口腔扁平苔藓T细胞受体BV基因的优势取用初探

目的:研究口腔扁平苔藓(oral lichen planus,OLP)患者损害组织中浸润的T细胞受体(T cell receptor,TCR)BV基因的取用格局。方法:采用实时定量聚合酶链反应,分析OLP患者损害组织中浸润的T细胞和外周血中T细胞TCR BV基因的取用格局。采用GraphPad Prism version 5.00软件对数据进行Mann-Whitney U检验。结果:43例OLP患者的损害组织对BV4、BV14和BV18基因的取用显著高于39例OLP患者外周血对这些BV基因的取用。结论:OLP患者损害组织浸润的T细胞优势取用BV4、BV14和BV18基因,提示带有BV4、BV14和BV18基因的T细胞参与了OLP的发病。     

Accumulation of oligoclonal T cells in the infiltrating lymphocytes in oral lichen planus

BACKGROUND: Identification of a disease-specific and possibly pathogenic T-cell receptor (TCR) in oral lichen planus (OLP) is one of the most important steps to reveal the pathogenic antigen recognized by the T cells and thereby elucidate the pathogenesis and etiology of OLP. METHODS: In buccal mucosa biopsy specimens and peripheral blood mononuclear cells (PBMC) from seven patients with OLP, the TCR V beta gene usage was examined by polymerase chain reaction-based and single-strand conformation polymorphism analyses. RESULTS: The V beta families expressed in the biopsy specimens were markedly heterogeneous, but they were restricted in comparison to those observed in the PBMC. The V beta families predominantly expressed in the biopsy specimens in comparison with the PBMC were still heterogeneous in individual patients and differed from patient to patient; however, V beta 2, V beta 6, and V beta 19 were commonly predominant in the biopsy specimens from more than half of the patients. Among the V beta families predominantly expressed in the biopsy specimens, the accumulation of T-cell clonotypes was observed in the majority of the V beta families including V beta 6 and V beta 19; however, it was not observed in the minority of the V beta families including V beta 2. CONCLUSIONS: These results suggest that unique T-cell populations bearing V beta 2, V beta 6, or V beta 19 gene products tend to expand in OLP lesions as a consequence of in situ stimulation with a restricted epitope of either a nominal antigen on the MHC molecule for the majority of the V beta families, even if only in minor populations, or of a common superantigen for the minority of the V beta families.     

辅助性T淋巴细胞极化相关细胞因子在口腔扁平苔藓中的研究进展

口腔扁平苔藓是一种病因不明的皮肤黏膜慢性免疫炎症性疾病,辅助性T淋巴细胞(Thcell)在口腔扁平苔藓的发病机制中起到了重要作用。近年来,口腔扁平苔藓中Th细胞的极化状态引起了较为广泛的关注。研究表明,Th细胞极化相关细胞因子的变化与口腔扁平苔藓的发病密切相关。下文就这些细胞因子在口腔扁平苔藓中的研究进展作一综述。     

Immunoelectron microscopic study of distribution of T cell subsets in oral lichen planus

Abstract – Monoclonal anti-CD4, anti-CD8, and anti-GD18 antibodies were applied in avidinbiotin-peroxidase complex staining using a pre-embedding immunoelectron microscopy technique. Although most of the local T cells in situ were of CD4⁺ subtype, local CD8⁺ cells generally had a lower nucleus/cytoplasm ratio and contained more cell organelles than CD4⁺ cells. This suggests a local activation of CD8⁺ subpopulation, rather than activation of the numerically predominant CD4⁺ cells. Topographical analysis disclosed that all lymphocytes, regardless of location, were CD18⁺ and that most of the CD8 ⁺ cells were located subbasally and intraepithelially, whereas CD4⁺ cells often occurred in small clusters deeper down in the subepithelial lymphocyte-rich band. Furthermore, CD8⁺ cells were often in close contact with macrophages, whereas CD4⁺ cells were in some instances in direct contact with plasma cells. This indicates that CD4⁺ cells may be involved in T cell-dependent B cell-mediated immunoglobulin synthesis, whereas CD8⁺ cytotoxic lymphocytes and tissue macrophages may be involved in the local pathogenetic process leading to basement membrane alterations.     

TCR Vβ gene expression in lesional T lymphocyte cell lines in oral lichen planus

OBJECTIVE: To study Vβ gene expression in oral lichen planus (OLP) lesional T lymphocyte cell lines.
MATERIALS AND METHODS: Lesional T lymphocytes were isolated from eight OLP patients and cell lines established. The total RNA was extracted from these lymphocyte cell lines and reverse transcribed. cDNA was amplified by the polymerase chain reaction (PCR) using a panel of 26 Vβ-specific oligonucleotide primers followed by qualitative analysis of the electrophoresed reaction products.
RESULTS: Vβ 1, 2, 3, 5.1, 6.1–3, 7, 8, 9, 22. 23, and 24 were represented consistently in all of the OLP samples, Vβ11, 12, and 17 were consistently negative, while the other Vβ families (Vβ4, 5.2–3, 10, 13.1, 13.2, 14, 15, 16, 18, 19, 20, and 21) were variable. Vβ22 and 23 were the most strongly expressed in all patients.
CONCLUSIONS A limited T cell receptor (TCR) gene usage indicates a degree of oligoclonality within these lesional T lymphocyte cell lines from OLP. This implies that OLP may be an antigen-specific disease or linked to a limited number of superantigens.     

Intra-epithelial subpopulations of T lymphocytes and Langerhans cells in oral lichen planus

This study has addressed the question of whether there is selective recruitment and distribution of intra-epithelial leucocytes in lesions of oral lichen planus (OLP). T-lymphocyte subsets were examined in the epithelium and peripheral blood of patients and controls using flow cytometry and double immunofluorescence, and the relationship between keratinocyte intercellular adhesion molecule-1 (ICAM-1) expression with T-lymphocyte and Langerhans cell (LC) distribution was examined. The circulating 'memory'subset (CD45RO⁺) of T-helper cells (CD4⁺) was increased from 49.1% in controls to 65.7% in patients ( P = 0.005), while the 'naive'subset (CD45RA⁺), which was absent from control epithelium, comprised 24% of helper cells in OLP ( P =0.037) and all T-cell and LC counts were significantly raised in ICAM-1-expressing areas of epithelium. These data demonstrate changes in intra-epithelial T-lymphocyte and LC populations compared with normal oral mucosa and suggest there is selective recruitment in OLP. In addition, Keratinocyte ICAM-1 expression does appear to be associated with accumulation of infiltrating T lymphocytes and LC.     

Mast cell expression in oral lichen planus: A systematic review

Lichen planus is a common chronic, inflammatory, immune‐mediated mucocutaneous disorder affecting the skin and mucosa. The role of mast cells in the genesis of lichen planus has been debated. Establishing a definitive part played by mast cells and its degranulation would possibly provide a permanent, cost‐effective treatment modality for oral lichen planus (OLP). This review aims to study the expression of mast cells qualitatively and quantitatively in OLP. The research questions were framed to assess the mast cell count, localization within the epithelium basement membrane zone and degranulation of mast cells. We performed a systematic search of PubMed, Medline, Cochrane and Web of Science. We found a total of 120 studies from which 12 were found suitable for the review. There is a marked increase in the number of mast cells in OLP. The mast cells were seen in increased numbers in the epithelial and connective tissue junction at areas of basement membrane disruption. There was also an increase in the degranulation of mast cells. It is evident that there is an increase in the mast cell number in lichen planus and its subsequent degranulation.     

T细胞免疫和细胞凋亡在口腔扁平苔藓发病中的作用

目的通过探讨口腔扁平苔藓（OLP）中细胞凋亡情况及CD4＋、CD8＋T细胞和CD4/CD8比例的变化分析细胞免疫与细胞凋亡的关系,进一步了解OLP的发病机制。方法应用免疫组化SP法检测27例OLP组织中CD4＋、CD8＋T细胞的表达水平,并用原位末端转移酶标记法（TUNEL）定位检测17例OLP中细胞凋亡情况。结果OLP组固有层中CD4＋、CD8＋T细胞明显高于对照组（P＜0.05）,CD4/CD8值降低。OLP组中上皮内细胞凋亡指数高于对照组,固有层淋巴细胞凋亡指数明显低于对照组。结论OLP组固有层中CD4＋、CD8＋T细胞浸润的增加、CD4/CD8比值的变化及OLP中上皮细胞和固有层淋巴细胞凋亡异常,说明细胞免疫功能紊乱和细胞凋亡异常在OLP发病中起重要作用。     

Immune mechanisms in oral lichen planus

Oral lichen planus (OLP) is a T cell-mediated inflammatory disease of the oral mucosa that has been extensively researched over many years but as yet the mechanisms of pathogenesis are still not fully understood. Whilst the specific aetiological factors driving OLP remain ambiguous, evidence points to the development of a chronic, dysregulated immune response to OLP-mediating antigens presented by innate immune cells and oral keratinocytes leading to increased cytokine, chemokine and adhesion molecule expression. These molecules recruit T cells and mast cells to the diseased site and orchestrate a complex interplay between cells that culminates in keratinocyte cell death, mucosal basement membrane destruction and long-term chronicity of the disease. The main lymphocytes involved are thought to be CD8+ cytotoxic and CD4+ Th1 polarised T cells although recent evidence indicates the involvement of other Th subsets such as Th9, Th17 and Tregs, suggesting that a more complex immune cell relationship exists during the disease process. This review provides an overview of the immune mechanisms at play in OLP pathogenesis with particular emphasis on the role of the different Th subsets and how these recent discoveries may guide research towards identifying potential therapeutic targets.     

Langerhans cell distribution and keratinocyte expression of HLADR in oral lichen planus

Keratinocyte expression of the Class II major histocompatibility complex antigen HLADR, is seen in several inflammatory disorders of skin and mucosa, including lichen planus. The purpose of this study is to determine whether the distribution of Langerhans cells and their expression of CD4 in oral lichen planus is related to keratinocyte HLADR. The numbers of CD1- and CD4-positive Langerhans cells were compared in areas of keratinocyte HLADR and areas showing no expression in oral lichen planus and with normal oral mucosa. Cells were identified using an immunoalkaline phosphatase technique and numbers were expressed per mm epithelial surface length. In lichen planus, an increase both in the number of Langerhans cells and the numbers expressing CD4 were found in areas of keratinocyte HLADR expression compared with HLADR negative areas and with normal oral mucosa. There was no difference in the numbers of Langerhans cells or their expression of CD4 between HLADR-negative areas in LP and normal oral mucosa. These results show that the distribution of Langerhans cells is related to keratinocyte expression of HLADR and suggest that Langerhans cell entry may be enhanced in these areas. Whilst it is possible this enhancement is mediated by CD4/HLADR interaction, other molecules are also likely to be important in controlling Langerhans cell entry into oral mucosa.     

Expression of lymphocyte function-associated antigen 3 in oral lichen planus

OBJECTIVE: The expression pattern of lymphocyte function-associated antigen 3 (LFA-3) in the buccal mucosa of oral lichen planus (OLP) patients was compared to that of healthy controls to investigate the possible role of LFA-3 in cell interactions within OLP lesions.
MATERIALS AND METHODS: Samples of buccal mucosa from 17 clinically healthy individuals and 17 OLP lesions were analysed. Expression of LFA-3, CD2, CD3 and CD 14 was visualized by an immunoperoxidase technique and assessed microscopically.
RESULTS: In healthy buccal mucosa LFA-3 was expressed on keratinocytes, Langerhans cells within the epithelium and on endothelial cells in the lamina propria. In OLP patients a similar pattern of LFA-3 staining was observed. In addition, cytoplasmic LFA-3, without accompanying surface staining, was seen on a subpopul-ation of macrophage-like cells. Substantial amounts of LFA-3 also appeared to be associated with non-cellular components of the extracellular matrix within the inflammatory infiltrate.
CONCLUSIONS: We have obtained evidence for a previously undescribed localization of LFA-3 within macro-phages, and have observed that expression of LFA-3 is apparently elevated within OLP lesions. LFA-3 may play an important role in the pathogenesis of OLP.     

口腔扁平苔藓外周血中辅助性T细胞/调节性T细胞平衡与临床特征相关性分析

目的 了解口腔扁平苔藓（OLP）患者外周血中辅助性T细胞17（Th17）与调节性T细胞（Treg）的平衡变化,探讨它们在OLP发病机制中的作用及意义。方法 选取17例正常组和33例OLP患者（网纹型15例,糜烂型18例）的外周血,应用流式细胞术（FCM）检测Th17、Treg细胞的表达水平,实时荧光定量聚合酶链反应（qPCR）检测它们的转录因子维甲酸相关孤核受体γt（RORγt）和叉头状转录因子3（Foxp3）mRNA的表达。结果 OLP外周血中Th17、Treg细胞及RORγt、Foxp3 mRNA表达均升高（P<0.05）,但Treg细胞和Foxp3 mRNA表达在OLP两型间差异无统计学意义。Th17/Treg比值在OLP中升高（P<0.05）,其中糜烂型OLP显著高于正常组及网纹型OLP（P<0.01）,但网纹型OLP与正常组相比差异无统计学意义。Spearman相关分析显示Th17细胞和Th17/Treg比值与体征计分、RAE计分存在正相关关系（r=0.66,P=0.00;r=0.66,P=0.00;r=0.52,P=0.00;r=0.50,P=0.00）;同时Th17细胞与Treg细胞也存在正相关关系（r=0.39,P=0.03）。结论 OLP外周血中Th17和Treg细胞以及它们的比例均增高,Th17/Treg失衡在糜烂型OLP的发病过程中起了一定作用。     

口腔扁平苔藓中RANTES的表达及其与肥大细胞的关系

目的:探讨RANTES及肥大细胞(MC)在口腔扁平苔藓(OLP)发生发展过程中的作用及意义。方法:采用SABC免疫组化法检测30例OLP黏膜组织及10例正常口腔黏膜组织中RANTES的表达及MC的数量,根据Bresalier半定量公式计算RANTES的平均染色强度。结果:RANTES的平均染色强度在正常对照组低于OLP非糜烂型组低于OLP糜烂型组,三组之间两两差异均有统计学意义(P<0.05)。MC密度正常对照组低于OLP非糜烂型组低于OLP糜烂型组,三组之间的两两差异均有统计学意义(P<0.05)。MC密度与RANTES表达呈正相关(r=0.850,P<0.01)。结论:MC及RANTES可能参与了OLP的致病机制,且两者间有相关性。     

Distribution of interferon-y mRNA-positive cells in oral lichen planus lesions

The present study investigated the potential involvement of interferon-γ (IFN-γ)-producing cells in the pathogenesis of oral lichen planus (OLP). On biopsies from 10 OLP patients, an in situ hybridization technique was employed to determine the topographic al distribution of cells expressing IFN-γ mRNA. It was estimated that approximately 1% or fewer lesional cells were IFN-γ mRNA-positive. These cells were mainly encountered lining the basal membrane in a majority of the patients, or were in a few cases circumscribing the infiltrate, but were more seldon localized to the center of the lesion. A slightly higher, but not statistically significant number of phytohemagglutinin (PHA)-induced IFN-γ-producing cells, in vitro , was found in blood from 11 other OLP patients compared with blood from matched controls. Equal concentration of IFN-γ in supernatants from PHA-stimulated blood cells were detected in the two groups. Similarly, the IFN-γ response towards C. albicans was alike in OLP and in healthy control (HC) blood cells, indicating normal immunological memory function in the OLP patients. A small set of cells with spontaneous IFN-γ production was found in OLP and in HC peripheral blood. The data suggest that T-lymphocyte activation and cytokine production act locally and are not reflected in peripheral blood. The localization of the IFN-γ mRNA-positive cells indicates that the antigenic peptides are presented at the periphery of the mononuclear cell infiltrate. Furthermore, the low frequency of IFN-γ mRNA-positive cells in the lesions suggests that the disease is maintained by a small number of antigen-specific T cells.     

Apoptosis in oral lichen planus

Apoptotic cell death may be a contributory cause of basal cell destruction in oral lichen planus (OLP). Therefore. the purpose of this study was to investigate the rate of apoptosis in OLP and the expression of two proteins (FasR and FasL) regulating this process. Biopsies from 18 patients with histologically diagnosed OLP were investigated, with comparison to normal oral mucosa of healthy persons. For visualisation of DNA fragmentation, the TUNEL method was used. In order to characterise the infiltrating cell population (CD3. CD4, CD8) and expression of FasR and FasL, we used an immunohistochemical technique. The results showed that T cells dominated in the subepithelial cell infiltrate. Within the epithelium the apoptotic cells were confined to the basal cell layer, and more apoptotic cells were seen in areas with basal cell degeneration and atrophic epithelium. There was a prominent expression of FasR/FasL in OLP. with a rather uniform distribution throughout the inflammatory cell infiltrate. In the epithelium, the FasR/FasL expression was more abundant in the basal cell area compared to the suprabasal cell layer. In conclusion, apoptosis within the epithelium is significantly increased in situ in OLP compared to normal oral mucosa, and seems to be related to the epithelial thickness.     

Role of distinct CD4+ T helper subset in pathogenesis of oral lichen planus

Oral lichen planus (OLP) is one of the most common chronic inflammatory oral mucosal diseases with T‐cell‐mediated immune pathogenesis. In subepithelial and lamina propria of OLP local lesions, the presence of CD4⁺ T helper (CD4⁺ Th) cells appeared as the major lymphocytes. These CD4⁺ T lymphocytes can differentiate into distinct Th cell types such as Th1, Th2, Treg, Th17, Th22, Th9, and Tfh within the context of certain cytokines environment. Growing evidence indicated that Th1/Th2 imbalance may greatly participate into the cytokine network of OLP immunopathology. In addition, Th1/Th2 imbalance can be regulated by the Treg subset and also greatly influenced by the emerging novel CD4⁺ Th subset Th17. Furthermore, the presence of novel subsets Th22, Th9 and Tfh in OLP patients is yet to be clarified. All these Th subsets and their specific cytokines may play a critical role in determining the character, extent and duration of immune responses in OLP pathogenesis. Therefore, we review the roles of distinct CD4⁺ Th subsets and their signature cytokines in determining disease severity and susceptibility of OLP and also reveal the novel therapeutic strategies based on T lymphocytes subsets in OLP treatment.     

Expression of RANTES and CCR1 in oral lichen planus and association with mast cell migration

BACKGROUND: T lymphocytes and mast cells infiltrate the lamina propria in oral lichen planus (OLP). Chemokines and their receptors are involved in T cell and mast cell migration and accumulation during the inflammatory process. METHODS: In the present study, we investigated the role of RANTES and its receptors in OLP using immunohistochemistry, RT-PCR and an in vitro chemotaxis assay. RESULTS: RANTES and CCR1 were expressed on T cells and mast cells in OLP, while OLP lesional T cell supernatants stimulated CCR1 mRNA expression in a human leukemia mast cell line (HMC-1). TNF-alpha stimulated CCR1, CCR4 and CCR5 mRNA expression in the same cell line. OLP lesional T cell supernatants stimulated HMC-1 migration, which was partly inhibited by anti-RANTES antibody. CONCLUSIONS: The present study shows, for the first time, the distribution of RANTES and CCR1 in OLP. It is hypothesized that RANTES and CCR1 may play important roles in mast cell trafficking and related events in OLP.     

DNA ploidy in oral lichen planus,determined by image cytometry

J Oral Pathol Med (2010) 39 : 206–211 Background: The objective of this study was to use image cytometry to determine the degree and frequency of DNA ploidy in biopsies of reticular and atrophic‐erosive oral lichen planus and to analyze 14 karyometric measurements of the nuclei of epithelial cells from each specimen. Methods: A total of 40 slides were analyzed, each of them representing one biopsy of one oral lichen planus (OLP) lesion from each one of the 40 patients (cases) studied. Specimens were embedded in paraffin and comprised 20 slides of reticular oral lichen planus (group R) and 20 slides of atrophic‐erosive oral lichen planus (group AE). Results: Group R, the reticular lichen samples, had 18 diploid cases and two aneuploid cases. Group AE, the atrophic‐erosive lichen samples, had 10 diploid cases, one tetraploid case, and nine aneuploid cases. Of the 14 karyometric measurements of the nuclei of OLP epithelial cells analyzed, the group R mean values for mean density and minimum density were significantly greater than the group AE mean values, and mean roundness in group AE was significantly greater than in group R (t‐test: P < 0.05). Conclusions: The most common degree of DNA ploidy in OLP lesions was diploidy. Comparing the two groups (chi‐square test of association P = 0.021) demonstrated that diploidy was associated with the reticular clinical form of OLP, while aneuploidy was associated with the atrophic‐erosive clinical form of oral lichen planus.