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Epidermal Langerhans cells (LCs) are the first dendritic cells to encounter skin pathogens. However, their function has recently been challenged, especially in the initiation of T-cell responses to viral antigens. We have previously reported that fresh immature human LCs express mRNA encoding TLR3. Here we analyze the response of highly purified human LCs to poly(I:C), a synthetic mimetic of viral dsRNA recognized by TLR3. We show that LCs exposed for 2 days to poly(I:C) under serum-free conditions up-regulated co-stimulatory molecules, a process associated with increased allostimulatory capacity. Furthermore, poly(I:C) significantly enhanced LC survival and induced them to produce CXCL10, IL-6, and IL-12 p40. Bioactive IL-12 p70, IL-1beta, IL-15, IL-18, and IL-23 were never detected, even after CD40 ligation. LC incubation in the presence of bafilomycin completely reversed the effect of poly(I:C) on LC phenotypic activation and survival, indicating that endosomal TLR3 is involved in this process. Most interestingly, we report here that poly(I:C)-treated LCs favored alloreactive CD4(+) T-cell differentiation toward a Th1 profile and concomitant differentiation of IL-10-producing CD4(+) T cells that might limit, at another time, the inflammatory response and subsequent tissue damage.

Dendritic cells (DC) are APCs essential for the development of primary immune responses. In pluristratified epithelia, Langerhans cells (LC) are a critical subset of DC which take up Ags and migrate toward lymph nodes upon inflammatory stimuli. TLR allow detection of pathogen-associated molecular patterns (PAMP) by different DC subsets. The repertoire of TLR expressed by human LC is uncharacterized and their ability to directly respond to PAMP has not been systematically investigated. In this study, we show for the first time that freshly purified LC from human skin express mRNA encoding TLR1, TLR2, TLR3, TLR5, TLR6 and TLR10. In addition, keratinocytes ex vivo display TLR1-5, TLR7, and TLR10. Accordingly, highly enriched immature LC efficiently respond to TLR2 agonists peptidoglycan and lipoteichoic acid from Gram-positive bacteria, and to dsRNA which engages TLR3. In contrast, LC do not directly sense TLR7/8 ligands and LPS from Gram-negative bacteria, which signals through TLR4. TLR engagement also results in cytokine production, with marked differences depending on the PAMP detected. TLR2 and TLR3 ligands increase IL-6 and IL-8 production, while dsRNA alone stimulates TNF-alpha release. Strikingly, only peptidoglycan triggers IL-10 secretion, thereby suggesting a specific function in tolerance to commensal Gram-positive bacteria. However, LC do not produce IL-12p70 or type I IFNs. In conclusion, human LC are equipped with TLR that enable direct detection of PAMP from viruses and Gram-positive bacteria, subsequent phenotypic maturation, and differential cytokine production. This implies a significant role for LC in the control of skin immune responses.

Gangliosides are ubiquitous, membrane-associated, glycosphingolipids, the composition and production of which is altered in many tumour cells. They have been shown to inhibit the in vitro generation and differentiation of dendritic cells (DCs) from progenitors, but their effect on human tissue-residing DCs is yet to be investigated. In the present study, we analysed the effect of GM3 and GD3 gangliosides purified from human melanoma tumours on the phenotypic and functional maturation of human epidermal Langerhans cells (LCs), the first immune barrier against the tumour cells. We showed that both gangliosides impaired spontaneous LC maturation induced by a short in vitro culture, as assessed by significant down-regulation of co-stimulation (CD40, CD54, CD80, CD86) and maturation markers (CD83, CCR7), which correlated to an impaired ability of the cells to mount allogeneic T cell proliferation. Furthermore, the ganglioside-treated cells displayed less ability to migrate towards CCL19/macrophage inflammatory protein 3 beta, the chemokine that specifically binds CCR7 and mediates LC migration to lymph nodes. Lastly, we showed that both GM3 and GD3 gangliosides enhance LC spontaneous apoptosis. Globally, these in vitro results might explain, at least in part, the altered number and distribution of LCs in melanoma-bearing patients. They underscore a new mechanism for gangliosides to impede the host immune response by inducing LC dysfunction in the tumour microenvironment.

Dendritic cells (DC) play a central role in immunity/tolerance decision, depending on their activation/maturation state. TNF-alpha is largely produced in the skin under inflammatory conditions. However, it still remains to be defined how TNF-alpha modulates the activation status of human LC, the most specialized DC controlling skin immunity. Here, we reported that fresh immature LC, highly purified from healthy human skin and exposed for two days to TNF-alpha under serum-free conditions, expressed up-regulated level of co-stimulatory molecules (CD40, CD54, CD86), maturation markers (CD83, DC-LAMP), CCR7 lymph node homing receptor, and down-regulated Langerin level, in a dose-dependent manner. This mature phenotype is closely associated with enhanced LC allostimulatory capacity. Furthermore, TNF-alpha significantly increased the number of viable LC and decreased their spontaneous apoptosis. More importantly, TNF-alpha induced LC to produce both IFN-gamma-inducible-protein IP-10/CXCL10, a Th1-attracting chemokine and IL-12 p40. Bioactive IL-12 p70 was never detected, even after additional CD40 stimulus. The results implicate LC as an effective target through which TNF-alpha may up- or down-regulate the inflammatory skin reactions.

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.

Sugar receptors are being increasingly implicated in host-pathogen interactions because of their specific recognition of carbohydrates of microorganisms. The aim of this study was to identify sugar receptors expressed on the surface of human epidermal Langerhans cells (LC). To this end, binding of a panel of fluorescent neoglycoproteins to human epidermal LC was analyzed by quantitative flow cytofluorometry after standardization with calibrated beads. We demonstrate that fresh human LC are the only cells isolated from healthy epidermis which express a membrane receptor specific for fucose-bovine serum albumin (BSA) and mannose-BSA. Quantitative analysis of mannose-BSA or fucose-BSA binding showed non-linear Scatchard plots, denoting the presence of high and moderate affinity binding on the LC surface. The binding parameters of these two ligands were not significantly different. Mannan, the yeast mannose-rich polysaccharide, fucose-BSA, mannose-BSA and free fucose are strong competitors of the three known ligands of the mannose receptor, i.e. fucose-BSA, mannose-BSA and fluorescein isothiocyanate dextran. The amount of mannose-BSA and fucose-BSA bound to LC was 1.5-fold higher at 37 degrees C than at 4 degrees C, suggesting an internalization process. Antibodies raised against the human macrophage mannose receptor strongly stained CD1a-positive LC but not CD1a-negative population. Taken together, our data demonstrate that fresh human LC are the only cells in the epidermis to express a fucose-mannose receptor on their surface.