The effect of resolvin D1n-3 DPA on primary oral epithelial cell migration in vitro.

Specialized pro-resolving lipid mediators (SPMs) are known for their anti-inflammatory and pro-resolving actions. The aim of the present study was to find new functions of the SPM resolvin D1n-3 DPA (RvD1n-3 DPA) on oral epithelial cells. As a starting point, we used a dataset obtained by RNA high-throughput sequencing of oral epithelial cells exposed to TNF-α and RvD1n-3 DPA versus TNF-α alone. GOrilla enrichment analysis showed that the actin cytoskeleton was significantly overrepresented after adjustment for multiple hypothesis testing. As actin, amongst others, is closely related to cell migration, we then explored whether RvD1n-3 DPA can modulate oral epithelial cell migration. To this end, we used an in vitro cell migration model, including TNF-α treatment, to mimic an inflammatory cell state. The analysis revealed that RvD1n-3 DPA increased oral epithelial cell migration in the presence but not in the absence of TNF-α. Addition of RvD1n-3 DPA also induced F actin accumulation around the cell nucleus, indicating that RvD1n-3 DPA potentially can mediate processes of intracellular transport. This indicates that this lipid mediator may be a promising therapeutic candidate in oral mucosal wound healing.

RvD1n-3 DPA Downregulates the Transcription of Pro-Inflammatory Genes in Oral Epithelial Cells and Reverses Nuclear Translocation of Transcription Factor p65 after TNF-α Stimulation.

Specialized pro-resolving mediators (SPMs) are multifunctional lipid mediators that participate in the resolution of inflammation. We have recently described that oral epithelial cells (OECs) express receptors of the SPM resolvin RvD1n-3 DPA and that cultured OECs respond to RvD1n-3 DPA addition by intracellular calcium release, nuclear receptor translocation and transcription of genes coding for antimicrobial peptides. The aim of the present study was to assess the functional outcome of RvD1n-3 DPA-signaling in OECs under inflammatory conditions. To this end, we performed transcriptomic analyses of TNF-α-stimulated cells that were subsequently treated with RvD1n-3 DPA and found significant downregulation of pro-inflammatory nuclear factor kappa B (NF-κB) target genes. Further bioinformatics analyses showed that RvD1n-3 DPA inhibited the expression of several genes involved in the NF-κB activation pathway. Confocal microscopy revealed that addition of RvD1n-3 DPA to OECs reversed TNF-α-induced nuclear translocation of NF-κB p65. Co-treatment of the cells with the exportin 1 inhibitor leptomycin B indicated that RvD1n-3 DPA increases nuclear export of p65. Taken together, our observations suggest that SPMs also have the potential to be used as a therapeutic aid when inflammation is established.

Expression and function of resolvin RvD1n-3 DPA receptors in oral epithelial cells.

Chronic inflammatory responses can inflict permanent damage to host tissues. Specialized pro-resolving mediators downregulate inflammation but also can have other functions. The aim of this study was to examine whether oral epithelial cells express the receptors FPR2/ALX and DRV1/GPR32, which bind RvD1n-3 DPA , a recently described pro-resolving mediator derived from omega-3 docosapentaenoic acid (DPA), and whether RvD1n-3 DPA exposure induced significant responses in these cells. Gingival biopsies were stained using antibodies to FPR2/ALX and DRV1/GPR32. Expression of FPR2/ALX and DRV1/GPR32 was examined in primary oral epithelial cells by qRT-PCR, flow cytometry, and immunofluorescence. The effect of RvD1n-3 DPA on intracellular calcium mobilization and transcription of beta-defensins 1 and 2, and cathelicidin was evaluated by qRT-PCR. FPR2/ALX and DRV1/GPR32 were expressed by gingival keratinocytes in situ. In cultured oral epithelial cells, FPR2/ALX was detected on the cell surface, whereas FPR2/ALX and DRV1/GPR32 were detected intracellularly. Exposure to RvD1n-3 DPA induced intracellular calcium mobilization, FPR2/ALX internalization, DRV1/GPR32 translocation to the nucleus, and significantly increased expression of genes coding for beta-defensin 1, beta-defensin 2, and cathelicidin. This shows that the signal constituted by RvD1n-3 DPA is recognized by oral keratinocytes and that this can strengthen the antimicrobial and regulatory potential of the oral epithelium.

The prognostic role of combining Krüppel-like factor 4 score and grade of inflammation in a Norwegian cohort of oral tongue squamous cell carcinomas.

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor involved in inflammation, cancer development, and progression. However, the relationship between KLF4, inflammation, and prognosis in oral cancer is not fully understood. KLF4 expression levels were examined in a multicenter cohort of 128 oral squamous cell carcinoma (OSCC) specimens from the tongue (OTSCC) using immunohistochemistry. In two external KLF4 mRNA datasets (The Cancer Genome Atlas/The Genotype-Tissue Expression Portal), lower KLF4 mRNA expression was found in OSCC and head and neck squamous cell carcinomas (HNSCC) than in control oral epithelium. These data indicate that down-regulation of KLF4 mRNA is linked to OSCC/HNSCC progression. Using Cox-multivariate analysis, a significantly favorable 5-year disease-specific survival rate was observed for a subgroup of patients with a combination of high levels of KLF4 expression and inflammation. OSCC cell lines exposed to IFN-γ showed a significant upregulation of nuclear KLF4 expression, indicating a link between inflammation and KLF4 expression in OSCC. Overall, the current data suggest a functional link between KLF4 and inflammation. The combination of high KLF4 nuclear expression and marked/moderate stromal inflammation might be useful as a favorable prognostic marker for a subgroup of OTSCC patients.

Origin of langerin (CD207)-expressing antigen presenting cells in the normal oral mucosa and in oral lichen planus lesions.

The number of langerin-expressing antigen-presenting cells is higher in oral lichen planus than in normal oral mucosa. However, langerin may be expressed by several functionally different lineages of antigen presenting cells (APCs), and this has important implications for our understanding of the pathogenesis of oral lichen planus. The aim of this study was to determine the origin of the langerin-expressing APCs. To this end, we examined oral mucosal biopsies from healthy persons and patients with oral lichen planus using multicolor immunofluorescence. In normal oral mucosa, a substantial fraction of Langerhans cells expressed Ki-67, indicating that steady-state oral mucosal Langerhans cells are at least partially maintained by self-renewal. In oral lichen planus, the numbers of Langerhans cells were higher but proliferation was not altered, indicating that the higher cell numbers appeared to depend on recruited dendritic cell (DC)-precursors. Moreover, we found a markedly higher number of langerin+ APCs within the lamina propria of oral lichen planus lesions. Such cells did not display monocyte- or macrophage markers, but rather showed a phenotype compatible with tissue-elicited IRF4+ cDC2. Detailed understanding of how the oral mucosal APC network is regulated and the functional capacities of the different ontogenies may identify novel treatment targets for oral lichen planus.

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-α6ß4, 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.

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.

The oral commensal Streptococcus mitis activates the aryl hydrocarbon receptor in human oral epithelial cells.

Streptococcus mitis (S. mitis) is a pioneer commensal bacterial species colonizing many of the surfaces of the oral cavity in healthy individuals. Yet, not much information is available regarding its interaction with the host. We used examination of its transcriptional regulation in oral keratinocytes to elucidate some of its potential roles in the oral cavity. Transcription factor analysis of oral keratinocytes predicted S. mitis-mediated activation of aryl hydrocarbon receptor (AhR). Activation and functionality of AhR was confirmed through nuclear translocation determined by immunofluorescence microscopy and real-time polymerase chain reaction with reverse transcription analysis of CYP1A1, the hallmark gene for AhR activation. Addition of Streptococcus mutans or Streptococcus gordonii did not induce CYP1A1 transcription in the keratinocyte cultures. Introduction of an AhR-specific inhibitor revealed that S. mitis-mediated transcription of CXCL2 and CXCL8 was regulated by AhR. Elevated levels of prostaglandin E2 (enzyme-linked immunosorbent assay) in supernatants from S. mitis-treated oral epithelial cells were also attenuated by inhibition of AhR activity. The observed AhR-regulated activities point to a contribution of S. mitis in the regulation of inflammatory responses and thereby to wound healing in the oral cavity. The concept that the oral commensal microbiota can induce AhR activation is important, also in view of the role that AhR has in modulation of T-cell differentiation and as an anti-inflammatory factor in macrophages.

Lysophosphatidic acid induces expression of genes in human oral keratinocytes involved in wound healing.

OBJECTIVE: Epithelial cells participate in wound healing by covering wounds, but also as important mediators of wound healing processes. Topical application of the phospholipid growth factor lysophosphatidic acid (LPA) accelerates dermal wound healing and we hypothesized that LPA can play a role in human oral wound healing through its effects on human oral keratinocytes (HOK). DESIGN: HOK were isolated from gingival biopsies and exposed to LPA. The LPA receptor profile, signal transduction pathways, gene expression and secretion of selected cytokines were analyzed. RESULTS: HOK expressed the receptors LPA1, LPA5 and LPA6 and LPA activated the ERK1/2, JNK and p38 intracellular pathways, substantiated by secretion of IL-6 and IL-8. The early (2h) and intermediate (6h) gene expression profiles of HOK after LPA treatment showed a wide array of regulated genes. The majority of the strongest upregulated genes were related to chemotaxis and inflammation, and became downregulated after 6h. At 6h, genes coding for factors involved in extracellular matrix remodeling and re-epithelialization became highly expressed. IL-36γ, not earlier known to be regulated by LPA, was strongly transcribed and translated but not secreted. CONCLUSIONS: After stimulation with LPA, HOK responded by regulating factors and genes that are essential in wound healing processes. As LPA is found in saliva and is released by activated cells after wounding, our results indicate that LPA has a favorable physiological role in oral wound healing. This may further point towards a beneficial role for application of LPA on oral surgical or chronic wounds.

The Role of Nerve Growth Factor (NGF) and Its Precursor Forms in Oral Wound Healing.

Nerve growth factor (NGF) and its different precursor forms are secreted into human saliva by salivary glands and are also produced by an array of cells in the tissues of the oral cavity. The major forms of NGF in human saliva are forms of pro-nerve growth factor (pro-NGF) and not mature NGF. The NGF receptors tropomyosin-related kinase A (TrkA) and p75 neurotrophin receptor (p75NTR) are widely expressed on cells in the soft tissues of the human oral cavity, including keratinocytes, endothelial cells, fibroblasts and leukocytes, and in ductal and acinar cells of all types of salivary glands. In vitro models show that NGF can contribute at most stages in the oral wound healing process: restitution, cell survival, apoptosis, cellular proliferation, inflammation, angiogenesis and tissue remodeling. NGF may therefore take part in the effective wound healing in the oral cavity that occurs with little scarring. As pro-NGF forms appear to be the major form of NGF in human saliva, efforts should be made to study its function, specifically in the process of wound healing. In addition, animal and clinical studies should be initiated to examine if topical application of pro-NGF or NGF can be a therapy for chronic oral ulcerations and wounds.

Phenotypically non-suppressive cells predominate among FoxP3-positive cells in oral lichen planus.

BACKGROUND: Oral lichen planus (OLP) is a common T-cell-dominated oral chronic inflammatory disease occurring in periods of remission, quiescence, activity with pronounced inflammation, and acute ulceration. Cell infiltrates in OLP contain varying numbers of CD4+ T cells expressing the transcription factor FoxP3. FoxP3+ CD4+ T cells are, however, a heterogeneous cell population containing suppressive and non-suppressive cells, and their distribution in infiltrates from OLP is unknown. METHODS: Biopsies were taken from normal oral mucosa (n = 8) and OLP lesions (n = 19), and a set of in situ methods for the determination of the functional phenotype of FoxP3+ CD4+ T cells was applied. RESULTS: Numbers of FoxP3+ CD4+ T cells were highest in the atrophic form of the disease, yet low in the ulcerative form. The main FoxP3+ CD4+ T-cell population observed was FoxP3+ CD45RA- CD25+ CD45RO+ and CD15s- , a phenotype delineating a non-suppressive subset. Numbers of cells with an actively suppressing phenotype (FoxP3+ CD45RA- CD25+ CD45RO+ and CD15s+ ) were, however, about twice as high in reticular lesions as compared with the atrophic form. Many FoxP3+ CD4+ T cells expressed T-bet, the hallmark transcription factor for IFN-γ-producing T cells, indicating that they may enhance immune and inflammatory responses rather than suppress them. CONCLUSIONS: The absence of actively suppressing FoxP3+ CD4+ T cells may in part explain why OLP is a remarkably persisting condition, in spite of the presence of substantially high numbers of FoxP3+ CD4+ T cells. The findings emphasize that it is crucial to examine not only numbers but also functional phenotype of FoxP3+ CD4+ T cells in human tissues.

Lysophosphatidic acid regulates adhesion molecules and enhances migration of human oral keratinocytes.

Oral keratinocytes are connected via cell-to-cell adhesions to protect underlying tissues from physical and bacterial damage. Lysophosphatidic acids (LPAs) are a family of phospholipid mediators that have the ability to regulate gene expression, cytoskeletal rearrangement, and cytokine/chemokine secretion, which mediate proliferation, migration, and differentiation. Several forms of LPA are found in saliva and gingival crevicular fluid, but it is unknown how they affect human oral keratinocytes (HOK). The aim of the present study was therefore to examine how different LPA forms affect the expression of adhesion molecules and the migration and proliferation of HOK. Keratinocytes were isolated from gingival biopsies obtained from healthy donors and challenged with different forms of LPA. Quantitative real-time RT-PCR, immunocytochemistry, and flow cytometry were used to analyze the expression of adhesion molecules. Migration and proliferation assays were performed. Lysophosphatidic acids strongly promoted expression of E-cadherin and occludin mRNAs and translocation of E-cadherin protein from the cytoplasm to the membrane. Occludin and claudin-1 proteins were up-regulated by LPA. Migration of HOK in culture was increased, but proliferation was reduced, by the addition of LPA. This indicates that LPA can have a role in the regulation of the oral epithelial barrier by increasing the expression of adhesion molecules of HOK, by promotion of migration and by inhibition of proliferation.

Distribution of nerve growth factor, pro-nerve growth factor, and their receptors in human salivary glands.

Nerve growth factor (NGF) is a pluripotent mediator that is present in a range of human tissues. Nerve growth factor was originally considered important only in neuronal homeostasis and pathophysiology, but later it was also implicated in the pathophysiology of inflammation, epithelial differentiation, and wound healing. In this study, the distribution of nerve growth factor beta (NGF-ß) and pro-NGF, and their receptors - tyrosine kinase A (TrkA) and p75(NTR) - was examined in human parotid, submandibular, sublingual, and labial salivary glands by immunohistochemistry. Intercalated, striated, and collecting-ducts in all gland types showed strong staining for pro-NGF but only weak cytoplasmic or sparse nuclear staining for NGF-ß. Tyrosine kinase A was strongly expressed in the ducts of all gland types, whereas p75(NTR) expression was mainly confined to collecting ducts. In acini, no or only weak cytoplasmic staining was found for all markers, and some nuclei stained positive for NGF-ß, pro-NGF, and TrkA. Western blotting of saliva showed secretion of several forms of pro-NGF, while no mature NGF-ß was detected. Salivary pro-NGF may play a role in oral wound healing.

Expression of the T-cell-specific adapter protein in oral epithelium.

The multifunctional T-cell-specific adapter protein (TSAd) was originally described in T cells but is also expressed in epithelial cells from the respiratory tract and in endothelium. In this study, we found expression of TSAd messenger RNA (mRNA) and protein in both human and murine oral mucosal epithelium as well as in human primary oral keratinocyte cell cultures. In TSAd(-/-) mice, the mucosa and skin appeared macroscopically normal, but severe disturbances were observed in the fine structures of the basal membrane and intercellular epithelial spaces upon analysis using transmission electron microscopy. Oral epithelial cells from TSAd(-/-) mice displayed decreased migration compared with cells from wild-type mice, whereas overexpression of TSAd in a human epithelial cell line resulted in impaired proliferation. This study is the first to show that TSAd is expressed in normal oral mucosa, that it is important for the normal ultrastructural morphology of the epithelium and the basal membrane, and that it is involved in the migration and proliferation of oral keratinocytes.

Induction of invasion in an organotypic oral cancer model by CoCl2, a hypoxia mimetic.

Invasion is a hallmark of malignancy. The aim of this study was to develop an in vitro model that can be used for experimental studies of cancer cell invasion. The organotypic oral cancer model was constructed by growing oral squamous cell carcinoma (OSCC) cells on a collagen matrix in which normal human fibroblasts were incorporated. Immunohistochemical staining of the model showed that the expression of invasion-related molecules such as phosphorylated extracellular signal-regulated kinases 1 and 2 (p-ERK1/2), cyclooxygenase-2 (COX-2), p75(NTR), and hepatocyte growth factor receptor (Met) was similar to that seen in OSCC. Treatment of the model with cobalt chloride (CoCl(2)) to mimic hypoxic conditions increased cancer cell invasion, defined as the appearance of cancer cell islands protruding into the matrix. Models treated with CoCl(2) showed increased expression of p75(NTR) and laminin-5 in the cancer cells, and a more pronounced fragmentation of collagen IV in the basal membrane area, in contrast to models that were left untreated. The results indicate that the present model is well suited for studies on cancer cell invasion in the matrix and that the addition of CoCl(2) on day 3 of the experiment is indicated because it markedly increases the invasion and improves the model.

Aquaporin 11 in the developing mouse submandibular gland.

Several aquaporins (AQPs) have been detected in mature and embryonic mammalian salivary glands (AQP1 and AQP3-AQP8). However, AQP11 has, to our knowledge, never before been described in salivary glands, but is known to be important in, for example, kidney development in mice. We therefore thought it relevant to investigate if AQP11 was present during salivary organogenesis. The submandibular salivary gland (SMG) from CD1 mice was studied during prenatal development and early postnatal development, and also in young adult male and female mice. The expression trend of the AQP11 transcript was detected using the reverse transcription-polymerase chain reaction (RT-PCR), and the temporal-spatial pattern was observed using in situ hybridization. The AQP11 transcript was first detected at embryonic day 13.5 and showed a more or less constitutive expression trend during the prenatal and early postnatal SMG development. Spatial studies demonstrated that the AQP11 transcript was present in the developing and mature duct structures at all stages studied. In the end pieces, the AQP11 transcript was reduced during glandular development. Our results point to an important role for AQP11 during salivary gland development.

NGF and its receptors TrkA and p75NTR in the epithelium of oral lichen.

BACKGROUND: Nerve growth factor (NGF) can through its receptors TrkA and p75(NTR) convey signals for cell survival, differentiation and death. The aim of this study was to examine whether NGF can play a role in the pathology of oral lichen (OL). METHODS: Sections from biopsies taken from patients with erythematous (ERY) OL and from volunteers with normal oral mucosa (NOM) were immunostained with antibodies against NGF, proNGF, TrkA, phosphorylated Trk, p75(NTR) and phosphorylated Akt (pAkt) and expression of RNA coding for proNGF/NGF was investigated by in situ hybridization. RESULTS: Both in ERY OL and NOM, cytoplasmic staining for NGF was seen in granular and upper spinous cell layers of the epithelium, whereas proNGF staining was seen in all epithelial cell layers. In situ hybridization showed that the proNGF protein was produced in the same cell layers. In OL, strong cytoplasmic stainings for TrkA and activated Trk (pTrk) were observed in all epithelial cell layers while these stainings were only weak in NOM. Basal keratinocytes in OL showed no or only weak cytoplasmic staining for p75(NTR), but in NOM there was a clear cell membrane staining. In OL, strong cytoplasmic and intermittent nuclear staining for pAkt was observed in spinous, granular and superficial layers, while basal and parabasal keratinocytes were negative. This staining was weak or absent in the entire epithelium of NOM. CONCLUSIONS: TrkA upregulation and activation in OL is one of the pathways that can activate pAkt and thereby rescue epithelial cells from untimely cell death.

Nerve growth factor beta/pro-nerve growth factor and their receptors in normal human oral mucosa.

Nerve growth factor beta (NGF-beta) and its precursor proNGF are important for the differentiation and survival of neurons and dermal keratinocytes. The aim of this study was to determine the role that NGF might play in the differentiation and wound healing of oral mucosa. Cultured normal human oral mucosal keratinocytes expressed mRNA for NGF-beta/proNGF and for their receptors TrkA and p75(NTR). Lysates from cultured oral mucosal keratinocytes did not contain detectable amounts of mature 14-kDa NGF-beta but did contain several NGF proforms with molecular weights between 32 and 114 kDa. Culture medium from oral mucosal keratinocytes contained 75 kDa proNGF. The addition of NGF-beta significantly enhanced the proliferation of oral mucosal keratinocyte cultures and in vitro scratch closure. Immunostaining of biopsies from normal oral mucosa showed the presence of proNGF in all epithelial layers. NGF staining was observed in the granular and upper spinous cell layers. TrkA immunoreactivity was detected in basal and parabasal cells, with weak to moderate staining in spinous and granular cell layers. p75(NTR) staining was seen in basal cell layers. These findings indicate that NGF-beta/proNGF have mitogenic and motogenic effects on oral mucosal keratinocytes and therefore may aid in the healing of oral wounds. Differential expression of NGF and NGF receptors throughout the epithelium suggests a role in epithelial differentiation.

Survival signalling in keratinocytes of erythematous oral lichen planus.

BACKGROUND: Keratinocytes in oral lichen (OL) planus have been shown to be exposed to potentially cell death-inducing factors such as tumour necrosis factor-alpha (TNF-alpha) and FasL, produced by the cells of the inflammatory infiltrate and by the keratinocytes themselves. Mostly, however, the lesions do not show ulceration, the clinical manifestation of substantial keratinocyte death. The aim of this study was to find support for the contention that there is activation of protecting anti-apoptotic mechanisms in keratinocytes in a form of chronic OL (erythematous OL; ERY OL), simultaneously with the pathological cell death signals. METHODS: Biopsies from patients with normal oral mucosa (NOM) or with ERY OL were compared by immunohistological staining. RESULTS: In ERY OL keratinocytes, both the pro-apoptotic FADD and the anti-apoptotic molecules p-IKK, NF-kappaB/p50, FLIP(L), cIAP-1 and cIAP-2 were strongly upregulated when compared with NOM. There were no significant differences in the staining patterns for active caspase-3 and caspase-8 with only few positive cells for both enzymes. CONCLUSIONS: The presently observed marked increase in expression of anti-apoptotic molecules in ERY OL epithelium may counteract the pro-apoptotic assault and rescue the epithelium from rampant cell death and thereby clinical ulceration.

Identity of TUNEL-positive cells in the oral buccal epithelium of normal mucosa and lichen lesions.

BACKGROUND: In situ detection of DNA fragmentation by TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end labeling (TUNEL) is a widely used technique to identify apoptotic cells in the terminal phases of cell death. Several studies have shown that there are statistically increased numbers of TUNEL(+) cells within the epithelium of oral lichen (OL). It was suggested that this indicates an increased rate of apoptosis among basal and suprabasal keratinocytes in OL epithelium. The aim of this study was to identify the TUNEL(+) cells in the epithelium of erythematous (ERY) OL and normal oral mucosa (NOM). METHODS: Sections of biopsies from NOM and ERY OL were processed for TUNEL combined with immunostaining for pan-cytokeratin or for cell markers specifically expressed by different leukocytes. RESULTS: In NOM, TUNEL(+) keratinocytes were almost exclusively seen in the outermost epithelial layers. This labeling was absent in ERY OL. In the basal and lower spinous layers, more TUNEL(+) cell nuclei were seen in ERY OL as compared with NOM, in accordance with previous studies. The present observations show, however, that only very few of these cells were keratinocytes, but rather were CD4(+) lymphocytes and CD68(+) macrophages. There was no difference between the numbers of TUNEL(+) keratinocytes in basal and lower spinous layers in ERY OL and NOM epithelium. No intraepithelial CD8(+) lymphocytes, Langerhans cells, or mast cells were found to be TUNEL(+). CONCLUSION: The findings indicate that the pathologic changes in ERY OL epithelium cannot be explained by increased prevalence of terminal keratinocyte cell death identified by TUNEL.