The immunohistology of CD40 in human oral epithelium in health and disease

BACKGROUND: CD40 has a role in the regulation of immune responses, cell proliferation and migration, and apoptosis. Little is known of its distribution in oral mucosal pathology. METHODS: Oral keratinocyte lines were tested for CD40 protein by Western blotting. Immunohistochemistry was used to stain paraffin sections of oral mucosa in health and in inflammatory, reactive, dysplastic and malignant disease. RESULTS: Western blotting confirmed the presence of CD40 in oral keratinocytes. CD40 was generally expressed by keratinocytes in the basal layer, with variable parabasal expression. Langerhans cells also stained positively. Expression was lost in nine of 33 (27%) epithelial dysplasias, seven of which were severe. Eighty-one percent of well, 69% of moderately and 50% of poorly differentiated oral squamous cell carcinomas (OSCC) expressed CD40. Overall, 45 of 65 (69%) OSCC were positive. The pattern of expression was unrelated to tumour differentiation. CONCLUSION: CD40 expression by basal and parabasal oral keratinocytes is physiological. Expression is lost in approximately one-third of oral epithelial dysplasias and OSCC. The significance of such loss remains unknown, but may be related to immunological or other abnormalities of keratinocyte homeostasis.     

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.     

Pathogenesis of oral lichen planus – a review

J Oral Pathol Med (2010) 39 : 729–734 Oral lichen planus (OLP) is a T‐cell‐mediated chronic inflammatory oral mucosal disease of unknown etiology. OLP presents as white striations, white papules, white plaques, erythema, erosions, or blisters affecting predominantly the buccal mucosa, tongue and gingiva. Both antigen‐specific and non‐specific mechanisms are hypothesized to be involved in the pathogenesis of oral lichen planus (OLP). Antigen‐specific mechanisms in OLP include antigen presentation by basal keratinocytes and antigen‐specific keratinocyte killing by CD8⁺ cytotoxic T cells. Non‐specific mechanisms include mast cell degranulation and matrix metalloproteinase activation in OLP lesions. These mechanisms may combine to cause T cell accumulation in the superficial lamina propria, basement membrane disruption, intra‐epithelial T cell migration and keratinocyte apoptosis in OLP. The various hypotheses proposed for pathogenesis of oral lichen planus are discussed in this review.     

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.     

Probiotics: A non-conventional therapy for oral lichen planus

Oral lichen planus (OLP) is a common T-cell mediated chronic inflammatory disease. Although the etiology is still unclear, present studies suggest that the composition of the oral microbiota and psychological problems are implicated in the etiology of OLP. The pathogenesis of OLP includes mainly antigen-specific and non-specific mechanisms. Antigen-specific mechanisms involve T-cell activation following antigen presentation and apoptosis of basal keratinocytes triggered by CD8⁺ cytotoxic T cells, while non-specific mechanisms consist of matrix metalloproteinase over-expression and mast cell degranulation in OLP lesions. Therapies for OLP are mainly used to control symptoms and a specific cure is not yet available. Probiotics are capable of modulating the immune response in a strain-specific manner. They are able to alleviate microbial infection and suppress T-cell activation, infiltration and proliferation, as well as suppress keratinocyte apoptosis and nuclear factor-kappa B signaling. Furthermore, probiotics can also modulate the production of inflammatory cytokines and microRNAs, inhibit MMP-9 expression and mast cell degranulation, and ameliorate psychological problems, all of which are involved in the pathogenesis of OLP. Therefore, we hypothesize that probiotics may be applicable to OLP as a safe, inexpensive and non-conventional therapy.     

Expression of keratins 8, 18, and 19 in epithelia of atrophic oral lichen planus

Keratins form intermediate filaments of the cytoskeleton in keratinocytes and have roles in cell structure, signaling, intracellular transport, and cell death. Oral lichen planus (OLP) is an oral inflammatory disease with derangements in basal keratinocytes and disruption of the basal membrane. Here, we focused on epithelial expression of keratins 8, 18, and 19 because these proteins are known to modulate cell death. Biopsies were taken from buccal oral mucosa of persons with normal oral mucosa (n = 10) or atrophic OLP (n = 10). Cultured normal oral keratinocytes (n = 4) showed expression of mRNA and protein for keratins 8, 18, and 19. Immunohistochemistry showed consistent staining for keratins 8 and 18 in basal keratinocytes of normal oral mucosa. In OLP, staining for keratin (K)8 was mostly negative and staining for K18 was weak. Keratin 19 was expressed irregularly in most biopsies of normal oral mucosa and not at all in OLP. Several mononuclear leukocytes in the cellular infiltrate showed membrane staining for K8 and K18. Positive staining for K16 confirmed partial collapse of the basal cell layer in OLP. The basal cell niche in OLP therefore appeared to be partly populated with keratinocytes demonstrating a higher degree of differentiation (K8⁻ K18⁻ K19⁻ K16⁺); consequently, such areas may be more susceptible to the action of cell death factors released from the cell infiltrate as a result of lacking the protective, normal keratin present in the basal epithelial cell layer of normal oral mucosa.     

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。     

The high expression level of programmed death‐1 ligand 2 in oral lichen planus and the possible costimulatory effect on human T cells

J Oral Pathol Med (2011) 40 : 525–532 Background: Oral lichen planus (OLP) is a T‐cell‐mediated chronic autoimmune disease whose precise etiology is unknown. The recently identified costimulatory programmed death‐1 (PD‐1) molecule and its ligands, PD‐L1 and PD‐L2, have been identified as CD28‐B7 family molecules and constitute a regulatory pathway of potential therapeutic use in immune‐mediated diseases. Methods: We examined the expression of two ligands of PD‐1 at both the protein and gene level in the focal mucosa and peripheral blood of OLP patients using immunohistochemistry and real‐time PCR. Next, we used the PD‐L2.Ig fusion protein and observed its effects on T cells, which were co‐cultured with IFN‐γ‐treated keratinocytes (KCs) in the presence of PHA. Results: We found that the expression of PD‐L2 at both the gene and protein level was statistically different in peripheral blood and local lesion tissue of patients with OLP compared to the normal controls. The proliferation ability of T cells and the expression level of IFN‐γ in the supernatant of the above co‐culture model were significantly augmented (P < 0.05). PD‐L2.Ig fusion protein significantly aggravated the apoptosis of T cells, inhibited the proliferation of T cells and decreased the release levels of IL‐2 and IFN‐γ in the model (P < 0.05). Conclusion: These data show that the increased expression of PD‐L2, as a costimulatory molecule, may have an important modulatory function on the local immune responses of OLP in vivo.     

Role of apoptosis in erosive and reticular oral lichen planus exhibiting variable epithelial thickness

Oral lichen planus (OLP) is a chronic inflammatory disease with different clinical types. Reticular and erosive forms are the most common. Although the cause of OLP remains speculative, many findings suggest auto-immune involvement, mediated by T lymphocytes against the basal keratinocytes. Inflammation, mechanical trauma or toxic agents can affect the epithelial homeostasia. Increased apoptosis may cause a decrease in epithelial thickness reflecting in the activity of the lesion. The objective of this study was to evaluate the occurrence of apoptosis and epithelial thickness in reticular and erosive forms of OLP. 15 samples of OLP each type (reticular and erosive) plus 10 of healthy mucosa were collected and processed. After morphometry, the apoptotic index and epithelial thickness were obtained. TUNEL and M30 CytoDEATH immunohistochemical assay were used to validate the morphologic criteria used. Apoptosis in the erosive OLP was significantly more intense than in the reticular type and both forms of OLP presented more apoptosis than the healthy oral mucosa. Healthy oral mucosa was thicker than both OLP forms and thicker in OLP reticular form than in the erosive one. The clinical differences between reticular and erosive forms of OLP are related to variations in epithelial thickness and in intensity of apoptosis.     

Activation of nuclear factor-kappa B correlates with tumor necrosis factor-alpha in oral lichen planus: a clinicopathologic study in atrophic-erosive and reticular form

Backgroud:  Nuclear factor-kappa B (NF-κB) is believed to be involved in the pathogenesis of various inflammatory diseases, including oral lichen planus (OLP). The objective of the present study was to investigate the possible relationship between NF-κB activation and expression of tumor necrosis factor-alpha (TNF-α) in OLP and their expression pattern in relation to several clinical features.
Methods:  Thirty OLP cases were divided into atrophic-erosive form (14 cases) and reticular form (16 cases) according to their clinical manifestations. The expression of NF-κB p65 and TNF-α of both two groups were investigated by immunohistochemical staining, and the percentage of positive cells was calculated in each case. Biopsies of 10 normal oral mucosa (NOM) also underwent the same procedure as controls.
Results:  Nuclear factor-kappa B p65 nuclear staining was found in nuclei of basal and suprabasal epithelial keratinocytes in OLP, however, no positive staining was found in NOM. Positive TNF-α staining was detected in cytoplasm of basal epithelial keratinocytes in OLP, and only scattered staining was detected in NOM. Expression of NF-κB p65 and TNF-α were significantly different with respect to clinical forms and lesion sites ( P  < 0.05), except for genders ( P  > 0.05) in 30 OLP cases. NF-κB nuclear staining positively correlated ( r  = 0.676, P  < 0.01) with TNF-α overexpression in OLP.
Conclusions:  Nuclear factor-kappa B activation and its correlation with overexpression of TNF-α may play an important role in pathogenesis of OLP. There might be a positive regulatory loop between NF-κB and TNF-α, which may contribute to inflammation in OLP; NF-κB may also protect epithelial keratinocytes from excessive apoptosis.     

Composition of hemidesmosomes in basal keratinocytes of normal buccal mucosa and oral lichen planus

Oral lichen planus (OLP) is a chronic inflammatory disease displaying ultrastructural disturbances in epithelial hemidesmosomes. The expression of several key hemidesmosomal components in OLP as well as in normal buccal mucosa is, however, unknown. The aim of the study was therefore to examine intracellular and extracellular components involved in hemidesmosomal attachment, in OLP (n = 20) and in normal buccal mucosa (n = 10), by immunofluorescence. In normal buccal mucosa, laminin-α3γ2, integrin-α6β4, CD151, collagen α-1(XVII) chain, and dystonin showed linear expression along the basal membrane, indicating the presence of type I hemidesmosomes. Plectin stained most epithelial cell membranes and remained unphosphorylated at S4642. In OLP, most hemidesmosomal molecules examined showed disturbed expression consisting of discontinuous increases, apicolateral location, and/or intracellular accumulation. Plectin showed S4642-phosphorylation at the basement membrane, and deposits of laminin-α3 and laminin-γ2 were found within the connective tissue. The disturbed expression of hemidesmosomal proteins in OLP indicates deficient attachment of the basal cell layer, which can contribute to detachment and cell death of basal keratinocytes seen in the disease.     

4-1BB、CD8在口腔扁平苔藓组织中的表达研究

目的 检测共刺激信号4-1BB及CD8在口腔扁平苔藓(oral lichen planus,OLP)组织中的表达,探讨OLP病损中4-1BB介导CD8⁺ T细胞免疫应答的潜在机制。方法 选取31例OLP患者(糜烂型15例、非糜烂型16例),15例健康成人(对照组)为研究对象,收集其临床及病理资料,采用免疫组织化学法检测局部组织中4-1BB、CD8的表达情况。结果 OLP患者局部组织中4-1BB及CD8的表达高于对照组(P<0.05),且两者表达呈正相关(r=0.389,P<0.05),糜烂型OLP患者中CD8、4-1BB表达高于非糜烂型(P<0.05)。在OLP不同的临床亚组中,VAS评分、REU评分及基底细胞损伤程度均表现出差异(P<0.05)。Spearman相关性分析结果显示,CD8与基底细胞损伤程度呈正比(P<0.05),CD8、4-1BB分别与VAS评分、REU评分存在正相关性(P<0.05)。结论 OLP患者局部组织中4-1BB表达升高,其与疾病活动进展程度呈正相关,4-1BB可能通过调控CD8⁺ T细胞参与OLP的发生发展。     

Analysis of apoptosis-associated genes and pathways in oral cancer cells

BACKGROUND: Cancer cells can resist apoptosis that is induced by stimuli such as detachment or differentiation, but may be more susceptible to apoptosis when exposed to chemotherapeutic drugs. The pattern of gene expression that produces this phenotype is unknown. METHODS: We compared gene expression patterns of normal human epidermal keratinocytes (NHEK cells) and the oral cancer cell line Tu183 when the cells were exposed to different apoptosis-inducing stimuli. RESULTS: Pathway analysis revealed that the phenotype difference could be best explained by the simultaneous existence of both proapoptosis and antiapoptosis signals in the cancer cells. Microarray analysis, supported by immunoblotting, showed that one gene that was likely to be involved in the proapoptosis signal was TNFRSF5, which encodes the receptor CD40. When Tu183 cells were exposed to the CD40 ligand they showed apoptosis, while NHEK cells did not. CONCLUSIONS: The effects of different apoptotic stimuli on normal cells and oral cancer cells can be explained by expression of proapoptosis genes, including the gene that encodes CD40.     

Expression of adhesion and activation molecules in human buccal epithelial cell lines and normal human buccal epithelium in situ

An in vitro culture system may be used to analyse interactions between T cells and epithelium. We have analysed two human buccal squamous epithelial cell lines: SqCC/Y1, derived from a buccal carcinoma, and SVpgC2a, an SV-transformed healthy buccal squamous epithelium. Under serum-free culture conditions, the cell lines resemble normal buccal squamous epithelium in situ in their expression of MHC class I, CD29 (beta1 integrin), CD40, CD44, CD54 (ICAM-1), CD58 (LFA-3), CD95 (Fas), and E-cadherin. Furthermore, when the SVpgC2a cell line was treated with T-cell derived cytokines, upregulation of CD40 expression was observed when the cells were treated with IL-4, whereas IFN-gamma caused upregulation in expression of CD40, CD54 and MHC class II. These buccal squamous epithelial cell lines, therefore, provide a good tool for analysing interactions between epithelium and T cells in vitro.     

Oral lichen planus: Focus on etiopathogenesis

Lichen planus is a chronic mucocutaneous inflammatory disease, which frequently affects the oral mucosa of white females over 40 years old. Its aetiology remains uncertain and the pathogenesis is still the object of much speculation. The present paper presents the most well known antigens, and describes the action of different cells and proteins associated with the development of that disease, as well as the possible agents involved with its malignant transformation. Different external agents, especially virus, and internal agents, like stress, and the heat shock protein antigen expression, associated or not, can alter the basal keratinocytes of the oral mucosa making them susceptible to apoptosis by CD8⁺ cytotoxic T cell as well as activate matrix metalloproteinase and mast cell degranulation, which produce a great range of inflammatory mediators and cytokines determining the clinical onset of the disease. Regarding carcinogenesis, since it is a complex process and presents multifactorial origin, it is believed that there may be a synergism between intrinsic, such as inflammation mediators, and extrinsic agents (tobacco, alcohol, viral infections) for the OLP malignant transformation to occur. However, further studies are needed to better understand the origin, pathogenesis and process of malignant transformation of OLP.     

Membrane-bound CD40 ligand on T cells from mice injected with lipopolysaccharide accelerates lipopolysaccharide-induced osteoclastogenesis

Yokoyama M, Ukai T, Ayon Haro ER, Kishimoto T, Yoshinaga Y, Hara Y. Membrane‐bound CD40 ligand on T cells from mice injected with lipopolysaccharide accelerates lipopolysaccharide‐induced osteoclastogenesis. J Periodont Res 2011; 46: 464–474. © 2011 John Wiley & Sons A/S Background and Objective: T cells infiltrate the inflammatory site of periodontitis and consequently stimulate the loss of periodontal bone. We previously reported that T cells from lipopolysaccharide (LPS)‐injected mice (LPS‐T cells) accelerated osteoclastogenesis in the presence of LPS. Ηowever, the detailed mechanism of this acceleration is still unclear. In this study, we analyzed the mechanism of osteoclastogenesis accelerated by LPS‐T cells. Material and Methods: We examined the mechanism of osteoclastogenesis acceleration. First, to determine the effect of cell‐to‐cell contact, we co‐cultured T cells and bone marrow macrophages, prestimulated with RANKL for 48 h (R‐BMMs), in the presence of LPS for 24 h, in a Transwell. Second, to determine the effect of CD40 ligand (CD40L), we co‐cultured T cells and R‐BMMs in the presence of LPS and anti‐CD40L immunoglobulin. Third, we examined the effect of recombinant mouse CD40L (rCD40L) in the presence of LPS in vitro and in vivo. Lastly, we examined the expression of membrane‐bound CD40L (mCD40L) by fluorescence‐activated cell sorting (FACS). Results: Blocking cell‐to‐cell contact between LPS‐T cells and R‐BMMs completely inhibited the acceleration of osteoclastogenesis. Anti‐CD40L immunoglobulin also completely inhibited the acceleration of osteoclastogenesis. Moreover, rCD40L accelerated osteoclastogenesis in the presence of LPS in vitro and in vivo. Finally, the expression of mCD40L on LPS‐T cells was higher than that on T cells isolated from mice not injected with LPS. Conclusion: The results demonstrate that CD40L accelerates osteoclastogenesis in the presence of RANKL and LPS. The results also suggest that mCD40L on LPS‐T cells accelerates osteoclastogenesis.     

Expression of TGF-beta1, Smad7 and cell apoptosis in epithelium of oral lichen planus

OBJECTIVE: To examine the expression of TGF-beta1, Smad7 and cell apoptosis in oral lichen planus (OLP) and to evaluate the possible pathogenesis of oral lichen planus. METHODS: Immunohistochemical technique was used to study the expression of TGF-beta1 and Smad7 in the epithelia cells of 17 OLP cases and 7 normal oral mucosa (NOM). TUNEL was used for detecting the cell apoptosis in 17 OLP cases and 7 NOM. RESULTS: TGF-beta1 was moderately positive in the epithelia cells of OLP. All the epithelia cells in OLP showed strong cytoplasmic staining. The expression of TGF-beta1 and Smad7 were significantly increased in OLP compared with that in NOM (P < 0.05). Cell apoptotic index (AI) was remarkably increased in epithelia cells in OLP cases, and the cell apoptosis was localized in basal and suprabasal epithelial layers. There was a positive correlation between TGF-beta1 expression and cell apoptosis in the epithelia of OLP (r = 0.69, P <0.05). CONCLUSIONS: High expression of TGF-beta1 and Smad7 in the epithelia of OLP suggests that TGF-beta1-Smad7 signal pathway was disturbed in oral lichen planus. The imbalance of TGF-beta1-Smad7 pathway may contribute to the mechanisms of cell apoptosis of epithelial cells in OLP.     

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.     

Apoptosis and cell cycle arrest in oral lichen planus Hypothesis on their possible influence on its malignant transformation

The quantitative importance of cell cycle arrest and apoptosis mechanisms in oral lichen planus (OLP) was analysed in order to assess the cell response to T lymphocyte aggression and establish a hypothesis on the influence of these phenomena in the malignant transformation process. The TUNEL assay and immunohistochemical methods were used to detect caspase-3, bax, and p21 in 32 tissue samples of oral mucosa with OLP and in 20 samples of normal oral mucosa. Positivity for TUNEL, caspase-3 and p21 was significantly more frequent in cases than in controls (p<0.001). Both TUNEL and caspase-3 positivity was significantly greater in the basal versus suprabasal layer (p=0.004 and 0.052, respectively). The basal and suprabasal expression of p21 was significantly higher in cases with a more intense liquefaction degeneration (p<0.01). There was no significant difference in basal expression of bax between cases and controls. The quantitative importance of apoptosis was small in OLP. Epithelial cells attacked in OLP have a very low response to apoptosis and cell cycle arrest mechanisms, which may produce an epithelial substrate that favours malignant transformation.     

Expression of caspase-3 and structural changes associated with apoptotic cell death of keratinocytes in oral lichen planus

OBJECTIVE: Apoptosis appears to be the mode of cell death by which damaged cells are removed from the lesional tissue. The aim of this study was to examine keratinocyte apoptosis and caspase-3 (CPP32) expression in oral lichen planus (OLP). MATERIALS AND METHODS: Paraffin-embedded samples of OLP (n = 30) and normal oral mucosa (NOM; n = 5) were prepared for haematoxylin-eosin (H & E), immunohistochemistry and electron microscopy. The number of apoptotic cells and the proportion of total cells that were either apoptotic (apoptotic index; AI) or mitotic (mitotic index; MI) were assessed in H & E stained sections. An immunostaining-intensity-distribution index (IIDI; proportion of stained cells x staining intensity) was used to assess CPP32 immunoreactivity. RESULTS: Results showed a significant increase in the number of apoptotic cells in OLP (P < 0.001). In OLP, all apoptotic bodies were found in the basal and prickle epithelial layers. Compared with NOM, the AI was significantly greater in atrophic (P < 0.05), reticular (P < 0.001) and plaque-like (P < 0.01) OLP. The MI was significantly greater in plaque-like OLP (P < 0.01). The proportion of CPP32-positive cells and the IIDI were significantly greater in all forms of OLP compared with NOM (P < 0.05). No difference in CPP32 expression was evident between clinical forms of OLP. Electron microscopy confirmed the light microscopic finding of apoptosis. CONCLUSION: Keratinocyte apoptosis and caspase-3 expression co-localized to the basal and parabasal epithelial layers, suggesting that proliferating epithelial cells may be targeted for destruction in OLP. Differences in epithelial AI and MI may underlie the various clinical and histological appearances of OLP.