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M J Staquet

Publications and source records attributed to M J Staquet.

At least 19 recordsLinked to original sources
PubMed2004

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.

Antigens, CD↗
PubMed2004

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.

Allergens↗
PubMed2003

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.

Blotting, Western↗
PubMed2003

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.

Cell Culture Techniques↗
PubMed2001

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.

Antigens, CD34↗
PubMed2001

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.

Actins↗
PubMed2001

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.

Basement Membrane↗
PubMed2000

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.

Basement Membrane↗
PubMed1999

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.

Acrolein↗
PubMed1997

Integration of Langerhans cells into a pigmented reconstructed human epidermis.

The majority of in vitro reconstructed human epidermis is composed of keratinocytes only. Recently, the introduction of melanocytes into epidermal reconstructs has enlarged their field of application. The completion of reconstructed epidermis by introducing Langerhans cells remained an important challenge because Langerhans cells, unlike the other epidermal cell types, cannot be subcultured and expanded. To solve this problem, we used cord blood-derived CD34+ hematopoietic progenitors. Seeding these cells, after induction of their differentiation by granulocyte macrophage-colony stimulating factor and tumor necrosis factor-alpha, onto a reconstructing epidermis, composed of keratinocytes and melanocytes, gives rise to a pigmented epidermis with melanocytes in the basal layer and resident epidermal Langerhans cells located suprabasally. Interestingly, the same result was obtained by co-seeding a mixture of keratinocytes, melanocytes, and nondifferentiated CD34+ hematopoietic progenitors on the dermal equivalent, indicating that keratinocytes provide the environmental conditions for hematopoietic progenitors to differentiate into resident epidermal Langerhans cells, expressing major histocompatibility complex class II molecules, CD1a antigen, and Birbeck granules.

Antigens, CD34↗
PubMed1997

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.

Antigens, CD1↗
PubMed1996

Expression and function of B7-1 (CD80) and B7-2 (CD86) on human epidermal Langerhans cells.

In addition to T cell receptor triggering, activation of T cells requires costimulatory signals that have been shown to be mainly initiated through CD28. We analyzed the expression and function of the two ligands for CD28, B7-1 (CD80) and B7-2 (CD86), on human Langerhans cells (LC), the antigen-presenting cells from epidermis. Human LC freshly isolated from epidermis (fLC) expressed significant level of B7-2, which was increased upon a short culture in vitro. In contrast, B7-1 was undetectable on fLC but appeared at the cell surface after a 3-day culture in vitro. Pre-incubation of 18-h cultured LC with anti-B7-2 monoclonal antibodies (mAB) was sufficient to abrogate the binding of CTLA4-Ig fusion protein, while a combination of both mAB against B7-1 and B7-2 was necessary to obtain a complete inhibition of CTLA4-Ig binding on 3-day cultured LC, showing the absence of a third CTLA4 ligand. The function of B7-1 and B7-2 on human LC has been analyzed by adding mAb at the beginning of mixed epidermal cell lymphocyte reactions. Anti-B7-2 mAb and CTLA4-Ig, but not anti-B7-1 mAb, strongly inhibited allogenic. as well as recall antigen-induced T cell proliferation supported by fLC or 3-day cultured LC. Collectively, these results demonstrate that B7-2 is the major ligand for CD28/CTLA4 at the LC surface and that it plays a crucial role in human LC co-stimulatory function with little, if any, dependence of B7-1 expression.

Antigen Presentation↗
PubMed1995

Expression of ICAM-3 on human epidermal dendritic cells.

Three counter-receptors for LFA-1 of the immunoglobulin family have been discovered: ICAM-1, ICAM-2, and ICAM-3. Despite their homologies, their patterns of expression suggest specialized roles. The finding that ICAM-3 is much better expressed than other LFA-1 ligands on monocytes and resting T cells, and that this discovery may be important in the initiation of immune responses prompted us to search for the expression of ICAM-3 by human epidermal Langerhans cells (LC). Six out of eight different ICAM-3 monoclonal antibodies were found to be reactive with epidermal LC. Immunoelectron-microscopy staining revealed that 100% of freshly-isolated, typical Birbeck granules containing LC expressed ICAM-3. After one day and three days of culture, 100% of LC still expressed ICAM-3, but the staining intensity was decreased by 58% and 76% respectively. Immunoprecipitation of 125I surface-labeled LC with anti-ICAM-3 antibodies revealed a polypeptide with apparent M(r) of 122,000-125,000. To determine whether ICAM-3 was involved in LC function, mixed epidermal cell-lymphocyte reactions were performed with freshly isolated LC in the presence of various concentrations of different anti-ICAM-3 antibodies. Among the different antibodies tested, HP2/19 and CBR-IC3/1 were found to partially block the reaction in a dose-dependent manner, suggesting that ICAM-3 represents a new molecule involved in the initiation of the immune response driven by epidermal LC.

Antibodies, Monoclonal↗
PubMed1995

Expression of the antigen recognized by mAb GB36 in normal skin and in skin tumours.

GB36, a mouse monoclonal antibody (mAb) raised against an epithelial antigen of the human trophoblast, reacts with the epithelial basement membrane of chorionic villi; it does not react with the invasive extravillous cytotrophoblast. Expression and characterization of the antigen of GB36 (designated GBA36) were investigated in normal keratinocytes by immunoprecipitation, immunofluorescence and immunoelectron microscopic studies. Immunoprecipitation experiments demonstrated that the proteins identified on keratinocytes by mAb GB36 and a rat mAb anti-integrin alpha 6 (GoH3) were the same. Using immunofluorescence and immunoelectron microscopic methods, GBA36 was localized on the cell membrane facing the epithelial basal lamina of basal keratinocytes. GBA36 distribution in benign and malignant skin tumours was evaluated by immunostaining methods (immunofluorescence and immunoperoxidase). Analysis of tumours revealed that whereas benign epithelial tumours and intradermal naevi displayed high levels of GBA36, the expression of this antigen decreased progressively in spinocellular and basal cell carcinomas, and in cutaneous melanomas in relation to invasiveness. During cell transformation, GBA36 undergoes quantitative alterations, and expression is down-regulated. Although the functional relevance of these changes remains unknown, the correlation of decreased GBA36 expression with tumour progression may indicate a role for altered integrin expression in tissue invasion by human skin carcinoma and melanoma.

Adult↗
PubMed1995

Role of specific successive contacts between extracellular matrix proteins and epidermal Langerhans cells in the control of their directed migration.

To migrate from epidermis to regional lymph nodes, antigen-bearing epidermal Langerhans cells (LC) must move through extracellular matrix (ECM) of various composition. The present study was designed to contrast the ability of basement membrane (BM)- and dermis-ECM components to successively stimulate the adhesion of normal human epidermal LC, in vitro. For this purpose, we used highly enriched LC suspensions (70%-80%), allowed them to attach to one ECM substrate, and then studied the readhesion properties of these recovered ECM-attached LC to the same and different ECM substrates. Each of four ECM molecules (laminin (LM), fibronectin (FN), type I and type IV collagen) was tested in pairs. Readhesion of recovered LM and type IV collagen-attached cells did not affect readhesion to FN and type I collagen, indicating that the interaction of LC with the BM components can be normally followed by interaction with the dermis-ECM molecules. In contrast, readhesion of recovered FN-, type I collagen-, and type IV collagen-attached cells to LM was significantly reduced. The findings indicate that following contact to BM components, epidermal LC are able to attach to ECM proteins present in the dermis, whereas once they have made contact with ECM present in the dermis, they reduce their binding capacity to the BM laminin, suggesting the contact with the dermal components could prevent LC from reentering the epidermal compartment. Binding to LM and FN was also shown to induce a decline in the expression of CD1a, known as a specific marker restricted to epidermal LC.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗