Pattern-recognition receptors in pulp defense.

Initial sensing of infection is mediated by germline-encoded pattern-recognition receptors (PRRs), the activation of which leads to the expression of inflammatory mediators responsible for the elimination of pathogens and infected cells. PRRs act as immune sensors that provide immediate cell responses to pathogen invasion or tissue injury. Here, we review the expression of PRRs in human dental pulp cells, namely, receptors from the Toll-like (TLR) and Nod-like NLR families, by which cells recognize bacteria. Particular attention is given to odontoblasts, which are the first cells encountered by pathogens and represent, in the tooth, the first line of defense for the host. Understanding cellular and molecular mechanisms associated with the recognition of bacterial pathogens by odontoblasts is critical for the development of therapeutic strategies that aim at preventing excessive pulp inflammation and related deleterious effects.

O34-pathogen sensing by human odontoblasts.

Human odontoblasts are neural crest-derived, dentin-producing mesenchymal cells aligned at the periphery of the dental pulp. They become exposed to cariogenic oral bacteria as these progressively demineralise enamel then dentin to gain access to the pulp. Due to their situation at the dentin-pulp interface, odontoblasts are the first cells encountered by invading pathogens and/or their released components, and represent, in the tooth, the first line of defence for the host. Previous studies have shown that odontoblasts are able to sense pathogens and elicit innate immunity. In particular, they express several pathogen recognition receptors of the Toll-like receptor (TLR) and nucleotide-binding oligomerisation domain (NOD) families, which allow them to recognize specific bacterial and viral components. So far, most studies aiming at elucidating the role of odontoblasts in the dental pulp innate response have focused on Gram-positive bacteria, as these largely dominate the carious microflora in initial and moderate dentin caries lesions. In vitro, odontoblasts were found to be sensitive to Gram-positive bacteria-derived components, mainly lipoteichoic acid which is recognized through cell membrane TLR2. Our studies have shown that engagement of odontoblast TLR2 by LTA triggers TLR2 and NOD2 up-regulation, NF-B nuclear translocation, production of various chemokines including CCL2, CXCL1, CXCL2, CXCL8 and CXCL10, while promoting immature dendritic cell recruitment. Conversely, LTA down-regulates major dentin matrix components, including collagen type I and dentin sialophosphoprotein, as well as TGF-b1, a known inducer of dentin formation. We provide here additional data showing the fine localization of NOD2 in healthy dental pulps, as well as differential regulation of TLR2, TLR4, NOD2, CCL2 and CXCL8 genes by LTA and the synthetic TLR2 agonists Pam2CSK4 and Pam3CSK4. It appears from the aforementioned data that odontoblast-triggered immune events constitute potential targets for interrupting the signaling cascades which lead to excessive immune response and necrosis in the dental pulp tissue challenged with cariogenic bacteria. In particular, preventing Gram-positive bacteria recognition or signal transduction by pattern recognition receptors may represent a valuable strategy to achieve this goal. Future studies in the field will pave the way for designing novel therapeutic agents which modulate odontoblast behaviour to promote pulp healing and repair.

Different roles of odontoblasts and fibroblasts in immunity.

Odontoblasts and fibroblasts are suspected to influence the innate immune response triggered in the dental pulp by micro-organisms that progressively invade the human tooth during the caries process. To determine whether they differ in their responses to oral pathogens, we performed a systematic comparative analysis of odontoblast-like cell and pulp fibroblast responses to TLR2-, TLR3-, and TLR4-specific agonists (lipoteichoic acid [LTA], double-stranded RNA, and lipopolysaccharide [LPS], respectively). Cells responded to these agonists by differential up-regulation of chemokine gene expression. CXCL2 and CXCL10 were thus increased by LTA only in odontoblast-like cells, while LPS increased CCL7, CCL26, and CXCL11 only in fibroblasts. Supernatants of stimulated cultures increased migration of immature dendritic cells compared with controls, odontoblast-like cells being more potent attractants than fibroblasts. Analysis of these data suggests that odontoblasts and pulp fibroblasts differ in their innate immune responses to oral micro-organisms that invade the pulp tissue.

HUGO (FNDC3A): a new gene overexpressed in human odontoblasts.

Previously, we established a subtractive cDNA library enriched in odontoblast-specific genes and hypothesized that new, previously unidentified, markers would be present, associated with the odontoblast phenotype. In this paper, we report the first characterization of a new gene we have named HUGO, and its associated deduced protein sequence. This gene expression is under the control of two alternative promoters, resulting in the synthesis of two proteins, one of which, HUGO2, is included in the other, HUGO1. HUGO proteins are mainly composed of a proline-rich region at the N-terminus, 8 type III-fibronectin modules, and a transmembranous helix at the C-terminus. In odontoblasts, the proteins are located in Golgi vesicles. However, they display a broader expression pattern, since they are also expressed by nerve fibers in the dental pulp and other tissues (e.g., trachea, brain, kidney), as demonstrated by immunohistochemistry and qPCR, respectively. Their location in odontoblasts suggests a role in collagen and glycosaminoglycan synthesis.

Expression and localisation of alphav integrins in human odontoblasts.

Integrin alphabeta heterodimers mediate adhesion to the extracellular matrix and at cell-cell contacts and initiate intracellular signalling cascades in response to a variety of inductive factors. Apart from the expression of alphavbeta3 that we have previously reported, little is known about the expression of integrins in odontoblasts. Here, we investigated the expression of alphav-binding beta integrin subunits in healthy human dental pulp in vivo and in odontoblasts differentiated in vitro. Reverse transcription/polymerase chain reaction analysis revealed the expression of alphav, beta1, beta5 and beta8 integrin mRNA, but not beta6, in whole pulp cells. Flow cytometry showed that the alphav and beta1 subunits were the most intensely expressed. Immunohistochemistry demonstrated that the beta1 subunit was localised in newly differentiated odontoblasts in the root and in mature odontoblasts in the crown, including their intradentinal cell processes. The alphav chain was predominantly expressed by mature odontoblasts and alphavbeta5 was only observed in mature odontoblasts. In vitro differentiated odontoblasts expressed genes for alphav, beta1 and beta5, but not for beta6 and beta8. A comparison of integrin profiles between cultured pulp cells and in vitro differentiated odontoblasts revealed that odontoblast maturation was characterised by a significant increase in the expression of alphav and beta1 subunits and alphavbeta5 integrin. The beta8 subunit was detected in nerve cells only. Histological analysis of teeth from alphav knockout mice showed no obvious structural modification in the odontoblast layer. Thus, human mature odontoblasts express alphavbeta3, alphavbeta5 and perhaps alphavbeta1 integrins, with the possible presence of alpha-beta1 pairs. The roles that these molecules play in the exchange of information throughout the odontoblast layer remain to be determined.

Alpha v beta 3 integrin expression in human odontoblasts and co-localization with osteoadherin.

Integrins are heterodimeric transmembrane receptors which promote cell adhesion, thus contributing to the maintenance of tissue organization in both normal and pathological conditions. To characterize the way odontoblasts may interact with other cells and the extracellular matrix in human teeth, we studied expression of alpha v beta 3 integrin, a putative receptor for osteoadherin. We showed that alpha v beta 3 integrin expression was restricted to odontoblasts, blood vessels, and small rounded cells in sound and carious pulp. Odontoblast staining intensity increased from the apical to the cusp region. Osteoadherin staining was strong in the whole odontoblast layer (with a slight decrease in the cusp region) and in predentin. Odontoblasts differentiating in vitro were stained with the anti-alpha v beta 3 integrin antibody, first at the level of intercellular contacts, then throughout the cell membrane. These results suggest that the alpha v beta 3 integrin could play a role in interodontoblast adhesion and odontoblast binding to the surrounding predentin/dentin/pulp matrix, possibly through osteoadherin.

Moderate skin sensitizers can induce phenotypic changes on in vitro generated dendritic cells.

In the present study, we analyzed the phenotypic alterations induced by several allergens on immature dendritic cells (DC), with the aim to develop a potential in vitro alternative for predicting the sensitizing potential of chemicals. DC were generated from human monocytes cultured in the presence of GM-CSF, IL-4 and TGF-beta1 and treated for 2 or 4 days with different chemicals. Surface marker expression (HLA-DR, CD1a, CD40, CD54, CD83, CD86, CCR7 and E-cadherin) was analyzed by flow cytometry. Results showed that a 2-day treatment with the representative allergens DNCB and NiSO(4) induced significant changes of most antigens while other chemicals such as balm of Peru (strong allergen), kathon (moderate allergen), cinnamic aldehyde (mild allergen) or the irritant SLS had no significant effect. In contrast, the 4-day treatment with allergens substantially improved the results. Indeed, despite a large variability according to the donors, the number of modified antigens was significantly higher with all the tested chemicals, except kathon, as compared to that observed with the irritant SLS. The present study indicates that, in this model, the screening of mild or moderate allergens requires both the consideration of many antigens and a prolonged time of incubation with the chemicals.

Novel protein kinase C and matrix metalloproteinase inhibitors of vegetable origin as potential modulators of Langerhans cell migration following hapten-induced sensitization.

BACKGROUND: Migration and maturation of epidermal dendritic cells, the Langerhans cells (LC), are central events in the initiation of the cutaneous immune response. LC migration from skin to draining lymph nodes is regarded as an indispensable step for the early phase of antigen-specific sensitization. Among the several agents which influence the ability of LC to migrate, previous studies have revealed that matrix metalloproteinases (MMPs) and protein kinase C (PKC) contribute to promoting LC migration. In this work, we studied the effect of two recently developed PKC and MMPs inhibitors of vegetable origin on the migration of in vitro activated LC. METHODS: The migratory capacity of epidermal and in vitro generated LC was assessed using a reconstituted basement membrane assay (Matrigel), mimicking the prerequisite passage through the dermal-epidermal basement membrane on the way to the lymph nodes. RESULTS: Contact with chemical allergens, Bandrowski's base or 2,4-dinitrobenzenesulfonic acid (DNBS), triggered migration. In the presence of PKC inhibitors, D-erythro-sphingosine and OX100, or an inhibitor of MMPs, LU105, allergen-induced migration of LC was strongly decreased. The association between OX100 and LU105 was more efficient in modulating the migration of activated LC compared to each molecule tested separately. CONCLUSIONS: These results showed that PKC and MMPs inhibitors act in synergy to inhibit the migration of activated epidermal dendritic cells in vitro. They underscore the role of PKC and MMPs inhibitors and suggest they may be of relevance for therapeutically regulating epidermal dendritic cell migration in inflammatory dermatoses.

Withdrawal of TNF-alpha after the fifth day of differentiation of CD34+ cord blood progenitors generates a homogeneous population of Langerhans cells and delays their maturation.

Human cord blood CD34+ progenitors cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) generate a heterogeneous population of dendritic cells (DC), including Langerhans cells (LC). This combination of cytokines has been shown to be crucial for differentiation into LC. After day 5 of culture, TNF-alpha has been maintained in the medium in most studies despite the observation of spontaneous maturation of LC after day 12. Five-day samples of in vitro differentiated LC were cultured in parallel with or without TNF-alpha. The absence of TNF-alpha was shown to: (1) slow down proliferation without triggering apoptotic cell death, (2) enhance the percentage of LC, (3) delay or abrogate the expression of CD83, CD86, HLA-DR and CD208 molecules, and (4) maintain endocytosis by receptor and macropinocytosis. The withdrawal of TNF-alpha abrogated the spontaneous synthesis of matrix metalloproteinases. At day 12, TNF-alpha-deprived LC were less efficient in allogeneic T cell activation than LC cultivated with TNF-alpha. These data indicate that the suppression of TNF-alpha after day 5 maintains cells in an immature state and provides a population with 80% of LC at day 12.

Comparative studies on the secretion and activation of MMPs in two reconstructed human skin models using HaCaT- and HaCaT-ras-transfected cell lines.

Matrix metalloproteinases play an important role in tissue regeneration, wound healing and tumor invasion. Our previous studies have shown a higher motility of HaCaT-ras-transfected cells compared with HaCaT or normal human keratinocytes (NHK) in correlation with a higher secretion of MMP-2 (72 kDa) or MMP-9 (92 kDa), according to the medium used for cell cultures. Presently, the expression and activity of MMPs were investigated in two reconstructed skin models, using a dead de-epidermized dermis (DED) or a dermal substitute including living fibroblasts. In all experiments, MMP-9 was essentially secreted by NHK and to a greater extent by HaCaT cells. Its active form (86 kDa) was only detected in both reconstructed skin models according to keratinocyte differentiation. MMP-2 was mainly secreted by living fibroblasts included in the dermal substitute skin model. In this case, its activation was up-regulated when HaCaT cell lines were seeded onto the dermal substitute according to their culture at air/liquid interface as shown for MMP-9. The collagenase MMP-1 and stromelysin-1 (MMP-3), susceptible to activate pro-MMP-2 and -9, respectively, were detected in their inactive form by ELISA. MMP-1 was expressed in both models but MMP-3 required the presence of living fibroblasts. Their activities were not detected using specific fluorogenic substrates. In the skin equivalent model using HaCaT, the extensive secretion and activation of MMP-2 and MMP-9 could explain the defect observed in basal membrane reconstruction, suggesting a direct interaction of HaCaT with fibroblasts.

Use of human reconstructed epidermis to analyze the regulation of beta-defensin hBD-1, hBD-2, and hBD-3 expression in response to LPS.

Defensins have been identified as key elements of innate immunity against microbial infections. In the present study, human beta-defensin-2 (hBD-2) mRNA and peptide expression were evaluated by RT-PCR and Western blotting in normal human keratinocytes, in function of their stage of differentiation. In proliferating, non-differentiating keratinocytes generated in serum-free, low-calcium medium, a very low hBD-2 mRNA expression was found. A significantly higher expression was detected in high-calcium cultivated keratinocytes grown either as monolayers or as multilayers under submerged conditions. In an air-liquid interface culture of keratinocytes, allowing epidermis to be reconstructed, hBD-2 mRNA expression level was significantly higher than in the other conditions and displayed inter-individual variability as observed in native epidermis. The peptide was detected only in reconstructed epidermis. These results indicate that hBD-2 gene expression in normal human keratinocytes is dependent upon their stage of differentiation. The level of expression of hBD-1 mRNA was lower and that of hBD-3 was higher than that of hBD-2 in reconstructed epidermis. Exposure of reconstructed epidermis to bacterial lipopolysaccharide (LPS) resulted in an average 4-fold increase in hBD-2 mRNA 18 h after challenge, but not of hBD-1 and hBD-3 gene expression. These results show the selective regulation of hBD-2-encoding gene in an organotypic epidermal model, in response to LPS. They also provide evidence that in vitro reconstructed epidermis represents a useful model for studying regulation of expression of beta-defensins after skin challenge with pathogenic microorganisms in conditions as close as possible to the in vivo situation.

Human melanoma cells inhibit the earliest differentiation steps of human Langerhans cell precursors but failed to affect the functional maturation of epidermal Langerhans cells.

Tumour-derived factors suppress differentiation and function of in vitro generated DC. Here, we investigate the effect of two melanoma clones differing in their invasive and metastatic properties on the generation and/or functional maturation of human epidermal LC. LC were generated from CD34(+) cord blood progenitors under GM-CSF/TNF-alpha/TGF-beta 1. CD34(+) cells were co-cultured with or without melanoma cells using Transwell dishes. After 11 days of co-culture, CD34(+)-derived cells display a non-adherent undifferentiated morphology, a high level of monocytic CD14 marker, a down-regulated expression of LC markers (CD1a, E-cadherin) and DC markers (CD40, CD80, CD54, CD58, CD83, CD86, HLA-DR, HLA-class I). These cells were less potent than control LC in inducing allogeneic T cell proliferation. The generation of the CD14(+) population was correlated with a decrease in the CD1a(+) population, without any statistical differences between the two clones. Melanoma cells diverted the differentiation of CD34(+) cells towards a dominant CD14(+) population only if the progenitors were in an early growth phase. IL-10, TGF-beta 1 and VEGF were not responsible for these effects, as assessed by using blocking antibodies. By contrast, co-culture of fresh epidermal LC with melanoma cells did not affect their phenotype and function. Our data demonstrate that melanoma cells inhibit the earliest steps of LC differentiation, but failed to affect the functional maturation of epidermal LC. This suggests that melanoma cells participate in their own escape from immunosurveillance by preventing LC generation in the local cutaneous microenvironment.

Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations.

The comparison of the gene expression profiles between two subpopulations of melanoma cells (1C8 and T1C3) derived from the tumor of one patient by cDNA array revealed differences in GAPDH and beta-actin gene levels. These two housekeeping genes were up-regulated in invasive T1C3 melanoma cells compared to noninvasive 1C8 cells. Since cDNA array results were not confirmed by conventional RT-PCR throughout the exponential phase of amplification, we performed duplex relative RT-PCR using ribosomal 18S RNA as internal standard including competimer technology. Statistical analyses provided significant evidence that invasive T1C3 melanoma cells exhibited a twofold higher mRNA level of both GAPDH and beta-actin than noninvasive 1C8 cells. This study demonstrates that the duplex relative RT-PCR procedure including ribosomal 18S RNA as internal standard and competimer technology is precise for RNA quantification and is tailored for cDNA array validation. Our data provide molecular evidence that cellular subpopulations of the same pathological origin are highly heterogeneous and extend the concept that the selection of an appropriate internal control for comparative mRNA analysis should be adapted to each model of human cancers.

A combination of MIP-3alpha and TGF-beta1 is required for the attraction of human Langerhans precursor cells through a dermal-epidermal barrier.

Signals regulating the traffic of Langerhans cell precursors from blood to the epidermis are not yet fully understood. The observations that TGF-beta1 is of unique importance in Langerhans cells (LC) ontogeny and that macrophage inflammatory protein-3alpha (MIP-3alpha) is able to attract LC within the epidermis, prompted us to study the effect of MIP-3alpha and TGF-beta1 on the migration of LC precursors. The migratory capacity of immature dendritic cells (DC) was assessed using a reconstituted basement membrane assay (Matrigel), mimicking the prerequisite passage through the dermal-epidermal basement membrane on the way into the epidermis. DC differentiated from cord blood CD34 cells in the presence of GM-CSF plus TNF-alpha were subjected to migration using modified Boyden chambers. Day-6 DC progenitors migrated in a dose-dependent fashion in response to MIP-3alpha, and CD1alpha+ LC precursors responded preferentially to the chemokine. Immature DC did not respond strongly to TGF-beta1 alone in migration assays, but up to 68% of the cells migrated in response to MIP-3alpha plus TGF-beta1. Among them, at least 50% expressed CD1a and E-cadherin and can be considered LC precursors. The allostimulatory function of these cells was significantly more potent than that which migrated in response to MIP-3alpha alone. Our results show that a significant proportion of immature DC is able to migrate through a dermal-epidermal basement membrane equivalent. In the presence of TGF-beta1, the DC which respond to MIP-3alpha have the phenotype and the functional capacity of epidermal LC. Our findings underline the role of MIP-3alpha and TGF-beta1 in attraction and localization of immature LC within the epidermis under normal conditions.

Penetration of human metastatic melanoma cells through an authentic dermal-epidermal junction is associated with dissolution of native collagen types IV and VII.

The events occurring during the penetration of melanoma cells through the dermal-epidermal junction, which is the first crucial step in the process of metastasis, are poorly understood, partly because no suitable tissue models exist. In the in vitro model reported here, two melanoma clones (T1C3, which generates lung metastases in experimental animals, and IC8, which does not) derived from a single parental cell line were co-seeded with normal allogenic keratinocytes onto acellular human de-epidermized dermis with preserved intact basement membrane and cultured for up to 1 month at an air-liquid interface. Histological, immunohistochemical and ultrastructural studies showed that melanoma cells from the metastatic clone (T1C3), but not from the non-metastatic clone (IC8), penetrated the dermal-epidermal junction to invade the dermis after 3 weeks of culture. Local invasion was associated with the dissolution of the native epidermal basement membrane collagens type IV and VII. Confocal laser scanning microscopy analysis demonstrated that numerous T1C3 cells were able to colonize the interstitial dermis and to rapidly penetrate empty dermal cavities. Our model represents a significant technical advance over others currently available since: (i) the organized three-dimensional architecture of the native dermal-epidermal junction is preserved; (ii) the active invasion process coincides with the dissolution of native components of the epidermal basement membrane, i.e. collagen types IV and VII; and (iii) the ability of melanoma cells to cross the dermal-epidermal junction correlates with their metastatic potential. This model provides a valuable tool for the study of the time-course of the cellular and molecular events that occur during the earliest steps of invasion in cutaneous melanoma. It also offers new opportunities to study the possible role of the keratinocyte environment in melanoma invasion.

Fragrance and contact allergens in vitro modulate the HLA-DR and E-cadherin expression on human epidermal Langerhans cells.

BACKGROUND: Epidermal Langerhans cells (LCs) play a critical role in the induction of contact hypersensitivity. The LCs leave the skin, move to the regional lymph nodes and present the allergens embedded in the HLA-DR molecule to naive T-lymphocytes. To allow LC emigration from the epidermis, E-cadherin must be downregulated. In this study, we have examined the early events that occur in the human epidermis after exposure to three strong contact sensitizers and two commonly used fragrances by examining alterations of E-cadherin and HLA-DR expression. METHODS: To determine whether E-cadherin and HLA-DR levels were modulated by allergens, flow cytometry was utilized to evaluate E-cadherin and HLA-DR expression on human epidermal LCs exposed to the different chemicals for 4 h at 37 degrees C. RESULTS: In vitro stimulation with the contact sensitizers isoeugenol, cinnamaldehyde, 2,4, 6-trinitrobenzenesulfonic acid, Bandrowski'sbase, or p-phenylene diamine resulted in a dose-dependent decrease of HLA-DR expression on the surface of LCs without affecting the number of positive cells. These contact allergens induced a downregulation of E-cadherin expression as well as a significant decrease of the percentage of E-cadherin-positive cells. Incubation with an irritant, sodium lauryl sulfate, did not significantly change HLA-DR and E-cadherin expression. CONCLUSIONS: Based on the alteration of E-cadherin and HLA-DR expression of human LCs under short-term exposure conditions, there was a clear difference between contact sensitizers and a well-characterized irritant. For the first time, the ability of fragrance allergens in dipropylene glycol, a widely used vehicle in fragrance and cosmetic industries, was demonstrated to induce human LC phenotypic alterations. In combination with a series of in vitro tests, this rapid and simple method should help to detect the sensitizing potential of a substance to be applied onto the human skin as an alternative to animal testing.

Fibronectin upregulates in vitro generation of dendritic Langerhans cells from human cord blood CD34+ progenitors.

Several studies have demonstrated that dendritic cells can be generated in vitro from CD34+ hematopoietic progenitor cells. In vivo, dendritic cells are found in many tissues and reside in direct proximity to extracellular matrix proteins. Because extracellular matrix proteins affect differentiation and location of cells in tissues, this study was designed to investigate potential effects of extracellular matrix proteins on differentiation of dendritic cells. Dendritic cells were generated from CD34+ human cord blood cells in the presence of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha for 6 d and subsequently cultured for an additional 6-d period on tissue culture plates coated with various extracellular matrix proteins. Among the extracellular matrix proteins tested, exposure to fibronectin stimulated dendritic cell/Langerhans cell differentiation as indicated by the 50% increase of the number of cells expressing the Birbeck granule-associated marker Lag and displaying numerous Birbeck granules. Adhesion on fibronectin was shown to be specifically mediated by the integrin alpha5beta1. Because laminin and collagen were unable to cause similar changes in Langerhans cell development, these results suggest that fibronectin may cause changes affecting cellular differentiation of progenitors. Hematopoietic progenitors may exhibit maturational regulated differences in response to both matrix molecules and cytokines. The influence of combined signals emanating from a supportive microenvironment, specific integrins, and particular cytokines in the differentiation of Langerhans cells is discussed.