J Oral Pathol Med (2014) 43: 691–695 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
doi: 10.1111/jop.12182
wileyonlinelibrary.com/journal/jop
Serum Th1, Th2 and Th17 cytokine profiles and alphaenolase levels in recurrent aphthous stomatitis Kemal Ozyurt1, Ahmet C ß elik2, Mehmet Sayarlıoglu3, Emine Colgecen4, Rahime Incı5, Tuba Karakas5, 6 Mehmet Kelles , Gozde Y. Cetin7 1
Dermatology Clinic, Kayseri Education and Research Hospital, Kayseri, Turkey; 2Department of Biochemistry, Sutcu Imam University, Medical School, Kahramanmaras, Turkey; 3Department of Rheumatology, Ondokuz Mayıs University, Medical School, Samsun, Turkey; 4 Department of Dermatology, Bozok University, Medical School, Yozgat, Turkey; 5Department of Dermatology, Sutcu Imam University, Medical School, Kahramanmaras, Turkey; 6Department of Otorhinolaryngology, Sutcu Imam University, Medical School, Kahramanmaras, Turkey; 7Department of Rheumatology, Sutcu Imam University, Medical School, Kahramanmaras, Turkey
BACKGROUND: All aspects of aetiopathogenesis of recurrent aphthous stomatitis (RAS) have not been elucidated. RAS and Behcßet’s disease (BD) have clinical and immunological characteristics in common. Although T17 cytokines and alpha-enolase have been shown to play effective roles in BD and many other autoinflammatory diseases recently, their roles in RAS have not been studied extensively. In the present study, we investigated levels of several Th1, Th2 and Th17 pathways related cytokines and alpha-enolase to elucidate pathogenesis of RAS and to obtain data about possible treatment alternatives for the condition. METHODS: Serum interleukin-1, interleukin-13, interleukin-17, interleukin-18, interferon gamma and alphaenolase levels in 24 patients with RAS, 30 patients with BD and 20 healthy controls were measured. RESULTS: Serum interleukin-1, interleukin-13, interleukin-17, interleukin-18, interferon gamma and alpha-enolase levels were higher in patients with RAS and patients with BD than in healthy controls (P < 0.005). CONCLUSION: Like Th1 and Th2 cells, Th17 cells were found to be effective in pathogenesis of RAS. In addition, alpha-enolase, the levels of which were high, may play an important role in etio-pathogenesis of RAS. Further studies to be designed in the light of these findings are required to shed light on pathogenesis and treatment of the condition. J Oral Pathol Med (2014) 43: 691–695 Keywords: alpha-enolase; Behcßet’s recurrent aphthous stomatitis; Th17
disease;
interleukin-17;
Correspondence: Dr Kemal Ozyurt, Dermatoloji Klinigi, Kayseri Egitim ve Arastırma Hastanesi, Melikgazi, Kayseri, Turkey. Tel: +903523368884, Fax: +903523368857, E-mail: [email protected] Accepted for publication February 11, 2014
Introduction Recurrent aphthous stomatitis (RAS) is a frequently encountered disease of the oral mucosa. Recurrent oral ulcers are the main diagnostic sign of Behcßet’s disease (BD). In addition to oral and genital ulcers, eyes, joints and the nervous system can also be involved in BD. Although genetic susceptibility, infections, various autoantibodies and immune complexes are incriminated for both RAS and BD, aetiopathogenesis in these conditions have not been understood completely yet (1–3). Cellular immunity involves an important part of pathogenesis in RAS and BD. CD4 + effector T cells in cellular immune responses can be divided into two, i.e. Th1 and Th2. Both groups of cells are known to be effective in RAS (3–5). In recent years, a new CD4 + cell which produces interleukin (IL)-17 and is called Th17 has attracted attention. In contrast to previous arguments, it has been understood that Th1 cells do not have a marked effect on autoimmune, allergic and microbial diseases and that IL-17 has a major role in these diseases. Based on experimental studies, it has been suggested that rheumatoid arthritis and multiple sclerosis are considered Th-17-related diseases, but not Th1-related conditions. IL-17 together with IL-21 and IL-22 controls immunity, inflammation and infections on mucosal surfaces and plays a role in vascular pathologies (6–10). The balance between Th17 and stimulated T regulatory (Treg) cells and cytokines like IL-23 and IL-17 have been shown to play an important role in inflammatory processes in BD, which have not been able to be explained by the Th1/Th2 paradigm (10–13). To our best knowledge, this is the first study to investigate Th17 cytokines in peripheral blood from cases of RAS. In this study, we examined IL-1, a natural cellular cytokine, IL-18 and and IFN-c, Th1 cytokines, and IL-13, a Th2 cytokine, in addition to IL-17, a Th17-related cytokine of cellular immunity in patients with RAS. We investigated these cytokines in patients with BD and healthy controls as well and attempted to study the role of Th17 in pathogenesis
Cytokine profiles in aphthous stomatitis Ozyurt et al.
692
of RAS. The fact that a Th17 cytokine, IL-17 is effective in mucosal immunity was the main factor directing us towards conducting this study (9, 14). In addition, it is striking that Th17 has a considerable role in BH, which has clinical and pathological aspects similar to RAS, and inflammatory diseases of the guts (15). Protein alpha-enolase (AE) is a plasminogen receptor on endothelial cell receptors and also exists on surfaces of streptococci and acts like a heat shock protein, which indicate a role of AE in autoimmune and inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, vaculitis and Crohn’s disease (16). Antiendothelial cell antibodies include a group of heterogeneous antibodies to various proteins on endothelial cell surfaces (17). Lee et al. (18) first reported that anti-AE antibody (AAEA), an anti-endothelial cell antibody, was found in sera from patients with BD and suggested that AAEA is a diagnostic marker for BD. To our knowledge, there have not been studies on AE in patients with RAS. AE can be an important marker for RAS which has the same immune-pathological features as many inflammatory diseases such as BH and Crohn’s disease.
Materials and methods Sampling The study included 24 patients with RAS, 30 patients with BD and 20 healthy individuals. The patients were selected from those presenting to Dermatology Outpatient Clinic of _ Medical School of Kahramanmarasß S€ utc߀ u Imam University and healthy individuals were selected from personnel working and students studying at the medical school. The prerequisite for both the patients and the healthy individuals is that they should not have an infection during collection of blood samples and the prerequisite for the healthy individuals was that they should not take medicine. Written informed consent was taken from all the participants and approval was obtained from the local ethical committee. RAS and BD were diagnosed by the dermatologist according to Lehner criteria and (19) and International Work Group criteria (20), respectively. The patients not fulfilling Lehner criteria or having an infection or other oral ulcers were not included in the study. Data about age, gender and duration, clinical signs and treatment of the diseases were recorded. Measurement of serum IL-1, IL-13, IL-17, IL-18, IFN-c and AE IL-1, IL-13, IL-17, IL-18, IFN-c and AE were analysed in the Department of Biochemistry with enzyme-linked immunosorbent assay (ELISA) by using commercial kits (Shanghai Sunred Biological Technology Co., Ltd, Shanghai, China), an automatic ELISA microplate reader (Thermo Scientific, Vantaa, Finland) and a computer programme (Thermo Scientific). Sensitivities and assay ranges are 0.917 pg/ml and 2–400 pg/ml for IL-1 kit (Catalogue No: 201-12-0078), 0.413 pg/ml and 0.5-100 pg/ml for IL-13 (Catalogue No: 201-12-0099), 12.013 pg/ml and 15–1000 pg/ml for IL-17 (Catalogue No: 201-12-0143), 0.537 ng/l and 0.6–100 ng/l for IL-18 (Catalogue No: 201-12-0148), 1.706 ng/l and 2–600 ng/l for IFN-c (Catalogue No: 201-12-0106) and J Oral Pathol Med
0.245 ng/ml and 0.5–60 ng/ml for a-enolase (Catalogue No: 201-12-0961) respectively. Specificity for each of the kits intra-assay CV was
doi: 10.1111/jop.12182
wileyonlinelibrary.com/journal/jop
Serum Th1, Th2 and Th17 cytokine profiles and alphaenolase levels in recurrent aphthous stomatitis Kemal Ozyurt1, Ahmet C ß elik2, Mehmet Sayarlıoglu3, Emine Colgecen4, Rahime Incı5, Tuba Karakas5, 6 Mehmet Kelles , Gozde Y. Cetin7 1
Dermatology Clinic, Kayseri Education and Research Hospital, Kayseri, Turkey; 2Department of Biochemistry, Sutcu Imam University, Medical School, Kahramanmaras, Turkey; 3Department of Rheumatology, Ondokuz Mayıs University, Medical School, Samsun, Turkey; 4 Department of Dermatology, Bozok University, Medical School, Yozgat, Turkey; 5Department of Dermatology, Sutcu Imam University, Medical School, Kahramanmaras, Turkey; 6Department of Otorhinolaryngology, Sutcu Imam University, Medical School, Kahramanmaras, Turkey; 7Department of Rheumatology, Sutcu Imam University, Medical School, Kahramanmaras, Turkey
BACKGROUND: All aspects of aetiopathogenesis of recurrent aphthous stomatitis (RAS) have not been elucidated. RAS and Behcßet’s disease (BD) have clinical and immunological characteristics in common. Although T17 cytokines and alpha-enolase have been shown to play effective roles in BD and many other autoinflammatory diseases recently, their roles in RAS have not been studied extensively. In the present study, we investigated levels of several Th1, Th2 and Th17 pathways related cytokines and alpha-enolase to elucidate pathogenesis of RAS and to obtain data about possible treatment alternatives for the condition. METHODS: Serum interleukin-1, interleukin-13, interleukin-17, interleukin-18, interferon gamma and alphaenolase levels in 24 patients with RAS, 30 patients with BD and 20 healthy controls were measured. RESULTS: Serum interleukin-1, interleukin-13, interleukin-17, interleukin-18, interferon gamma and alpha-enolase levels were higher in patients with RAS and patients with BD than in healthy controls (P < 0.005). CONCLUSION: Like Th1 and Th2 cells, Th17 cells were found to be effective in pathogenesis of RAS. In addition, alpha-enolase, the levels of which were high, may play an important role in etio-pathogenesis of RAS. Further studies to be designed in the light of these findings are required to shed light on pathogenesis and treatment of the condition. J Oral Pathol Med (2014) 43: 691–695 Keywords: alpha-enolase; Behcßet’s recurrent aphthous stomatitis; Th17
disease;
interleukin-17;
Correspondence: Dr Kemal Ozyurt, Dermatoloji Klinigi, Kayseri Egitim ve Arastırma Hastanesi, Melikgazi, Kayseri, Turkey. Tel: +903523368884, Fax: +903523368857, E-mail: [email protected] Accepted for publication February 11, 2014
Introduction Recurrent aphthous stomatitis (RAS) is a frequently encountered disease of the oral mucosa. Recurrent oral ulcers are the main diagnostic sign of Behcßet’s disease (BD). In addition to oral and genital ulcers, eyes, joints and the nervous system can also be involved in BD. Although genetic susceptibility, infections, various autoantibodies and immune complexes are incriminated for both RAS and BD, aetiopathogenesis in these conditions have not been understood completely yet (1–3). Cellular immunity involves an important part of pathogenesis in RAS and BD. CD4 + effector T cells in cellular immune responses can be divided into two, i.e. Th1 and Th2. Both groups of cells are known to be effective in RAS (3–5). In recent years, a new CD4 + cell which produces interleukin (IL)-17 and is called Th17 has attracted attention. In contrast to previous arguments, it has been understood that Th1 cells do not have a marked effect on autoimmune, allergic and microbial diseases and that IL-17 has a major role in these diseases. Based on experimental studies, it has been suggested that rheumatoid arthritis and multiple sclerosis are considered Th-17-related diseases, but not Th1-related conditions. IL-17 together with IL-21 and IL-22 controls immunity, inflammation and infections on mucosal surfaces and plays a role in vascular pathologies (6–10). The balance between Th17 and stimulated T regulatory (Treg) cells and cytokines like IL-23 and IL-17 have been shown to play an important role in inflammatory processes in BD, which have not been able to be explained by the Th1/Th2 paradigm (10–13). To our best knowledge, this is the first study to investigate Th17 cytokines in peripheral blood from cases of RAS. In this study, we examined IL-1, a natural cellular cytokine, IL-18 and and IFN-c, Th1 cytokines, and IL-13, a Th2 cytokine, in addition to IL-17, a Th17-related cytokine of cellular immunity in patients with RAS. We investigated these cytokines in patients with BD and healthy controls as well and attempted to study the role of Th17 in pathogenesis
Cytokine profiles in aphthous stomatitis Ozyurt et al.
692
of RAS. The fact that a Th17 cytokine, IL-17 is effective in mucosal immunity was the main factor directing us towards conducting this study (9, 14). In addition, it is striking that Th17 has a considerable role in BH, which has clinical and pathological aspects similar to RAS, and inflammatory diseases of the guts (15). Protein alpha-enolase (AE) is a plasminogen receptor on endothelial cell receptors and also exists on surfaces of streptococci and acts like a heat shock protein, which indicate a role of AE in autoimmune and inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, vaculitis and Crohn’s disease (16). Antiendothelial cell antibodies include a group of heterogeneous antibodies to various proteins on endothelial cell surfaces (17). Lee et al. (18) first reported that anti-AE antibody (AAEA), an anti-endothelial cell antibody, was found in sera from patients with BD and suggested that AAEA is a diagnostic marker for BD. To our knowledge, there have not been studies on AE in patients with RAS. AE can be an important marker for RAS which has the same immune-pathological features as many inflammatory diseases such as BH and Crohn’s disease.
Materials and methods Sampling The study included 24 patients with RAS, 30 patients with BD and 20 healthy individuals. The patients were selected from those presenting to Dermatology Outpatient Clinic of _ Medical School of Kahramanmarasß S€ utc߀ u Imam University and healthy individuals were selected from personnel working and students studying at the medical school. The prerequisite for both the patients and the healthy individuals is that they should not have an infection during collection of blood samples and the prerequisite for the healthy individuals was that they should not take medicine. Written informed consent was taken from all the participants and approval was obtained from the local ethical committee. RAS and BD were diagnosed by the dermatologist according to Lehner criteria and (19) and International Work Group criteria (20), respectively. The patients not fulfilling Lehner criteria or having an infection or other oral ulcers were not included in the study. Data about age, gender and duration, clinical signs and treatment of the diseases were recorded. Measurement of serum IL-1, IL-13, IL-17, IL-18, IFN-c and AE IL-1, IL-13, IL-17, IL-18, IFN-c and AE were analysed in the Department of Biochemistry with enzyme-linked immunosorbent assay (ELISA) by using commercial kits (Shanghai Sunred Biological Technology Co., Ltd, Shanghai, China), an automatic ELISA microplate reader (Thermo Scientific, Vantaa, Finland) and a computer programme (Thermo Scientific). Sensitivities and assay ranges are 0.917 pg/ml and 2–400 pg/ml for IL-1 kit (Catalogue No: 201-12-0078), 0.413 pg/ml and 0.5-100 pg/ml for IL-13 (Catalogue No: 201-12-0099), 12.013 pg/ml and 15–1000 pg/ml for IL-17 (Catalogue No: 201-12-0143), 0.537 ng/l and 0.6–100 ng/l for IL-18 (Catalogue No: 201-12-0148), 1.706 ng/l and 2–600 ng/l for IFN-c (Catalogue No: 201-12-0106) and J Oral Pathol Med
0.245 ng/ml and 0.5–60 ng/ml for a-enolase (Catalogue No: 201-12-0961) respectively. Specificity for each of the kits intra-assay CV was
