In skin inflammation, vascular endothelial growth factor (VEGF) and CXCL-8/IL-8 play an important role and are produced by activated keratinocytes. Extracts from Ginkgo biloba leaves (GBE), widely used in phytotherapy, have been reported to exert antioxidant and anti-inflammatory properties in the skin. We therefore evaluated the effects of GBE on the release of VEGF and CXCL8/IL-8 by normal human keratinocytes (NHKs) activated by tumor necrosis factor α (TNFα). Moreover, as we previously showed that epigallocatechin-3-gallate (EGCG) reduces VEGF and CXCL8/IL-8 secretion in TNFα-activated NHKs, we also tested its effect in association with GBE. Our results showed that GBE exerted a potent inhibition on VEGF and CXCL8/IL-8 levels in activated cells. In association with EGCG, GBE down-regulated VEGF and CXCL8/IL-8 levels in a cumulative manner in TNFα-stimulated NHKs. These results suggest that GBE, alone or in association with EGCG may contribute to moderate inflammatory processes in skin diseases associated with angiogenesis.

Phenotypic activation of monocyte‐derived dendritic cells has been proposed as an in vitro alternative assay to discriminate potential sensitizers from irritants, but the sensitivity of the assay remains controversial. In this study, we first determined the dynamic range of expression of activation/maturation markers on human monocyte‐derived dendritic cells cultured in the presence or absence of transforming growth factor ß (TGFβ)? On day three of culture, most monocytes had already differentiated into dendritic cells that expressed low levels of costimulatory molecules especially in the presence TGFβ‐treatment of 3‐day‐old TGFβ‐treated monocyte‐derived dendritic cells with several chemicals at sub‐toxic concentrations induced significant phenotypic changes for all the strong and mild sensitizers tested, whereas the irritant sodium lauryl sulfate had no effect. However, a very large variability was observed among the experiments. Most interestingly, we could show here for the first time that at concentrations sub‐toxic for monocyte‐derived dendritic cells all the allergens tested induced monocyte apoptosis within 2 days of culture. In contrast, sodium lauryl sulfate displayed similar toxicity on monocytes and monocyte‐derived dendritic cells and these results were confirmed with other irritants such as benzoic acid or methylsalicylate. Although testing of far more chemicals is required, these results indicate that differential toxicity of chemicals to monocytes and monocyte‐derived dendritic cells could be a rapid, simple and valuable tool to differentiate sensitizers from irritants.

Recent studies by our group and others have disclosed the presence of ceramides in mitochondria, and the activities of ceramide synthase and reverse ceramidase in mitochondria have also been reported. Since a possible contamination with the ER (endoplasmic reticulum)-related compartment MAM (mitochondria-associated membrane) could not be ruled out in previous studies, we have re-investigated the presence of the enzymes of ceramide metabolism in mitochondria and MAM highly purified from rat liver. In the present paper, we show that purified mitochondria as well as MAM are indeed able to generate ceramide in vitro through both ceramide synthase or reverse ceramidase, whereas the latter enzyme activity is barely detectable in microsomes. Moreover, ceramide synthase activities were recovered in outer mitochondrial membranes as well as in inner mitochondrial membranes. Using radiolabelled sphingosine as a substrate, mitochondria could generate ceramide and phytoceramide. However, the in vitro sensitivity of ceramide synthase toward FB1 (fumonisin B1) in mitochondria as well as in MAM was found to depend upon the sphingoid base: whereas dihydrosphingosine N-acyltransferase was inhibited by FB1 in a concentration-dependent manner, FB1 actually activated the ceramide synthase when using sphingosine as a substrate. Acylation of sphingosine 1-phosphate and dihydrosphingosine 1-phosphate, generating ceramide 1-phosphate, was also shown with both subcellular fractions. Moreover, the same difference in sensitivity towards FB1 for the ceramide synthase activities was seen between the two phosphorylated sphingoid bases, raising the possibility that distinct base-specific enzymes may be involved as ceramide synthases. Collectively, these results demonstrate the involvement of mitochondria in the metabolism of ceramides through different pathways, thereby supporting the hypothesis that topology of ceramide formation could determine its function.

Many potential targets of human immunodeficiency virus type 1 (HIV-1) reside in the human reproductive tract, including dendritic cells (DC). The ability of these cells to replicate HIV-1 is dependent on many factors such as their differentiation/maturation stage. Nevertheless, precise mechanisms underlying the early steps of transmucosal infection are still unknown. Our purpose was to investigate DC/HIV-1 interactions in a subepithelial mucosal layer equivalent (SEMLE) reconstructed in vitro. We used mixed interstitial DC (IntDC)/Langerhans cell (LC)-like cell subpopulations generated in vitro from CD34(+) progenitors. These cells were either integrated in SEMLE or maintained in suspension. Experimental infections were performed with a type X4 strain (HIV-1(LAI)) and a type R5 strain (HIV-1(Ba-L)). Proviral DNA was detected by in situ polymerase chain reaction (PCR) and viral replication was quantified by measuring p24 core protein release in the culture media. Our results showed that SEMLE enable DC to retain immature stage and reproduce the tropic selection that occurs in vivo. Indeed, IntDC/LC were infected by both types of HIV-1 strains, regardless of the infection schedule, whereas only type R5 virus replicated in DC in the absence of T cell subsets. Furthermore, the ability of DC to replicate HIV-1(BaL) was lost after 14 days of culture unless the cells had previously been integrated in SEMLE. These results suggest that this 3D model maintains the ability of DC to replicate type R5 virus by delaying their maturation. In conclusion, this in vitro model mimics human submucosa and can be considered as relevant for studying the preliminary steps of transmucosal HIV-1 infection.

In skin inflammation, vascular endothelial growth factor (VEGF) and IL-8 play an important role and are produced by activated keratinocytes. Recently, some polyphenols have been reported to exhibit antiinflammatory and antiangiogenic properties. We therefore evaluated the effects of green tea, its major component epigallocatechin-3-gallate (EGCG) and an isoflavone derived from soybean (genistein) on the release of VEGF and IL-8 by activated normal human keratinocytes (NHK). NHK cultured in defined medium were stimulated for 48 h with the proinflammatory cytokine TNFalpha with the addition or not of different concentrations of polyphenols. Levels of VEGF and IL-8 were measured in cell supernatants by enzyme-linked immunosorbent assays. The different constituents tested inhibited keratinocyte proliferation without inducing apoptosis. They reduced in a dose-dependent manner the basal release and the upregulation of VEGF in NHK. Green tea and EGCG were also potent inhibitors of IL-8 release by TNFalpha-stimulated NHK, whereas genistein exerted only minor effects. These results underline the divergent pathways involved in the downregulation of VEGF and IL-8 by polyphenols in activated keratinocytes. They also suggest that polyphenols may contribute to moderate inflammatory processes in skin diseases associated with angiogenesis.

Although most glycosphingolipids (GSLs) are thought to be located in the outer leaflet of the plasma membrane, recent evidence indicates that GSLs and their precursor, ceramide, are also associated with intracellular organelles and, particularly, mitochondria. GSL biosynthesis starts with the formation of ceramide in the endoplasmic reticulum (ER), which is transported by controversial mechanisms to the Golgi apparatus, where stepwise addition of monosaccharides on to ceramides takes place. We now report the presence of GSL-biosynthetic enzymes in a subcompartment of the ER previously characterized and termed 'mitochondria-associated membrane' (MAM). MAM is a membrane bridge between the ER and mitochondria that is involved in the biosynthesis and trafficking of phospholipids between the two organelles. Using exogenous acceptors coated on silica gel, we demonstrate the presence of ceramide glucosyltransferase (Cer-Glc-T), glucosylceramide galactosyltransferase and sialyltransferase (SAT) activities in the MAM. Estimation of the marker-enzyme activities showed that glycosyltransferase activities could not be ascribed to cross-contamination of MAM by Golgi membranes. Cer-Glc-T was found to have a marked preference for ceramide bearing phytosphingosine as sphingoid base. SAT activities in MAM led to the synthesis of G(M3) ganglioside and small amounts of G(D3). G(M1) was also synthesized along with G(M3) upon incubation of the fraction with exogenous unlabelled G(M3), underlying the presence of other sphingolipid-specific glycosyltransferases in MAM. On the basis of our results, we propose MAM as a privileged compartment in providing GSLs for mitochondria.

MMP-9 (92 kDa) is the major gelatinase able to degrade collagen IV, secreted by keratinocytes that are actively involved in wound-healing or tumorigenesis. Since the invasive phenotype of cancers is dependent on MMP-9 expression, it appeared of interest to precisely characterize which signal transduction pathways activated by TNF-alpha are involved in MMP-9 up-regulation induced by TNF-alpha. In HaCaT cells, activation of MMP-9 occurs at the transcriptional level. Inhibition of the MAPK pathway using specific inhibitors of the Ras, Raf, MEK1/2, and Erk1/2 cascade was correlated with a marked inhibition of MMP-9 activity, as determined by gene and protein expression. MAPK pathway activation via TNF-alpha was confirmed by marked AP-1 activation detected in EMSA. Under our experimental conditions, p38 MAPK and SAPK/JNK pathways were not activated. Gene and protein expression of other MMPs that regulate MMP-9, such as MMP-1 and MMP-13, were also up-regulated by TNF-alpha and inhibited by UO126, providing evidence that the MAPK pathway plays a fundamental role in the regulation of MMP-9 secretion by keratinocytes. As TNF-alpha is known to be a main activator of NF-kappaB pathway, the effects of campthothecin and caffeic acid were investigated, such as, TNF-alpha campthothecin up-regulated MMP-9 activity but caffeic acid only weakly inhibited MMP-9 activation induced by TNF-alpha. However, NF-kappaB is activated as shown from immunostaining data, a nuclear staining and higher Western blotting expression of p50 and p65 NF-kappaB subunits were detected after TNF-alpha treatment. A higher specific signal was also detected in EMSA for TNF-alpha-treated cells.

Gangliosides are ubiquitous membrane-associated glycosphingolipids, which are involved in cell growth and differentiation. Most tumor cells synthesize and shed large amounts of gangliosides into their microenvironment, and many studies have unraveled their immunosuppressive properties. In the present study we analyzed the effects of GM3 and GD3 gangliosides, purified from human melanoma tumors, on the differentiation of monocyte-derived dendritic cells (MoDC). At concentrations close to those detected in the sera from melanoma patients, both gangliosides dose-dependently inhibit the phenotypic and functional differentiation of MoDC, as assessed by a strong down-regulation of CD1a, CD54, CD80, and CD40 Ags and impaired allostimulatory function on day 6 of culture. Furthermore, GM3 and GD3 gangliosides decreased the viable cell yield and induced significant DC apoptosis. Finally, addition of GD3 to differentiating DC impaired their subsequent maturation induced by CD154. The resulting DC produced low amounts of IL-12 and large amounts of IL-10, a cytokine pattern that might hamper an efficient antitumor immune response. In conclusion, the results demonstrate that gangliosides impair the phenotypic and functional differentiation of MoDC and induce their apoptosis, which may be an additional mechanism of human melanoma escape.

Background: GM-CSF, IL-4 and TGF-β1 can drive the differentiation of CD14+ monocytes towards the immature Langerhans (LC) dendritic cell (DC) pathway. Their in vivo epidermal LC counterparts are mainly identified by the langerin molecules which are cross-linked into Birbeck granules (BG) upon mannose residue activation. Objective: The IL-13 and IL-14 cytokines sharing similar anti-inflammatory/immune functions, we investigated whether IL-13 (plus GM-CSF/TGF-β1) can substitute for IL-4 to preferentially skew the CD14+ monocyte differentiation towards LC. Methods: CD14+ monocytes cultured in the presence of GM-CSF/TGF-β1/IL-13 (IL-13-DC) for 6 days were then compared to GM-CSF/TGF-β1/IL-4-generated LC (IL-4-DC) by studying their phenotype, ultrastructural and functional features. Results: IL-13, in synergy with GM-CSF/TGF-β1, induced CD14+ monocytes to differentiate into LC after a short TNF-α stimulation more efficiently than IL-4. IL-13-DC are more immature than IL- 4-DC, as shown by both their preserved expression of monocyte markers (CD14, CD68) and their strong capacity of FITC-dextran uptake. Conclusion: IL-13 in combination with GM-CSF/TGF-β1/TNF-α favors CD14+ monocyte differentiation into LC which display numerous BG.

Dendritic cells constitute a family of antigen presenting cells defined by their morphology and their capacity to initiate primary immune response. Langerhans cells are paradigmatic dendritic cells, described in 1868 by a young medical student, Paul Langerhans in Berlin. Langerhans cells are present with epithelial cells in the epidermis, bronchi and mucosae. After antigenic challenge, Langerhans cells migrate into the T cell areas of proximal lymph nodes where they act as professional antigen-presenting cells. Langerhans cells originate in the bone marrow and CD34+ hematopoïetic progenitors are present in cord blood or circulating blood. They are actively involved in skin lesions of allergic contact dermatitis or atopic dermatitis, in cancer immunosurveillance and are infected by HIV in AIDS. Since 1992, Langerhans cells may be generated in vitro from CD34+ cord blood or circulating blood progenitors by culture with GM-CSF and TNF alpha, as well as from peripheral blood monocytes by culture with GM-CSF, IL4 and TGF beta 1. The possibility to obtain from the blood, the circulating progenitors of dendritic cells and the subsequent possibility to harvest a large number of these cells through in vitro culture using growth factors, have given rise to several very interesting therapeutic perspectives, especially in the field of anti-cancer immunotherapy. In dermatology advanced studies have concerned malignant melanomas. Anti-melanoma immunization trials were performed in patients, through dendritic cells charged with melanoma antigens. Side effects appear to be limited. Injections of antigenically charged dendritic cells were performed subcutaneously, intravenously or in the lymph nodes. Positive clinical responses were obtained with, in some cases, complete remission of the metastasis. These results open a particularly interesting perspective in the field of cancer treatment.

BACKGROUND The safety and efficacy of sublingual immunotherapy have been demonstrated in moderate allergic asthma and seasonal rhinitis. However, not much is known about the precise mechanism of action of the allergen when it crosses the oral mucosa. OBJECTIVE To define this mechanism, we investigated the role of Langerhans' cells in the capture and internalization of allergens. METHODS We generated dendritic cells in vitro with the phenotypic characteristics of Langerhans-like dendritic cells (LLDCs) from cord blood CD34(+) progenitors. We used two recombinant major allergens: Bet v 1 and Phl p 1 labeled with FITC. RESULTS Internalization of allergens and control proteins was dose- and time-dependent and related to the immature state of the cells. LLDCs internalized allergens with a high efficiency in comparison with control molecules. Allergens were only internalized by macropinocytosis, as demonstrated by the use of various inhibitors. Addition of intracellular pH-modifying molecules indicated that only a part of the allergens was accumulated in acidic vesicles, whereas the majority remained in other cytoplasmic structures. Pulse-chase experiments calculated a half-life of 4 hours, suggesting that part of the molecules were not metabolized in the lysosome. Allergen internalization by LLDCs might be followed by processing in some experiments, as demonstrated by activation of autologous T lymphocytes in 4 of 9 experiments. CONCLUSION These elements showed that Langerhans' cells present in mucosa might play an active role in immune responses to allergens.

Dendritic cells (DCs) form a network comprising different populations that initiate and differentially regulate immune responses. Langerhans cells (LCs) represent a unique population of DCs colonizing epithelium, and we present here observations suggesting that macrophage inflammatory protein (MIP)-3alpha plays a central role in LC precursor recruitment into the epithelium during inflammation. (a) Among DC populations, MIP-3alpha was the most potent chemokine inducing the selective migration of in vitro-generated CD34(+) hematopoietic progenitor cell-derived LC precursors and skin LCs in accordance with the restricted MIP-3alpha receptor (CC chemokine receptor 6) expression to these cells. (b) MIP-3alpha was mainly produced by epithelial cells, and the migration of LC precursors induced by the supernatant of activated skin keratinocytes was completely blocked with an antibody against MIP-3alpha. (c) In vivo, MIP-3alpha was selectively produced at sites of inflammation as illustrated in tonsils and lesional psoriatic skin where MIP-3alpha upregulation appeared associated with an increase in LC turnover. (d) Finally, the secretion of MIP-3alpha was strongly upregulated by cells of epithelial origin after inflammatory stimuli (interleukin 1beta plus tumor necrosis factor alpha) or T cell signals. Results of this study suggest a major role of MIP-3alpha in epithelial colonization by LCs under inflammatory conditions and immune disorders, and might open new ways to control epithelial immunity.

The present study was aimed at determining immune protection factors (IPFs) for sunscreens. Human skin explants from donors of phototype II-III were treated, or not, with sunscreens with increasing sun protection factors (SPF 4, 8, 15 and 30), or their respective vehicles. Explants were submitted, or not, to increasing doses of UVB irradiation (312 nm). After an 18-h incubation at 37 °C, epidermal cells were recovered through trypsinization and tested in a mixed epidermal cell/T lymphocyte reaction. The UVB dose providing 50% immunosuppression (D50%) was determined graphically. We first demonstrated a large difference in the individual response to UVB, as assessed by the D50% in the absence of any topical treatment (mean 1615 ± 839 J/m 2 from 14 experiments with values ranging from 500 to 3200 J/m 2 ). For all the tested sunscreens, the D50% values were significantly higher than those obtained without sunscreens or with their respective vehicles ( P < 0.01), thus demonstrating their immunoprotective effect. IPFs were determined as the ratio of the D50% in the presence of sunscreen to that with vehicle alone. Although they displayed important individual variations, IPFs ranked according to the sunscreen SPFs.

To better characterize human dendritic cells (DCs) that originate from lymphoid progenitors, the authors examined the DC differentiation pathways from a novel CD71CD45RA1 progenitor population found among cord blood CD34 1 cells. Unlike CD7 2CD45RA1 and CD71CD45RA2 progenitors, this population displayed high natural killer (NK) cell differentiation capacity when cultured with stem cell factor (SCF), interleukin (IL)-2, IL-7, and IL-15, attesting to its lymphoid potential. In cultures with SCF, Flt3 ligand (FL), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)a (standard condition), CD71CD45RA1 progenitors expanded less (37vs 155-fold) but yielded 2-fold higher CD1a 1 DC percentages than CD72CD45RA1 or CD7 1CD45RA2 progenitors. As reported for CD34 1CD1a2 thymocytes, cloning experiments demonstrated that CD7 1CD45RA1 cells comprised bipotent NK/DC progenitors. DCs differentiated from CD7 2CD45RA1 and CD71CD45RA1 progenitors differed as to E-cadherin CD123, CD116, and CD127 expression, but none of these was really discriminant. Only CD7 1CD45RA1 or thymic progenitors differentiated into Lag1S1001 Langerhans cells in the absence of exogenous transforming growth factor (TGF)-b1. Analysis of the DC differentiation pathways showed that CD71CD45RA1 progenitors generated CD1a1CD142 precursors that were macrophage-colony stimulating factor (M-CSF) resistant and CD1a 2CD141 precursors that readily differentiated into DCs under the standard condition. Accordingly, CD71CD45RA1 progenitor-derived mature DCs produced 2to 4-fold more IL-6, IL-12, and TNF-a on CD40 ligation and elicited 3to 6-fold higher allogeneic Tlymphocyte reactivity than CD7 2CD45RA1 progenitor-derived DCs. Altogether, these findings provide evidence that the DCs that differentiate from cord blood CD341CD71CD45RA1 progenitors represent an original population for their developmental pathways and function. (Blood. 2000;96:3748-3756)

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°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.