Eduard Ling a,⁎, George Shubinsky b,c , Joseph Press a a Division of Pediatrics, Soroka University Medical Center, Rager Ave, Beer Sheva 84105, Israel Flow Cytometry Unit, Hematology Laboratory, Soroka University Medical Center, Beer Sheva, Israel Department of Microbiology and Immunology, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel b
c
Available online 28 August 2006
Abstract Introduction: Behcet's disease (BD) is a multi-system inflammatory disorder of poorly understood pathogenesis, which is characterized by oral aphtosis, genital ulcers and uveitis. Objective: To assess the role of CD3+CD4−CD8− double negative (DN) T cells in pathogenesis of Behcet's disease. Patients: Ten BD patients (age 12.2 ± 2.2 years, 7 in remission, 3 in exacerbation state) treated at the Pediatric Rheumatology unit of Soroka University Medical Center and 3 age-matched controls participated in the study. Methods: Peripheral blood lymphocytes of study subjects were isolated and stained with fluorescein-labeled anti-CD45, CD3, CD4, CD8 antibodies and analyzed by FACS assay. Results: Proportion of CD4−CD8− DN T cells was significantly increased in BD patients (n = 10) as compared to healthy controls (6.2 ± 3.4% vs. 3.2 ± 1.1% of total CD3+ cells, p b 0.05), this cell group was additionally enhanced in BD exacerbation, compared to patients in remission (10 ± 4.1% vs. 4.7 ± 1.2%, p b 0.05, respectively). DN T cells were significantly increased in BD patients in remission, compared to healthy controls (4.7 + 1.2% vs. 3.2 + 1.1% of total CD3+ cells, p b 0.05, respectively). Conclusions: Behcet's disease is characterized by increased proportion of CD3+CD4−CD8− double negative T cells in peripheral blood. Further studies, that include additional immunophenotyping and analysis of gene expression, aimed at characterization of these cells are currently underway. © 2006 Elsevier B.V. All rights reserved. Keywords: Behcet's disease; T cell; CD4−CD8− double negative
Contents 1. 2.
Introduction . . . . . Patients and methods . 2.1. Patients . . . . 2.2. Flow cytometry
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⁎ Corresponding author. Tel.: +972 8 640 0140; fax: +972 8 640 3089. E-mail address: [email protected] (E. Ling). 1568-9972/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2006.08.008
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3. Results . . . . . 4. Discussion. . . . Take-home messages . Acknowledgement . . References . . . . . .
E. Ling et al. / Autoimmunity Reviews 6 (2007) 237–240
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1. Introduction
2. Patients and methods
Behcet's disease (BD) is a multisystem inflammatory disorder with neural, vascular and articular manifestations which is classically characterized by a triad of symptoms that includes oral aphtosis, genital ulcers and uveitis and is mediated by occlusive vasculitis [1]. In Israel prevalence of BD is 12 to 10,000 and is presumably similar between Arab and Jewish populations [2] and between adults and children [3]. BD has been associated with various HLA genotypes, with HLA-B5 or B51 being most common among patients of Middle Eastern origin [4] and HLA-B12 and HLA-DR7 characteristic to BD patients residing in the UK and USA [5]. The tight association of BD with specific HLA genotypes gave rise to theories, suggesting the infectious nature of this disorder. Indeed, BD patients develop exaggerated cytokine and antibody responses to microbial HSP-60/65, certain Streptococcus spp. and Saccharomyces cerevisiae antigens [6–8]. HSV-1 infection of C57BL mice resulted in development of clinical and histologic signs specific for BD [9]. BD is characterized by multiple immune alterations. CD4+ cells recognizing retinal-S autoantigen were discovered in BD [10]. The presence of these cells might be a key factor in development of BD uveitis. Another autoantigen discovered in BD is α-tropomyosin [11]. Pathogenetical significance of autoimmune reaction to this protein remains to be elucidated. Immune response in Behcet's disease is skewed toward Th1 path with markedly increased serum levels of IL-2, IL-6, IL-8, IL12, TNF-α and IFN-γ [12]. T cells depletion in BD patients following administration of CAMPATH-1 antiCD52 monoclonal antibody resulted in rapid significant improvement of their clinical state, indicating crucial role of T cells in BD pathogenesis [13]. Analysis of T cell subsets in peripheral blood of BD patients revealed expansion of CD14+, CD8+/γδ, CD3+/γδ, CD56+/CD16+, CD4+/CD25+ and CD8+/ CD25+ cells [14]. Appearance of Behcet-like symptoms in a patient with CD4-lymphocytopenia was associated with accumulation of CD3+CD4−CD8−αβ+ doublenegative (DN) T cells [15]. In the present study we aimed to assess the role of CD3+CD4−CD8− DN T cells in pathogenesis of BD.
2.1. Patients
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239 239 239 240 240
Ten Behcet's disease patients, age 12.2 ± 2.2 years, 6 girls, 4 boys, treated at the Pediatric Rheumatology Unit of Soroka University Medical Center, Beer Sheva, Israel were enrolled in the study. Seven patients presented in remission, three patients presented in exacerbation of BD, manifested as oral aphtosis. Patients were diagnosed according to International Study Group Criteria (1990). The control group consisted of 3 age- and gendermatched children treated from non-inflammatory and non-infectious diseases at the Pediatric Emergency Room of the same institution. The study was approved by Institutional Ethics Committee of Ben-Gurion University, informed written consent was obtained from parents of each patient prior to enrollment into the study. 2.2. Flow cytometry Different subsets of T cells were counted in the sample of peripheral blood stained with FITC-conjugated monoclonal antibodies (Mab) against CD8, PEconjugated anti-CD4, ECD-conjugated anti-CD3 (both Dako-Cytomation A/S, Glostrup, Denmark), and PerCP-conjugated anti-CD45 Mab (BD Biosciences Becton, Dickinson and Company, San Jose, CA). T cells with different types of T-cell receptor (TCR) were counted in the samples stained with anti-TCRγ/δFITC, anti-TCRa/β-PE, anti-CD3-ECD (Immunotech, A Beckman Coulter Company, Marseille, France), and the mixture of PE-Cy5 conjugated anti-CD4 and anti-CD8 Mab. After staining with Mab, the RBC were removed from the blood samples by IQP Lysis reagent (IQ Products, Groningen, The Netherlands). The stained cells were analysed using EPICS XL-MCL flow cytometer (Beckman Coulter, Miami, FL) equipped with EXPO-32 ADC software. The cells were separated from cell debris by electronic gating on a side scatter (SS) vs. forward scatter (FS) plot, and lymphoid cells were gated on a SS vs. CD45 dot plot according to their typical properties. The fluorescence of cells stained with FITC-conjugated (FL1), PE-conjugated (FL2), ECD-conjugated (FL3), and TC (PerCP)-conjugated (FL4) Mab was measured
E. Ling et al. / Autoimmunity Reviews 6 (2007) 237–240
using logarithmic amplification. The samples of cells stained with irrelevant isotype-matched fluorescent antibodies were used to determine the level of background fluorescence. Percentages of positive cells with the fluorescence above the background level were evaluated for each specific Mab. Amounts of CD4+, CD8+, CD4+ CD8+ double-positive, and CD4−CD8− double-negative cells were calculated within gated CD3+ T lymphocytes. T lymphocytes with the different types of TCR were counted within gated CD3+ and CD4−CD8− cells. 3. Results Measurement of different subsets of T cells in peripheral blood of the children with BD showed that the proportion of CD3+CD4−CD8− T cells was significantly increased compared to control group children. Significant increase of CD4−CD8− DN T cells population was found in all (active disease and remission) BD patients as compared to healthy controls (6.2 ± 3.4% vs. 3.2 ± 1.1% of total CD3+ cells, p b 0.05). CD3+CD4−CD8− T cells population was significantly enhanced in patients with active disease, as compared with control group (10 ± 4.1% vs. 3.2 ± 1.1%, p b 0.05, respectively). Likewise, proportion of CD4−CD8− DN T cells population was significantly increased in patients with active disease (n = 3) compared to patients in remission (n = 7) — 10 ± 3.33% vs. 4.7 ± 1.2%, p b 0.05, respectively. CD4−CD8− DN T cells population was significantly increased in patients in remission, as compared with control group (4.7 ± 1.2% vs. 3.2 ± 1.1%, respectively, p b 0.05). No difference in expression of CD4+ and CD8+ cells between the study groups was found (Table 1). In a part of patients (n = 5, 1 healthy control and 4 in remission) we performed staining of CD3+ DN cells for αβ- and γδ-TCR. αβ-TCR positive cells constituted 26.3% of CD3+ DN cells, while γδ-TCR positive cells constituted remaining 73.7% (not shown). 4. Discussion In the present study we demonstrated increased proportion of CD3+CD4−CD8− DN T cells in peripheral
CD4+
CD8+
CD4+CD8+
CD4−CD8−
53.3 ± 5.7 51.2 ± 5.6 51.9 ± 4.1 58.1 ± 12.9
38.3 ± 7.9 40.6 ± 9.3 36.5 ± 8.8 41 ± 13.1
0.8 ± 0.6 1 ± 0.7 0.6 ± 0.1 0.7 ± 0.3
6.2 ± 3.4* 4.7 ± 1.2*⊥| 10 ± 4.1*⊥| 3.2 ± 1.1*⊥|
*p b 0.05, ⊥p b 0.05, |p b 0.05, Student's t-test.
blood of pediatric BD patients. Our data indicate that in BD patients both αβ- and γδ-positive populations of DN T cells are elevated. Our results, demonstrating increased content of DN γδ-T cells in BD patients are in accordance with recently reported expansion of γδ-T cells reactive to microbial flora colonizing the oral ulcers in peripheral blood of BD patients [16]. Of particular interest is αβ-TCR expressing DN T cells population which was also found to be relatively increased in BD patients in our study. CD3+CD4−CD8− αβ+ DN cells belong to a group of regulatory T cells (Tregs) that eliminate the undesired effector clone via Fas/FasL mediated apoptosis [17]. Resistance of lymphocytes to Fas-mediated apoptosis is a known feature of BD [18]. We speculate that DN Tregs observed in the present study accumulate in an attempt to control the pathological effector clone (CD8+ or DN γδ-T cells?). The tissue damage, observed in BD, might be inflicted by FasL-bearing Tregs as a result of a “bystander effect”. Indeed, accumulation of FasL bearing lymphocytes has been observed in skin lesions of BD patients [19]. Separate populations of CD3+CD4−CD8− DN cells are involved in antiviral and antibacterial defense [20,21]. One might propose that DN CD3+ cells accumulated in BD represent a missing link between infection and BD. Our results demonstrate that αβ+ DN T cells comprise approximately 2% of all circulating T cells in BD patients during exacerbation of the disease. This observation, coupled with earlier reports of lymphocytes resistance to Fas-mediated apoptosis and elevated cancer risk [22] in BD tempt one to entertain a possibility of a link between BD and autoimmune lymphoproliferative syndrome (ALPS). As a matter of fact, the mentioned above features of BD comply with the ALPS diagnostic criteria: αβ+ DN T cells comprise N 1% of all circulating T cells, disordered Fas-mediated apoptosis and propensity to malignancy [23]. In conclusion, the results of present study for the first time demonstrate possible involvement of CD4−CD8− double-negative Tcells in pathogenesis of Behcet's disease. It is noteworthy, however, that this was a preliminary report based on relatively few observations and a larger number of patients is required to confirm the present findings. Take-home messages
Table 1 Percentage of T cells with different surface markers
All patients Remission Exacerbation Control
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• BD is an autoinflammatory occlusive vasculitis • Behcet's disease is characterized by abnormal lymphocyte function • CD3+CD4−CD8− DN T cells accumulate in Behcet's disease, indicating their involvement in pathogenesis of this disease
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E. Ling et al. / Autoimmunity Reviews 6 (2007) 237–240
Acknowledgement The study was supported by the Grant from Residents' Fund, Faculty of Health Sciences, Ben Gurion University of the Negev to EL. References [1] Michealson JB, Chisari FV. Behcet's disease. Surv Ophtalmol 1982;20:19–203. [2] Krause I, Mader R, Sulkes J, Paul M, Uziel Y, Adawi M, et al. Behcet's disease in Israel: the influence of ethnic origin on disease expression and severity. J Rheumatol 2001;28:1033–6. [3] Uziel Y, Brik R, Padeh S, Barash J, Mukamel M, Harel L, et al. Juvenile Behcet's disease in Israel. Clin Exp Rheumatol 1998; 16:502–5. [4] Brautbar C, Chajek T, Ben-Tuvia S, Lamm L, Cohen T. A genetic study of Behcet disease in Israel. Tissue Antigens 1978;11:113–20. [5] Hegab S, Al-Mutawa S. Immunopathogenesis of Behcet's disease. Clin Immunol 2000;96:174–86. [6] Direskeneli H, Eksioglu-Demiralp E, Yavuz S, Ergun T, Shinnick T, Lehner T, et al. T cell responses to 60/65 heat shock protein protein derived peptides in Turkish patients with Behcet's disease. J Rheumatol 2000;27:708–13. [7] Cuchacovich M, Merina G, Yamamoto JH, Villarroel F, Saavedra T, Jofre S, et al. Behcet's disease patients present high levels of deglycosylated anti-lipoteichoic acid acid IgG and high IL8 production after lipoteichoic acid stimulation. Clin Exp Rheumatol 2005;23:S27–34. [8] Fresko I, Ugurlu S, Ozbakir F, Celik A, Yurdakul S, Hamuryudan V, et al. Anti-Saccharomyces cerevisiae antibodies (ASCA) in Behcet's syndrome. Clin Exp Rheumatol 2005;23:S67–70. [9] Sohn S, Lee E-S, Lee S. The correlation of MHC haplotype and development of Behcet's disease-like symptoms induced by herpes simplex virus in several inbred mice strains. J Dermatol Sci 2001;26:173–81. [10] Yamamoto JH, Fujino Y, Lin C, Nieda M, Juji T, Masuda K. S-antigen specific T cell clones from a patient with Behcet's disease. Br J Ophthalmol 1994;78:927–32. [11] Mahesh SP, Li Z, Buggage R, Mor F, Cohen IR, Chew EY, et al. α-Tropomyosin as a self-antigen in patients with Behcet's disease. Clin Exp Immunol 2005;140:368–75.
[12] Ben Ahmed M, Houman H, Miled M, Dellagi K, Louzir H. Involvement of chemokines and Th1 cytokines in the pathogenesis of mucocutaneous lesions of Behcet's disease. Arthritis Rheum 2004;50:2291–5. [13] Lockwood C, Hale G, Waldman H, Jayne D. Remission induction in Behcet's disease following lymphocyte depletion by the antiCD52 antibody CAMPATH-1. Rheumatology 2003;42:1539–44. [14] Treusch M, Vonthein R, Baur M, Günaydin I, Koch S, Stübiger N, et al. Influence of human recombinant interferon-α2a on altered lymphocyte subpopulations and monocytes in Behcet's disease. Rheumatology 2004;43:1275–82. [15] Venzor J, Hua Q, Bressler RB, Miranda CH, Huston DP. Behcet'slike syndrome associated with idiopathic CD4+ T-lymphocytopenia, opportunistic infections, and a large population of TCR αβ+ CD4− CD8− T cells. Am J Med Sci 1997;313:236–8. [16] Bank I, Duvdevani M, Livneh A. Expansion of γδ T-cells in Behcet's disease: role of disease activity and microbial flora. J Lab Clin Med 2003;141:33–9. [17] Fisher K, Voelkl S, Heymann J, Przybylski G, Mondal K, Laumer M, et al. Isolation and characterization of human antigen-specific TCRab+CD4−CD8− double-negative regulatory T cells. Blood 2005;105:2828–35. [18] Todaro M, Zerilli M, Triolo G, Iovino F, Patti M, AccardoPalumbo A, et al. NF-kB protects Behcet's disease T cells against CD-95 induced apoptosis up-regulating antiapoptotic proteins. Arthritis Rheum 2005;52:2179–91. [19] Wakisaka S, Takeba Y, Mihara S, Takeno M, Yamamoto S, Sakane T, et al. Aberrant Fas ligand expression in lymphocytes in patients with Behcet's disease. Int Arch Allergy Immunol 2002; 129:175–80. [20] Hossain M, Takimoto H, Ninomiya T, Yoshida H, Kishihara K, Matsuzaki G, et al. Characterization of CD4−CD8−CD3+ T-cell receptor-αβ+ T cells in murine cytomegalovirus infection. Immunology 2000;101:19–29. [21] Cowley S, Hamilton E, Frelinger J, Su J, Forman J, Elkins K. CD4−CD8− T cells control intracellular bacterial infections both in vitro and in vivo. JEM 2005;202:309–19. [22] Cengiz M, Altundag M, Zorlu A, Gűllű I, Özyar E, Atahan I. Malignancy in Behcet's disease: a report of 13 cases and a review of the literature. Clin Rheumatol 2001;20:239–44. [23] Holzelova E, Vonarbourg C, Stolzenberg MC, Arkwright PD, Selz F, Prieur AM, et al. Autoimmune lymphoproliferative syndrome with somatic Fas mutations. N Engl J Med 2004;351:1409–18.
Anti-signal recognition particle autoantibodies: marker of a necrotizing myopathy To elucidate the clinical importance of the anti-signal recognition particle (SRP) autoantibody in patients with myositis, Hengstman GJ. et. al. (Ann Rheum Dis 2006; 65: 1635-8) analyzed retrospective systemic assessment of the clinical, laboratory and histological characteristics of 23 anti-SRP-positive patients from six European centers. Data were compared with a large group of anti-SRP-negative patients with myositis published previously. Clinically, patients with anti-SRP autoantibodies often had a severe symmetric proximal muscle weakness resulting in marked disability, dysphagia and highly elevated levels of serum creatine kinase. Three patients had typical dermatomyositis rashes. The disease was associated with the occurrence of extramuscular signs and symptoms including interstitial lung disease. No association was found with an increased risk of cardiac involvement, and the disease carried a reasonably favorable prognosis with most patients responding to treatment. None of those patients had the typical histological features of myositis. Most muscle biopsy specimens showed the presence of necrotic muscle fibers and no inflammatory infiltrates. Thus, anti-SRP autoantibodies are associated with a syndrome of a necrotizing myopathy in the spectrum of immune-mediated myopathies that differs from typical polymyositis.
A meeting review
Increased proportion of CD3+CD4−CD8− double-negative T cells in peripheral blood of children with Behcet's disease Eduard Ling a,⁎, George Shubinsky b,c , Joseph Press a a Division of Pediatrics, Soroka University Medical Center, Rager Ave, Beer Sheva 84105, Israel Flow Cytometry Unit, Hematology Laboratory, Soroka University Medical Center, Beer Sheva, Israel Department of Microbiology and Immunology, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel b
c
Available online 28 August 2006
Abstract Introduction: Behcet's disease (BD) is a multi-system inflammatory disorder of poorly understood pathogenesis, which is characterized by oral aphtosis, genital ulcers and uveitis. Objective: To assess the role of CD3+CD4−CD8− double negative (DN) T cells in pathogenesis of Behcet's disease. Patients: Ten BD patients (age 12.2 ± 2.2 years, 7 in remission, 3 in exacerbation state) treated at the Pediatric Rheumatology unit of Soroka University Medical Center and 3 age-matched controls participated in the study. Methods: Peripheral blood lymphocytes of study subjects were isolated and stained with fluorescein-labeled anti-CD45, CD3, CD4, CD8 antibodies and analyzed by FACS assay. Results: Proportion of CD4−CD8− DN T cells was significantly increased in BD patients (n = 10) as compared to healthy controls (6.2 ± 3.4% vs. 3.2 ± 1.1% of total CD3+ cells, p b 0.05), this cell group was additionally enhanced in BD exacerbation, compared to patients in remission (10 ± 4.1% vs. 4.7 ± 1.2%, p b 0.05, respectively). DN T cells were significantly increased in BD patients in remission, compared to healthy controls (4.7 + 1.2% vs. 3.2 + 1.1% of total CD3+ cells, p b 0.05, respectively). Conclusions: Behcet's disease is characterized by increased proportion of CD3+CD4−CD8− double negative T cells in peripheral blood. Further studies, that include additional immunophenotyping and analysis of gene expression, aimed at characterization of these cells are currently underway. © 2006 Elsevier B.V. All rights reserved. Keywords: Behcet's disease; T cell; CD4−CD8− double negative
Contents 1. 2.
Introduction . . . . . Patients and methods . 2.1. Patients . . . . 2.2. Flow cytometry
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⁎ Corresponding author. Tel.: +972 8 640 0140; fax: +972 8 640 3089. E-mail address: [email protected] (E. Ling). 1568-9972/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2006.08.008
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3. Results . . . . . 4. Discussion. . . . Take-home messages . Acknowledgement . . References . . . . . .
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1. Introduction
2. Patients and methods
Behcet's disease (BD) is a multisystem inflammatory disorder with neural, vascular and articular manifestations which is classically characterized by a triad of symptoms that includes oral aphtosis, genital ulcers and uveitis and is mediated by occlusive vasculitis [1]. In Israel prevalence of BD is 12 to 10,000 and is presumably similar between Arab and Jewish populations [2] and between adults and children [3]. BD has been associated with various HLA genotypes, with HLA-B5 or B51 being most common among patients of Middle Eastern origin [4] and HLA-B12 and HLA-DR7 characteristic to BD patients residing in the UK and USA [5]. The tight association of BD with specific HLA genotypes gave rise to theories, suggesting the infectious nature of this disorder. Indeed, BD patients develop exaggerated cytokine and antibody responses to microbial HSP-60/65, certain Streptococcus spp. and Saccharomyces cerevisiae antigens [6–8]. HSV-1 infection of C57BL mice resulted in development of clinical and histologic signs specific for BD [9]. BD is characterized by multiple immune alterations. CD4+ cells recognizing retinal-S autoantigen were discovered in BD [10]. The presence of these cells might be a key factor in development of BD uveitis. Another autoantigen discovered in BD is α-tropomyosin [11]. Pathogenetical significance of autoimmune reaction to this protein remains to be elucidated. Immune response in Behcet's disease is skewed toward Th1 path with markedly increased serum levels of IL-2, IL-6, IL-8, IL12, TNF-α and IFN-γ [12]. T cells depletion in BD patients following administration of CAMPATH-1 antiCD52 monoclonal antibody resulted in rapid significant improvement of their clinical state, indicating crucial role of T cells in BD pathogenesis [13]. Analysis of T cell subsets in peripheral blood of BD patients revealed expansion of CD14+, CD8+/γδ, CD3+/γδ, CD56+/CD16+, CD4+/CD25+ and CD8+/ CD25+ cells [14]. Appearance of Behcet-like symptoms in a patient with CD4-lymphocytopenia was associated with accumulation of CD3+CD4−CD8−αβ+ doublenegative (DN) T cells [15]. In the present study we aimed to assess the role of CD3+CD4−CD8− DN T cells in pathogenesis of BD.
2.1. Patients
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Ten Behcet's disease patients, age 12.2 ± 2.2 years, 6 girls, 4 boys, treated at the Pediatric Rheumatology Unit of Soroka University Medical Center, Beer Sheva, Israel were enrolled in the study. Seven patients presented in remission, three patients presented in exacerbation of BD, manifested as oral aphtosis. Patients were diagnosed according to International Study Group Criteria (1990). The control group consisted of 3 age- and gendermatched children treated from non-inflammatory and non-infectious diseases at the Pediatric Emergency Room of the same institution. The study was approved by Institutional Ethics Committee of Ben-Gurion University, informed written consent was obtained from parents of each patient prior to enrollment into the study. 2.2. Flow cytometry Different subsets of T cells were counted in the sample of peripheral blood stained with FITC-conjugated monoclonal antibodies (Mab) against CD8, PEconjugated anti-CD4, ECD-conjugated anti-CD3 (both Dako-Cytomation A/S, Glostrup, Denmark), and PerCP-conjugated anti-CD45 Mab (BD Biosciences Becton, Dickinson and Company, San Jose, CA). T cells with different types of T-cell receptor (TCR) were counted in the samples stained with anti-TCRγ/δFITC, anti-TCRa/β-PE, anti-CD3-ECD (Immunotech, A Beckman Coulter Company, Marseille, France), and the mixture of PE-Cy5 conjugated anti-CD4 and anti-CD8 Mab. After staining with Mab, the RBC were removed from the blood samples by IQP Lysis reagent (IQ Products, Groningen, The Netherlands). The stained cells were analysed using EPICS XL-MCL flow cytometer (Beckman Coulter, Miami, FL) equipped with EXPO-32 ADC software. The cells were separated from cell debris by electronic gating on a side scatter (SS) vs. forward scatter (FS) plot, and lymphoid cells were gated on a SS vs. CD45 dot plot according to their typical properties. The fluorescence of cells stained with FITC-conjugated (FL1), PE-conjugated (FL2), ECD-conjugated (FL3), and TC (PerCP)-conjugated (FL4) Mab was measured
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using logarithmic amplification. The samples of cells stained with irrelevant isotype-matched fluorescent antibodies were used to determine the level of background fluorescence. Percentages of positive cells with the fluorescence above the background level were evaluated for each specific Mab. Amounts of CD4+, CD8+, CD4+ CD8+ double-positive, and CD4−CD8− double-negative cells were calculated within gated CD3+ T lymphocytes. T lymphocytes with the different types of TCR were counted within gated CD3+ and CD4−CD8− cells. 3. Results Measurement of different subsets of T cells in peripheral blood of the children with BD showed that the proportion of CD3+CD4−CD8− T cells was significantly increased compared to control group children. Significant increase of CD4−CD8− DN T cells population was found in all (active disease and remission) BD patients as compared to healthy controls (6.2 ± 3.4% vs. 3.2 ± 1.1% of total CD3+ cells, p b 0.05). CD3+CD4−CD8− T cells population was significantly enhanced in patients with active disease, as compared with control group (10 ± 4.1% vs. 3.2 ± 1.1%, p b 0.05, respectively). Likewise, proportion of CD4−CD8− DN T cells population was significantly increased in patients with active disease (n = 3) compared to patients in remission (n = 7) — 10 ± 3.33% vs. 4.7 ± 1.2%, p b 0.05, respectively. CD4−CD8− DN T cells population was significantly increased in patients in remission, as compared with control group (4.7 ± 1.2% vs. 3.2 ± 1.1%, respectively, p b 0.05). No difference in expression of CD4+ and CD8+ cells between the study groups was found (Table 1). In a part of patients (n = 5, 1 healthy control and 4 in remission) we performed staining of CD3+ DN cells for αβ- and γδ-TCR. αβ-TCR positive cells constituted 26.3% of CD3+ DN cells, while γδ-TCR positive cells constituted remaining 73.7% (not shown). 4. Discussion In the present study we demonstrated increased proportion of CD3+CD4−CD8− DN T cells in peripheral
CD4+
CD8+
CD4+CD8+
CD4−CD8−
53.3 ± 5.7 51.2 ± 5.6 51.9 ± 4.1 58.1 ± 12.9
38.3 ± 7.9 40.6 ± 9.3 36.5 ± 8.8 41 ± 13.1
0.8 ± 0.6 1 ± 0.7 0.6 ± 0.1 0.7 ± 0.3
6.2 ± 3.4* 4.7 ± 1.2*⊥| 10 ± 4.1*⊥| 3.2 ± 1.1*⊥|
*p b 0.05, ⊥p b 0.05, |p b 0.05, Student's t-test.
blood of pediatric BD patients. Our data indicate that in BD patients both αβ- and γδ-positive populations of DN T cells are elevated. Our results, demonstrating increased content of DN γδ-T cells in BD patients are in accordance with recently reported expansion of γδ-T cells reactive to microbial flora colonizing the oral ulcers in peripheral blood of BD patients [16]. Of particular interest is αβ-TCR expressing DN T cells population which was also found to be relatively increased in BD patients in our study. CD3+CD4−CD8− αβ+ DN cells belong to a group of regulatory T cells (Tregs) that eliminate the undesired effector clone via Fas/FasL mediated apoptosis [17]. Resistance of lymphocytes to Fas-mediated apoptosis is a known feature of BD [18]. We speculate that DN Tregs observed in the present study accumulate in an attempt to control the pathological effector clone (CD8+ or DN γδ-T cells?). The tissue damage, observed in BD, might be inflicted by FasL-bearing Tregs as a result of a “bystander effect”. Indeed, accumulation of FasL bearing lymphocytes has been observed in skin lesions of BD patients [19]. Separate populations of CD3+CD4−CD8− DN cells are involved in antiviral and antibacterial defense [20,21]. One might propose that DN CD3+ cells accumulated in BD represent a missing link between infection and BD. Our results demonstrate that αβ+ DN T cells comprise approximately 2% of all circulating T cells in BD patients during exacerbation of the disease. This observation, coupled with earlier reports of lymphocytes resistance to Fas-mediated apoptosis and elevated cancer risk [22] in BD tempt one to entertain a possibility of a link between BD and autoimmune lymphoproliferative syndrome (ALPS). As a matter of fact, the mentioned above features of BD comply with the ALPS diagnostic criteria: αβ+ DN T cells comprise N 1% of all circulating T cells, disordered Fas-mediated apoptosis and propensity to malignancy [23]. In conclusion, the results of present study for the first time demonstrate possible involvement of CD4−CD8− double-negative Tcells in pathogenesis of Behcet's disease. It is noteworthy, however, that this was a preliminary report based on relatively few observations and a larger number of patients is required to confirm the present findings. Take-home messages
Table 1 Percentage of T cells with different surface markers
All patients Remission Exacerbation Control
239
• BD is an autoinflammatory occlusive vasculitis • Behcet's disease is characterized by abnormal lymphocyte function • CD3+CD4−CD8− DN T cells accumulate in Behcet's disease, indicating their involvement in pathogenesis of this disease
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Acknowledgement The study was supported by the Grant from Residents' Fund, Faculty of Health Sciences, Ben Gurion University of the Negev to EL. References [1] Michealson JB, Chisari FV. Behcet's disease. Surv Ophtalmol 1982;20:19–203. [2] Krause I, Mader R, Sulkes J, Paul M, Uziel Y, Adawi M, et al. Behcet's disease in Israel: the influence of ethnic origin on disease expression and severity. J Rheumatol 2001;28:1033–6. [3] Uziel Y, Brik R, Padeh S, Barash J, Mukamel M, Harel L, et al. Juvenile Behcet's disease in Israel. Clin Exp Rheumatol 1998; 16:502–5. [4] Brautbar C, Chajek T, Ben-Tuvia S, Lamm L, Cohen T. A genetic study of Behcet disease in Israel. Tissue Antigens 1978;11:113–20. [5] Hegab S, Al-Mutawa S. Immunopathogenesis of Behcet's disease. Clin Immunol 2000;96:174–86. [6] Direskeneli H, Eksioglu-Demiralp E, Yavuz S, Ergun T, Shinnick T, Lehner T, et al. T cell responses to 60/65 heat shock protein protein derived peptides in Turkish patients with Behcet's disease. J Rheumatol 2000;27:708–13. [7] Cuchacovich M, Merina G, Yamamoto JH, Villarroel F, Saavedra T, Jofre S, et al. Behcet's disease patients present high levels of deglycosylated anti-lipoteichoic acid acid IgG and high IL8 production after lipoteichoic acid stimulation. Clin Exp Rheumatol 2005;23:S27–34. [8] Fresko I, Ugurlu S, Ozbakir F, Celik A, Yurdakul S, Hamuryudan V, et al. Anti-Saccharomyces cerevisiae antibodies (ASCA) in Behcet's syndrome. Clin Exp Rheumatol 2005;23:S67–70. [9] Sohn S, Lee E-S, Lee S. The correlation of MHC haplotype and development of Behcet's disease-like symptoms induced by herpes simplex virus in several inbred mice strains. J Dermatol Sci 2001;26:173–81. [10] Yamamoto JH, Fujino Y, Lin C, Nieda M, Juji T, Masuda K. S-antigen specific T cell clones from a patient with Behcet's disease. Br J Ophthalmol 1994;78:927–32. [11] Mahesh SP, Li Z, Buggage R, Mor F, Cohen IR, Chew EY, et al. α-Tropomyosin as a self-antigen in patients with Behcet's disease. Clin Exp Immunol 2005;140:368–75.
[12] Ben Ahmed M, Houman H, Miled M, Dellagi K, Louzir H. Involvement of chemokines and Th1 cytokines in the pathogenesis of mucocutaneous lesions of Behcet's disease. Arthritis Rheum 2004;50:2291–5. [13] Lockwood C, Hale G, Waldman H, Jayne D. Remission induction in Behcet's disease following lymphocyte depletion by the antiCD52 antibody CAMPATH-1. Rheumatology 2003;42:1539–44. [14] Treusch M, Vonthein R, Baur M, Günaydin I, Koch S, Stübiger N, et al. Influence of human recombinant interferon-α2a on altered lymphocyte subpopulations and monocytes in Behcet's disease. Rheumatology 2004;43:1275–82. [15] Venzor J, Hua Q, Bressler RB, Miranda CH, Huston DP. Behcet'slike syndrome associated with idiopathic CD4+ T-lymphocytopenia, opportunistic infections, and a large population of TCR αβ+ CD4− CD8− T cells. Am J Med Sci 1997;313:236–8. [16] Bank I, Duvdevani M, Livneh A. Expansion of γδ T-cells in Behcet's disease: role of disease activity and microbial flora. J Lab Clin Med 2003;141:33–9. [17] Fisher K, Voelkl S, Heymann J, Przybylski G, Mondal K, Laumer M, et al. Isolation and characterization of human antigen-specific TCRab+CD4−CD8− double-negative regulatory T cells. Blood 2005;105:2828–35. [18] Todaro M, Zerilli M, Triolo G, Iovino F, Patti M, AccardoPalumbo A, et al. NF-kB protects Behcet's disease T cells against CD-95 induced apoptosis up-regulating antiapoptotic proteins. Arthritis Rheum 2005;52:2179–91. [19] Wakisaka S, Takeba Y, Mihara S, Takeno M, Yamamoto S, Sakane T, et al. Aberrant Fas ligand expression in lymphocytes in patients with Behcet's disease. Int Arch Allergy Immunol 2002; 129:175–80. [20] Hossain M, Takimoto H, Ninomiya T, Yoshida H, Kishihara K, Matsuzaki G, et al. Characterization of CD4−CD8−CD3+ T-cell receptor-αβ+ T cells in murine cytomegalovirus infection. Immunology 2000;101:19–29. [21] Cowley S, Hamilton E, Frelinger J, Su J, Forman J, Elkins K. CD4−CD8− T cells control intracellular bacterial infections both in vitro and in vivo. JEM 2005;202:309–19. [22] Cengiz M, Altundag M, Zorlu A, Gűllű I, Özyar E, Atahan I. Malignancy in Behcet's disease: a report of 13 cases and a review of the literature. Clin Rheumatol 2001;20:239–44. [23] Holzelova E, Vonarbourg C, Stolzenberg MC, Arkwright PD, Selz F, Prieur AM, et al. Autoimmune lymphoproliferative syndrome with somatic Fas mutations. N Engl J Med 2004;351:1409–18.
Anti-signal recognition particle autoantibodies: marker of a necrotizing myopathy To elucidate the clinical importance of the anti-signal recognition particle (SRP) autoantibody in patients with myositis, Hengstman GJ. et. al. (Ann Rheum Dis 2006; 65: 1635-8) analyzed retrospective systemic assessment of the clinical, laboratory and histological characteristics of 23 anti-SRP-positive patients from six European centers. Data were compared with a large group of anti-SRP-negative patients with myositis published previously. Clinically, patients with anti-SRP autoantibodies often had a severe symmetric proximal muscle weakness resulting in marked disability, dysphagia and highly elevated levels of serum creatine kinase. Three patients had typical dermatomyositis rashes. The disease was associated with the occurrence of extramuscular signs and symptoms including interstitial lung disease. No association was found with an increased risk of cardiac involvement, and the disease carried a reasonably favorable prognosis with most patients responding to treatment. None of those patients had the typical histological features of myositis. Most muscle biopsy specimens showed the presence of necrotic muscle fibers and no inflammatory infiltrates. Thus, anti-SRP autoantibodies are associated with a syndrome of a necrotizing myopathy in the spectrum of immune-mediated myopathies that differs from typical polymyositis.