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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. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Human cord blood CD34+ progenitors cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) 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-α 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-α. The absence of TNF-α was shown to: (1) slow down proliferation without triggering apoptotic cell death, (2) enhance the percentage of LC, (3) delay or abrogat the expression of CD83, CD86, HLA-DR and CD208 molecules, and (4) maintain endocytosis by receptor and macropinocytosis. The withdrawal of TNF-α abrogated the spontaneous synthesis of matrix metalloproteinases. At day 12, TNF-α-deprived LC were less efficient in allogeneic T cell activation than LC cultivated with TNF-α. These data indicate that the suppression of TNF-α after day 5 maintains cells in an immature state and provides a population with 80% of LC at day 12. [ABSTRACT FROM AUTHOR]

Two monoclonal antibodies, K20 and 4B4, assigned to the CDW29 cluster of differentiation antigens, were shown to react with basal keratinocytes (BK). The aim of this study was to identify the antigens recognized by K20 and 4B4 on epidermal cells, and to determine whether they were identical to those found on lymphocytes. Basal keratinocyte-enriched cell suspensions were labelled with 125I and then 1% NP40 cell lysates were used for immunoprecipitation. Under reducing conditions, K20 and 4B4 irnmunoprecipitated from basal keratinocytes a broad MW 105 000 band and proteins of MW 145 000, 90 000 and 80 000. Under non-reducing conditions, each band was shifted down by approximately 5000 MW. Metabolic labelling studies demonstrated that the MW 145 000 and 105 000 subunits were synthesized by basal keratinocytes. On lymphoid cells, K20 and 4B4 are known to precipitate glycoprotein complexes made of a broad MW 130 000 protein band (β subunit) associated with a protein of MW 150 000 (α subunit) and proteins of MW 90 000 and 80 000 expressed in very low amounts. The MW 145 000 and 105 000 bands immunoprecipitated by K20 from basal keratinocytes correspond to the α and β subunits present on lymphoid cells. It has recently been demonstrated that K20 recognizes the common β subunit of the very late antigens family (VLA) and that 4B4 defines the helper-inducer subset of T lymphocytes. The present investigation provides evidence that basal keratinocytes share antigens of the VLA family with lymphoid cells and that they play an important role in the immune response in the skin-immune system. [ABSTRACT FROM AUTHOR]

We have used tritiated thymidinc (3HT) labelling and immunoperoxidase staining of 67 K polypeptide in human epidermis to study normal skin before and after irritation induced by application of croton oil (20% and 50% in olive oil). By this double labelling method, it was possible to identify, on the same skin section, DNA-synthesizing nuclei and epidermal cells which contained 67 K polypeptide. Our results clearly indicate that the germinative compartment of normal epidermis is a mixed population comprising basal and adjacent suprabasal cells; as a rule, absence of expression of 67 K polypeptide, which characterizes all basal cells, could not be regarded as a good marker to distinguish the germinative from the differentiating compartments. In mild primary irritation, the ratio of 3HT-labelled undifferentiated cells to keratinizing ones was similar to that observed in normal epidermis. In severe irritation, this homeostatic process was altered, as 3HT-Iabelled cells were mainly 67 K negative. [ABSTRACT FROM AUTHOR]

The role of cell-mediated immunity (CMI) in wart infection was demonstrated. The correlation with humoral immunity was also suggested. Patients with cell-mediated immune deficiency were found to be more susceptible to wart infection than those with humoral immune deficiency. [ABSTRACT FROM AUTHOR]

The identification of mononuclear cells extracted from various skin disorders has been investigated and the relative amount of B and T cells determined according to various assays such as E-rosette test, EAC-rosette test, FITC and peroxidase anti-immunoglobulin conjugates. It has been found that: (a) there were two different patterns of distribution of the cells in the infiltrates; one with a T/B ratio above 3 (closest to a delayed type reaction), the other with a T/B ratio below 2. (b) The morphological appearance of tissue lymphocytes differs from that of circulating lymphocytes by the presence of both membrane and cytoplasmic immunoglobulins. [ABSTRACT FROM AUTHOR]

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.

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.

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.

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.

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., (Copyright 2001 Academic Press.)

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.

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.