Simultaneous detection of IFN-gamma and IL-4 in lesional tissues and whole unstimulated saliva from patients with oral lichen planus

Background:  Although the roles of interferon (IFN)-gamma and interlekin (IL)-4 in oral lichen planus (OLP) have been described extensively in past decades, the available results are controversial. Moreover, few studies have utilized simultaneous detection of cytokines in local tissues and saliva to determine whether salivary cytokines could reflect the fact of local lesions.
Methods:  IFN-gamma and IL-4 were determined simultaneously in lesions and whole unstimulated saliva (WUS) from OLP patients with various clinical forms.
Results:  In OLP lesions, both IFN-gamma and IL-4 in erythematous/ulcerated OLP were higher significantly than that in control specimens. In WUS, however, only IFN-gamma of erythematous/ulcerated OLP was increased compared with control. Remarkably, IFN-gamma and IL-4 in WUS showed a more significant correlation to those in local tissues of all subjects.
Conclusion:  These results indicate that both IFN-gamma and IL-4 may play more important role in pathogenesis of erythematous/ulcerated OLP, and changes of these proinflammatory cytokines in WUS may reflect the status of the OLP lesion.     

Th1 cytokines in oral lichen planus

BACKGROUND: Cell-mediated immune responses in oral lichen planus (OLP) may be regulated by cytokines and their receptors. METHODS: In situ cytokine expression and in vitro cytokine secretion in OLP were determined by immunohistochemistry and ELISA. RESULTS: The majority of subepithelial and intraepithelial mononuclear cells in OLP were CD8+. In some cases, intraepithelial CD8+ cells were adjacent to degenerating keratinocytes. CD4+ cells were observed mainly in the deep lamina propria with occasional CD4+ cells close to basal keratinocytes. Mononuclear cells expressed IFN-gamma in the superficial lamina propria and TNF-alpha adjacent to basal keratinocytes. Basal keratinocytes expressed TNF-alpha as a continuous band. TNF R1 was expressed by mononuclear cells and basal and suprabasal keratinocytes. There was variable expression of TGF-beta1 in the subepithelial infiltrate while all intraepithelial mononuclear cells were TGF-beta1-. Keratinocytes in OLP stained weakly for TGF-beta1. Unstimulated OLP lesional T cells secreted IFN-gamma in vitro. TNF-alpha stimulation down-regulated IFN-gamma secretion and up-regulated TNF-alpha secretion. IL-4, IL-10 and TGF-beta1 secretion were not detected. CONCLUSIONS: These data suggest the development of a T helper 1 immune response that may promote CD8+ cytotoxic T-cell activity in OLP.     

中药养阴益气合剂对口腔扁平苔藓相关细胞因子影响的研究

目的观察中药养阴益气合剂对口腔扁平苔藓患者外周血中相关细胞因子水平的影响,探讨口腔扁平苔藓的免疫应答模式。方法选取阴虚血瘀证型口腔扁平苔藓患者40例,采用中药养阴益气法治疗3个月,检测治疗前、后NF-KB依赖性炎症介质IL-1β、IL-6、IL-8和Th1／Tb2代表性细胞因子IFN-γ、IL-10水平的变化。并选取18例健康志愿者,同时检测IL-1β、IL-6、IL,8、IFN-γ和IL-10水平,作为对照。结果中药养阴益气法治疗阴虚血瘀型口腔扁平苔藓总有效率达87．5％,治疗前,IL-6、IL-8表达水平明显高于对照组（P〈0．05）,治疗后,IL-8、IL-10与治疗前比较差异有显著性（P〈0．05）。结论口腔扁平苔藓的不同阶段患者的免疫状态是变化的,IL-8、IFN-γ/IL-10比值对于监测口腔扁平苔藓的病情可能更加敏感。中药养阴益气合剂可能通过调节Th1／Th2免疫平衡和阻断NF-KB信息通路的激活而发挥其治疗作用。     

OLP患者唾液和血清中骨桥蛋白等细胞因子的表达

目的：研究口腔扁平苔藓（OLP）患者唾液和血清中骨桥蛋白（OPN）等细胞因子的含量及其相互关系。方法：经病理学检查确诊的OLP患者26例,其中充血糜烂型12型,网纹/斑块型14例。酶联免疫吸附试验（ELISA）检测患者唾液和血清中OPN,肿瘤坏死因子-α（TNF-α）,转化生长因子-β1（TGF-β1）的含量。以26名健康志愿者作为正常对照。结果：OLP患者唾液和血清OPN,TNF-α含量与正常对照组相比均有升高（P〈0.05）;充血糜烂型OLP的TGF-β1唾液和血清水平显著高于网纹/斑块型OLP（P〈0.05）。三种抗体在唾液和血清中表现出明显的相关关系。结论：OPN在OLP患者唾液和血清中含量升高,唾液和血清中OPN含量具有相关性。唾液作为无创伤性检测标本有望有限地代替血清标本应用于OLP与口腔白斑的临床检测。     

糖皮质激素对口腔扁平苔藓辅助性T细胞平衡的影响

目的　明确口腔扁平苔藓 (OLP)患者的Th1 /Th2免疫应答模式,探讨糖皮质激素对OLP患者辅助性T细胞平衡的影响。方法　密度梯度离心法分离OLP患者和对照组外周血单个核细胞,分别用植物血凝素(PHA)和地塞米松刺激OLP患者外周血单个核细胞,采用酶联免疫吸附法,检测培养上清液中干扰素γ(IFN γ)和白细胞介素 4(IL 4)的含量;应用半定量逆转录聚合酶链反应技术,检测培养细胞中IFN γ和IL 4mRNA的表达水平。结果　外周血单个核细胞经PHA诱导培养,OLP患者组IFN γ的水平低于健康对照组 (P<0 05),IL 4的水平高于对照组,但差异无统计学意义(P>0 05),IFN γ/IL 4的比值低于对照组 (P<0 05 )。地塞米松可抑制IFN γ和IL 4的水平(P<0 01),且对IFN γ的抑制作用较IL 4更为显著。结论　OLP患者存在Th1 /Th2的平衡失调,为Th2占优势的免疫应答,糖皮质激素对OLP的Th1 /Th2细胞因子均有抑制作用。     

Oral submucous fibrosis patients have altered levels of cytokine production

Oral submucous fibrosis (OSF) is a pre-malignant fibrotic lesion of the mouth in betel quid chewers and is characterised by dense bands of collagen in the juxta-epithelial region preceded by inflammation. We have investigated the spontaneous and stimulated production of cytokines by peripheral blood mononuclear cells (PBMC) from OSF patients and compared them with genetically-related relatives, Indian and Caucasian control subjects. The cytokines studied included: interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). The results show: a) significant differences in the stimulated versus non-stimulated levels of IL-1beta, IL-6, IL-8 and TNF-alpha but not of IFN-gamma production by patients, and in the relatives' stimulated versus non-stimulated levels of IL-1beta, IL-6 and IFN-gamma; b) no difference in the spontaneous cytokine production between any two groups; and c) significant increases in the patients' stimulated cytokines compared to the Caucasian and Indian controls (P< or =0.050). These results demonstrate increased levels of proinflammatory cytokines and reduced anti-fibrotic IFN-gamma in patients with OSF, which may be central to the pathogenesis of OSF.     

Elevated serum levels of the apoptosis related molecules TNF-α, Fas/Apo-1 and Bcl-2 in oral lichen planus

BACKGROUND: The serum circulatory levels of apoptosis related molecules measured in patients with oral lichen planus (OLP) and healthy individuals in order to investigate possible alterations associated with the clinical forms of OLP. METHODS: Serum levels of tumor necrosis factor (TNF)-alpha, soluble Fas (sFas) and Bcl-2 studied by enzyme-linked immunosorbent assay in whole blood samples in 13 OLP reticular, 13 OLP atrophic-erosive form patients and 26 healthy subjects. RESULTS: Significantly elevated levels of TNF-alpha and sFas detected in OLP patients as compared with controls. Serum concentrations of Bcl-2 although increased in 17/26 patients, they were not statistically significant. Reticular OLP exhibited slightly elevated TNF-alpha and significantly elevated Bcl-2 serum levels, compared with erosive OLP. CONCLUSIONS: These data suggest that a putative dysfunction in the Fas/FasL mediated apoptosis might be involved in the OLP pathogenesis. A downregulation of Bcl-2 serum levels in the atrophic-erosive OLP may be associated with promotion of the disease activity.     

Distribution of interleukin-2, -4, -10, tumour necrosis factor-alpha and transforming growth factor-beta mRNAs in oral lichen planus.

In the present study, MRNA for the cytokines interleukin-2 (IL-2), IL-4, IL-10 tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor beta-1 (TGF-beta-1) were investigated in oral lichen planus (OLP) lesions using in situ hybridization with 35S-labelled oligonucleotide probes on frozen tissue sections. In addition, the expression of interferon-gamma (IFN-gamma), IL-10 and IL-4 mRNAs was analysed in cultured lesional T lymphocytes from oral lichen planus by polymerase chain reaction. Cells expressing mRNA for IL-2, IL-4, IL-10, TNF-alpha and TGF-beta 1 were found in all the biopsies studied. Approximately 1-2% of the total number of infiltrating cells in the lesions were positive for each of the different cytokine mRNAs. Most biopsies contained basement membrane-oriented, mRNA-positive cells. In the cultured T-cell lines, message for IFN-gamma was detected in all the patients, IL-10 in all but one, and IL-4 in just one of the seven patients investigated. The results suggest that mRNA for both pro- and anti-inflammatory cytokines, i.e., mixed T-helper 1 (TH1) and TH2 cytokine profiles, are generated simultaneously by a limited number of cells in chronic lesions of OLP.     

s-IgA and cytokine levels in whole saliva of Sjögren's syndrome patients before and after oral pilocarpine hydrochloride administration: a pilot study

Previous investigations have found elevated levels of s-IgA in the parotid saliva and normal levels in submandibular saliva of patients with Sjögren's syndrome (SS). Fox et al. also found elevated levels of cytokines (i.e., IL-2 and IL-6) in serum, salivary epithelial cells and parotid saliva of patients with SS. The oral administration of pilocarpine hydrochloride stimulates whole and parotid salivary flow. The purpose of this study was to determine the levels of s-IgA and IL-2 and IL-6 in whole saliva before and after administration of pilocarpine hydrochloride in SS subjects. Ten definitively diagnosed SS subjects were enrolled in the study, as were ten controls (C). The mean age was 57.2 years and all subjects were female. Whole unstimulated saliva (WUS) was collected by standard techniques for 5 min, after which the volume and flow rate were determined (mean WUS: SS = 0.047 vs C = 0.480 ml/min). Samples were centrifuged and the immunoglobulin analysis performed on the supernatants by immunoreactivity in a double-sandwich technique as previously described by Rudney et al. Cytokine analysis was performed similarly utilizing commercially available kits from R&;D Systems. The results as analyzed by pairwise t-tests revealed comparable levels of s-IgA in the saliva of the SS patients, as compared to controls at baseline (means±SEM: SS-IgA = 348.1±82.0 vs C-IgA = 284.0±65.1 μg/ml; NS ). Whole salivary flow was significantly increased (328%) in the SS subject group 60 min after the administration of 5 mg pilocarpine hydrochloride (means±SEM: 0.0472±0.017 vs 0.1546±0.054 ml/min; P<0.01). There was no significant change in the concentration of s-IgA in the SS subject group following the pilocarpine dose (means±SEM: SS-IgA = 439.9±121.2 μl/ml; P = NS). There were elevated levels of IL-2 in the saliva of four out of the ten and IL-6 in two out of the ten SS patients, as compared to controls (means±SEM: SS-IL-2 = 127.8±11.4 vs C-IL-2 = 30.8±1.6 pg/ml and SS-IL-6 = 41.4±7.1 vs C-11.6 ± 2.8 pg/ml). There was also a significant decrease in the concentration of IL-2 in the same four out of ten SS subjects following the pilocarpine dose (means±SEM: SS-IL-2 = 32.4±10.3; P<0.01). These preliminary results indicate that s-IgA levels do not change with increased salivary flow following the administration of pilocarpine hydrochloride in patients with Sjögren's syndrome. While cytokines are elevated in the whole saliva of some SS patients, a decrease in IL-2 concentration may occur with increased salivary flow.     

Serum cytokine levels in patients with oral mucous membrane disorders

Serum cytokine levels were examined in 18 oral squamous cell carcinoma (SCC), 26 lichen planus (OLP), 20 recurrent aphthous stomatitis (RAS), 8 herpetic gingivostomatitis (HGS), 16 pseudomembrane candidiasis (PMC) and 19 acute bacterial infection (ABI) cases. All SCC and most PMC patients possessed clear serum IL-3. No clear increase of IL-4 was observed in most cases though over 20 pg/ml were found in a few OLP, RAS and ABI. ABI exhibited the highest IL-6, and the cytokine level was lower in RAS, PMC, HGS and OLP in this order. Suppressed IL-6 activity was elevated with improvement of HGS lesion. TNF-alpha increased in 9 OLP, but the levels were below 100 pg/ml in all cases. Most SCC possessed higher GM-CSF activity than the controls. Increase of the cytokine corresponding with improvement of the oral lesion was seen in HGS, but not in OLP. From these results, each serum cytokine seems to reflect a characteristic pathophysiology of individual oral disorder.     

Oral and serum IL-6 levels in oral lichen planus patients

OBJECTIVE: The purpose of this study was to compare IL-6 levels in oral exfoliated mucosal cell samples and in serum in subjects with oral lichen planus versus controls.Study design Ten patients with ulcerative OLP, 10 with reticular OLP, and 10 control subjects were recruited at a University Oral Medicine Clinic. Using smear tissue culture brushes, oral samples were collected from lesional sites for OLP patients and from buccal mucosa for controls into vials with 300 muL PBS. After centrifugation, the supernatants were aspirated for cytokine ELISA assay and protein assay. Venous blood was processed to serum for ELISA assay. Oral IL-6 was expressed as both pg/mL and pg/mug protein, and serum IL-6 was expressed as pg/mL. RESULTS: The mean oral IL-6 levels were higher in the ulcerative OLP group (11.19 +/- 5.34 pg/mL) than in the reticular OLP (1.05 +/- 0.34 pg/mL) and control (0.52 +/- 0.29 pg/mL) groups. There were significant differences between ulcerative OLP and reticular OLP groups (P < .039), and between ulcerative OLP and control groups (P < .009). After the standardization of IL-6 concentration by protein, a significant difference in IL-6 concentration was shown only between the ulcerative OLP (0.0245 +/- 0.0121 pg/mug protein) and control (0.0023 +/- 0.0012 pg/mug protein) groups (P < .029). Similarly, the ulcerative OLP group showed a significantly higher serum IL-6 level than the control group (P < .021). CONCLUSION: Both oral and serum IL-6 levels were higher in patients with ulcerative OLP. An oral exfoliated cell technique may be a useful and sensitive method to measure IL-6 in patients with OLP as it provided results consistent with those found in peripheral blood.     

口腔扁平苔藓与NF-κB通路相关因子IL-8和RANTES的关系研究

目的探讨NF-κB信号通路相关细胞因子白细胞介素-8 (IL-8),受激活调节正常T细胞表达和分泌因子(RANTES)在口腔扁平苔藓(OLP)患者组和健康对照组中的表达,并分析IL-8、RANTES表达的相关性,探讨在口腔扁平苔藓发生发展过程中NF-κB信号传导通路的作用。方法选取30例OLP患者作为实验组,30例健康者为对照组,取其相应黏膜组织。应用RT-PCR法检测NF-κB相关因子IL-8、RANTES在OLP患者组与健康对照组中的表达,分析患者组与对照组的表达差异及二者表达相关性。结果 RT-PCR结果显示IL-8、RANTES在OLP两组中均有表达,但患者组的表达明显高于正常对照组(P<0.05)。OLP患者组中IL-8与RANTES的表达相关(P<0.05)。结论 OLP的发生发展可能与NF-κB引导的炎症通路及其介导的炎症因子IL-8、 RANTES相关,同时IL-8和RANTES在OLP的发病过程中可能具有协同致病作用。     

Cytokine production by keratinocytes and mononuclear infiltrates in oral lichen planus

Cytokine generation by tissue-infiltrating mononuclear cells (TIMC) and by keratinocytes (KC) was investigated in material obtained from the oral mucosal tissues of patients with oral lichen planus (OLP). Peripheral blood mononuclear cells (PBMC) and chronically inflamed and noninflamed gingival KC (CIG-KC, NOR-KC, respectively) were used as the controls. Compared to NOR-KC and CIG-KC, KC from OLP patients (OLP-KC) produced much more interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The OLP-KC superiority in the production of these cytokines was more prominent when the KC were cultured in the presence of interleukin-l beta (IL-1β), lipopolysaccharide and phorbol myristate acetate. OLP-KC also produced more monocyte-chemotactic factor(s) which were not inactivated by the antibodies against GM-CSF, macrophage colony-stimulating factor and monocyte chemoattractant protein-1. TIMC in OLP tissues (OLP-TIMC) were superior to PBMC in the generation of IL-6 and GM-CSF. OLP-TIMC were stimulated to produce more TNF-a by IL-1β, IL-6 and GM-CSF, more IL-6 by IL-1β and GM-CSF, and more GM-CSF by IL-1β and IL-6 than PBMC. When compared to cytokine generation in TIMC from the chronically inflamed gingivae, more interferon-gamma, IL-6 and TNF-α were generated by OLP-TIMC. These results indicate that KC play a critical role in OLP, producing cytokines including monocyte-chemotactic factor(s), and that the cytokines produced by TIMC and OLP-KC through autocrine and paracrine processes enhance the local inflammatory response.     

In vivo induction of proinflammatory cytokines in mouse tissue by Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans

Periodontitis is a chronic inflammatory disease initiated by a multitude of bacteria. Persistent infection leads to generation of various inflammatory mediators, resulting in tissue destruction and osteoclastic resorption of the alveolar bone. This study describes a novel in vivo murine calvarial model to assess the effects of oral pathogens on the expression of three proinflammatory cytokines [interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha] which are involved in bone resorption. We chose Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans as prototype oral pathogens. We also tested the effects of Streptococcus gordonii, an oral commensal supragingival microorganism, considered a non-pathogen. Live bacteria were injected into subcutaneous tissue overlying the parietal bone of mice calvaria for 6 days. At the end of the experimental period, tissues overlying the calvaria were removed and analyzed for proinflammatory cytokine expression by Northern blotting. Cytokine mRNA was not detected in the tissue over the calvaria of control animals. In contrast, P. gingivalis and A. actinomycetemcomitans elicited mRNA expression of all three cytokines, TNFalpha being the highest (TNFalpha > > IL-1beta > IL-6). P. gingivalis was more potent than A. actinomycetemcomitans in inducing cytokine expression. In contrast, S. gordonii induced only low levels of mRNA for IL-1beta and TNFalpha but no IL-6 mRNA induction. These results suggest that oral microorganisms with access to host tissues elicit a battery of proinflammatory cytokines. There were clear differences in profiles and, interestingly, a commensal bacterium also stimulated bone resorptive cytokine expression in host tissues.     

Perspective of cytokine regulation for periodontal treatment: fibroblast biology

Efforts to understand the pathogenesis of periodontal diseases have been underway for decades. Studies of immunological aspects in addition to the structural components of gingival fibroblasts showed that the fibroblasts actively participate in immune and inflammatory events in periodontal diseases. Future strategies for the prevention and treatment of periodontal diseases should biologically regulate fibroblast activities. These cells are surrounded by monocyte-derived proinflammatory cytokines such as interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and lymphocyte-derived interleukin-6 (IL-6) in inflamed gingival tissue. Recent anti-cytokine therapy for inflammatory diseases including rheumatoid arthritis aimed to inhibit the binding of cytokines to targeted cells such as fibroblasts and condrocytes. IL-1beta and TNF-alpha are thought to be therapeutic targets because these cytokines are essential for the initiation of inflammatory immune reactions and are produced for prolonged periods in inflammatory diseases. IL-6 is also a target, because it is abundantly present in inflammatory lesions and activates fibroblasts in the presence of soluble IL-6 receptor. In addition, these cytokines accelerate gingival fibroblasts to produce collagenolytic enzymes, resulting in tissue destruction. Soluble receptors for IL-1beta and TNF-alpha are suggested to be candidates for therapeutic molecules, but soluble receptor for IL-6 is suggested to be a factor-stimulating fibroblast. This paper will review the utilization of soluble receptors specific to inflammatory cytokines which potentially stimulate fibroblasts to regulate biological events involved in the pathogenesis of periodontal diseases.     

Diabetes modulates gene expression in the gingival tissues of patients with chronic periodontitis

AIM: This study evaluated whether diabetes modulates gene expression [interleukin (IL)-1beta, IL-1ra, IL-6, IL-8, IL-10; tumor necrosis factor (TNF)-alpha; interferon (IFN)-gamma, receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG)] in sites with periodontitis. MATERIALS AND METHODS: Gingival biopsies were harvested and divided into three groups--Control group: systemically and periodontally healthy subjects (n = 10); Periodontitis group: systemically healthy subjects diagnosed with chronic periodontitis (n = 20); Diabetes group: type 1 diabetic subjects, diagnosed with chronic periodontitis (n = 20). Total RNA was obtained and analyzed by quantitative polymerase chain reaction. RESULTS: Data analysis demonstrated that, except for OPG, mRNA levels for all factors were increased by inflammation (P < 0.001). Interleukin-1beta, IL-1ra, IL-6, IL-8, IFN-gamma, and RANKL mRNA levels were higher in the diabetic group when compared with the control non-periodontitis group (P < 0.05), whereas IL-10 and OPG were lower (P < 0.05). No difference was observed for TNF-alpha between diabetic and control groups (P > 0.05). Diabetes lowered IL-1beta, IL-8, IL-10, TNF-alpha, RANKL, and OPG mRNA levels in sites with comparable type of periodontitis (P < 0.001). Moreover, increased RANKL:OPG and IL-6:IL-10 ratios were found. CONCLUSION AND CLINICAL RELEVANCE: Taken together, these data suggest that decreased levels of IL-10 and OPG may play an important role in the periodontal breakdown in diabetic patients.     

口腔扁平苔藓患者血清IL-2、IL-4、IL-6水平与临床症状的相关性

目的 探讨不同类型口腔扁平苔藓(OLP)患者血清IL-2、IL-4、IL-6在OLP发病及治疗中的意义。方法 采用ELISA方法检测60例各型OLP患者血清IL-2、IL-4、IL-6的水平，记录患者的疼痛程度、充血和糜烂面积。结果 OLP患者血清IL-2、IL-4水平较对照组无显著差异，而各型OLP患者IL-6显著升高。糜烂型OLP患者IL-4水平显著高于对照。血清IL-2、IL-4水平与疼痛程度、充血面积和糜烂面积之间无相关性，而IL-6与三者呈正相关。结论 本研究支持自身免疫机制是OLP的发病机制之一的观点；在OLP的不同阶段患者的免疫状态是变化的；IL-6在OLP的发病中发挥了一定作用。血清IL-6水平也许可以作为动态监测OLP疾病进展的指标和疗效观察的指标。