The Roles Of OPN-Regulated CEACAM1 Expression In Immunopathogenesis Of Oral Lichen Planus

Oral lichen planus (OLP) is a common chronic inflammatory disorder of oral mucosa. Because of its uncertain etiology, no effective therapeutic approach, chronicity, liability to relapse and potentiality of malignant transformation, OLP is known as a difficult condition in oral medicine clinic. In particular, The 14th International Cancer Congress considered OLP as a premalignant condition, which put great pressure on the patients. Although the exact etiology of OLP is unknown in the majority of the cases, it is considered as a multifactorial process related to many factors, such as genetic influence, and immune, endocrine, psychological or infectious conditions. Among these, immune serves as the most important factor. It is widely accepted that OLP is a cell-mediated autoimmune disease. Pathological study demonstrated that abundant T lymphocytes infiltrated the oral mucosa, in which activated T cells trigger apoptosis of oral epithelial cells is an important mechanism for OLP. However, to date the molecular mechanisms underlying the abnormal infiltration of activated T cells and the apoptosis of oral epithelial cells in OLP remain unclear.Carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a member of Ig superfamily and carcinoembryonic antigen (CEA) family of molecules. In human, CEACAM1 is expressed by certain epithelial, endothelial, lymphoid and myeloid cells. It is a novel multifunctional protein, which is involved in homophilic intercellular adhesion and intracellular signaling events in normal as well as in cancer cells. It is well established that CEACAM1 protein has crucial roles in cell proliferation, angiogenesis, and insulin clearance. It has been recently recognized that CEACAM1 also plays an important role in modulating of innate and adaptive immune responses. Although abnormal expressions of CEACAM1 in several autoimmune diseases have been reported, the expression pattern of CEACAM1 in OLP is unknown. Furthermore, the roles of CEACAM1 in immunopathogenesis of T cell-mediated autoimmune diseases and the mechanisms underlying the dysregulation of CEACAM1 expressions in these conditions still need to be determined.Osteopontin (OPN) is an important extracellular matrix protein. It participates in a wide range of biological processes, including cell migration and adhesion, cell-mediated immunity, wound healing, bone remodeling, and metastasis of tumor. It has been reported that OPN was colocalized with CEACAM1 in the extravillous trophoblast of the human placenta and could enhance the invasion of CEACAM1-expressing placental cells. In the previous study, we have found that OPN was overexpressed in sera and in local oral mucosal epithelia in OLP patients and has potential suppressive effects on the apoptosis of activated T cells. However the mechanisms underlying the regulation of apoptosis by OPN remain unclear.In this study, we began by hypothesizing that CEACAM1 may be involved in regulating the T-cell-mediated immunity in OLP and may be correlated with OPN. Therefore we firstly designed experiments to determine the expression patterns of CEACAM1 on peripheral blood T cells and in local oral mucosal tissues in OLP patients. Secondly, we investigated whether the expressions of CEACAM1 on T cells and oral keratinocytes can be regulated by OPN. Furthermore, we performed experiments to explore the effects of CEACAM1 on apoptosis of human oral keratinocytes and activated T cells. It was hoped that this study will offer new insights into our understanding of the regulation and the roles of CEACAM1 in immunopathogenesis of OLP and other T cell-mediated autoimmune diseases. Part I The Expressions of CEACAM1 in Oral Lichen PlanusObjective To determine the expression patterns of CEACAM1 on peripheral blood T cells and in local oral mucosal tissues in OLP patients and to explore the role of CEACAM1 in immunopathogenesis of OLP.Methods 1. The detection of CEACAMl expressions on peripheral blood CD4+ T cells and on CD8+ T cells from OLP patients and healthy controls. Peripheral bloodsamples were obtained from patients with OLP and healthy adult controls. The cell surface expressions of CEACAM1 on peripheral blood CD4+ T and on CD8+ T cells were determined by flow cytometry.2. The detection of CEACAM1 expressions in local oral mucosal tissues from OLP patients and healthy controls. Oral mucosal tissue samples were obtained from patients with OLP and healthy adult controls. The expressions of CEACAM1 in local oral mucosal tissues were determined by immunohistochemistry.Results1. Cell surface expression of CEACAM1 on peripheral blood T cells. The mean percentage of CEACAM1+ population in CD8+T cells in OLP patients was 35.42%, which is about 7-fold higher than that in normal controls (4.70%) (p< 0.01). The mean percentage of CEACAM1+ population in CD4+ T cells in OLP patients was 9.65%, which is also significantly higher than that in normal controls (5.15%) (p< 0.05).2. CEACAM1 expression in local oral mucosal tissues. Overexpression of CEACAM1 was observed in the epithelia of OLP mucosas. The quantitative score (Fromowitz) of CEACAMl expression in oral mucosal epithelia from OLP patients was 4.98±1.61, which is statistically higher than that of controls (2.35±1.18) (p< 0.01). A band of abundant inflammatory mononuclear cells infiltrated the lamina propria of OLP mucosas. A fraction of the inflammatory mononuclear cells were positive for CEACAM1. The percentages of CEACAM1+ population in infiltrating inflammatory mononuclear cells from 43 OLP patients ranged from 5% to 60%, with a mean of 12%. Notably, the majority of the CEACAM1+ cells were located in the deeper layer of the infiltrating inflammatory cells band or nearby small vessels.ConclusionsCEACAM1 was abnormally expressed on peripheral blood T cells and in local oral mucosal tissues from OLP patients. This implied that CEACAM1 might play some important role in pathogenesis of OLP. Part II The Regulation of CEACAM1 Expression on T cells and on Human Oral Keratinocytes by OPN in VitroObjective In the previous study, we have found that OPN was overexpressed in sera and in local oral mucosal epithelia in OLP patients and has potential suppressive effects on the apoptosis of activated T cells. However the mechanisms underlying the regulation of apoptosis by OPN remain unclear. In Part I of this study, we found that CEACAM1 was abnormally expressed on peripheral blood T cells and in local oral mucosal tissues from OLP patients. In this part, we performed experiments to investigate whether the expressions of CE AC AMI on T cells and oral keratinocytes can be regulated by OPN, and whether CEACAM1 mediate the effect of OPN on immune response.Methods1. Isolation of T cells. Human peripheral blood was drawn from healthy adult volunteers under written informed consent. PBMC were prepared by Ficoll gradient centrifugation. Sorting of CD3+ T cells was performed by magnetic cell sorting (MACS) according to the manufacturer’s instructions. The purity of the cell population was> 96%, as determined by flow cytometry.2. Study on the regulation of CEACAM1 expression on primary T cells by OPN in vitro. Freshly purified T cells which served as primary T cells were cultured in RPMI 1640 media either in the absence or in the presence of OPN. Cells were cultured at 37℃in a 5% CO2 atmosphere for 72 hours and were then harvested. The expressions of CEACAM1 on T cells were analyzed by flow cytometry.3. Study on the regulation of CEACAM1 expression on activated T cells by OPN in vitro. Freshly purified T cells activated with IL-2, anti-CD3 mAb and anti-CD28 mAb were cultured in RPMI 1640 media either in the absence or in the presence of OPN. Cells were cultured at 37℃in a 5% CO2 atmosphere for 72 hours and were then harvested. Culture supernatants were collected by centrifugation and stored at -80℃before use. The expressions of CEACAM1 on T cells were analyzed by flow cytometry.4. Study on the regulation of CEACAM1 expression on Human Oral Keratinocytes (HOK) by OPN in vitro. HOK were cultured in Oral Keratinocyte Medium (OKM) at 37℃in a 5% CO2 atmosphere according to the manufacturer’s instructions. When the keratinocytes became semiconfluent, they were washed and incubated in fresh media for 48 hours either in the absence or in the presence of OPN. In T cells culture supernatants stimulation experiments, the culture supernatants of T cells stimulated with or without OPN were added to the respective cell cultures. For experiments to determine whether the up-regulation of CEACAM1 on HOK by the culture supernatants of OPN stimulated T cells may be attributed to the effect of IFN-y, the anti-IFN-y mAb or an isotype-matched control antibody was added to the respective culture supernatants before their addition to the HOK cultures. The expressions of CEACAM1 on HOK were analyzed by flow cytometry.5. Study on the effect of OPN on IFN-y secretion by activated T cells. Culture supernatants of T cells were assayed for IFN-y using a Quantikine ELISA kit according to the manufacturer’s instructions.Results1. Regulation of CEACAM1 expression on T cells by OPN in vitro. When the T cells were cultured in the presence of OPN, the expressions of CEACAM1 were significantly up-regulated on primary T cells (p<0.01), whereas they were significantly down-regulated on activated T cells (p<0.05), as compared with the T cells cultured in the absence of OPN.2. Regulation of CEACAM1 expression on HOK by OPN in vitro. OPN alone did not modulate the expressions of CEACAM1 on HOK. The culture supernatants of OPN stimulated T cells strongly induced the expressions of CEACAM1 on HOK, compared with the control supernatants (p<0.01). The induction of CEACAM1 expressions on HOK by the culture supernatants of OPN stimulated T cells was restrained by anti-IFN-γmAb significantly (p<0.05).3. Effect of OPN on IFN-γsecretion by activated T cells. The concentrations of IFN-y in the culture supernatants from OPN stimulated T cells were significantly higher than those in controls (p< 0.01).ConclusionsThe expressions of CEACAM1 on T cells in different states can be differently regulated by OPN. The culture supernatants of OPN stimulated T cells can induce the expression of CEACAM1 on HOK in an IFN-y-dependent manner. These data indicate that OPN may play an important role in disease development by regulating the expressions of CEACAM1 in OLP.PartⅢRole of CEACAM1 in Regulating the Apoptosis of Oral Keratinocytes and Activated T CellsObjective The data of the former two parts indicate that the abnormally overexpressed OPN is highly likely to play an important role in disease development by regulating the expressions of CEACAM1 in OLP. In this part, we performed experiments to evaluate the effect of anti-CEACAM1 mAb on the apoptosis of oral keratinocytes, activated T cells and cocultured T cells with HOK, and to explore the role of CEACAM1 in immunopathogenesis of OLP.Methods1. Study on the effect of OPN on the apoptosis of HOK. HOK were cultured in OKM at 37℃in a 5% CO2 atmosphere according to the manufacturer’s instructions. When the keratinocytes became semiconfluent, they were washed and incubated for 48 hours in fresh media, media with OPN, culture supernatants of T cells, culture supernatants of OPN stimulated T cells, or culture supernatants of OPN stimulated T cells, which were preincubated with anti-IFN-y mAb, respectively. The cells apoptosis of HOK were analyzed by flow cytometry.2. Study on the effect of anti-CEACAM1 mAb on apoptosis of HOK. HOK were cultured in OKM at 37℃in a 5% CO2 atmosphere according to the manufacturer’s instructions. When the keratinocytes became semiconfluent, they were washed and stimulated with IFN-γfor 48 hours in the presence of anti-CEACAM1 mAb or an isotype-matched control antibody. The cells apoptosis of HOK were analyzed by flow cytometry.3. Study on the effect of anti-CEACAM1 mAb on apoptosis of activated T cells. Freshly purified T cells activated with IL-2, anti-CD3 mAb and anti-CD28 mAb were cultured in RPMI 1640 media for 48 hours in the presence of anti-CEACAM1 mAb or an isotype-matched control antibody. The apoptosis of T cells were analyzed by flow cytometry.4. Study on the effect of anti-CEACAM1 mAb on apoptosis of cocultured T cells with HOK. HOK were cultured in OKM at 37℃in a 5% CO2 atmosphere. When keratinocytes became semiconfluent, they were washed and stimulated with IFN-γfor 48 hours, followed by treatment with anti-CEACAM1 mAb or an isotype-matched control antibody for 30 minutes. HOK cultured without stimulation by rhIFN-γwere used as a negative control. Then the cells were washed and cocultured for 48 hours with T cells which were activated by anti-CD3 mAb and anti-CD28 mAb. The apoptosis of T cells was analyzed by flow cytometry.Results1. Effect of OPN on the apoptosis of HOK. The apoptosis of HOK was significantly increased by the culture supernatants from OPN stimulated T cells (p< 0.01) and the incremental effect was restrained by anti-IFN-γmAb (p<0.05), which was paralleled by the expressions of CEACAM1 on these cells.2. Effect of anti-CEACAM1 mAb on apoptosis of HOK. IFN-γinduced the apoptosis of HOK (p<0.01), and the inducible effect was restrained by anti-CEACAM1 mAb significantly (p<0.01).3. Effect of anti-CEACAM1 mAb on apoptosis of activated T cells. The apoptosis of activated T cells was significantly suppressed by anti-CEACAM1 mAb (p<0.01).4. Effect of anti-CEACAM1 mAb on apoptosis of cocultured T cells with HOK. IFN-γstimulated HOK induced the apoptosis of the cocultured T cells (p<0.01). Preincubation with anti-CEACAM1 mAb significantly suppressed the apoptosis of the cocultured T cells (p< 0.05).ConclusionsCEACAM1 plays an important role in regulating the apoptosis of oral keratinocytes and activated T cells. Therefore, in pathogenesis of OLP, the abnormally upregulated OPN is highly likely to contribute to the abnormal apoptosis of oral keratinocytes and activated T cells by regulating the expression of CEACAM1.