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Targeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and β are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3β compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3β genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3β binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3β is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3β correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.

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 alpha (TNFalpha). Moreover, as we previously showed that epigallocatechin-3-gallate (EGCG) reduces VEGF and CXCL8/IL-8 secretion in TNFalpha-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 TNFalpha-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.

There is now strong evidence that the ultraviolet A (UVA) part of the solar spectrum contributes to the development of skin cancers. Its effect on the skin immune system, however, has not been fully investigated. Here, we analyzed the effects of UVA radiation on dermal dendritic cells (DDC), which, in addition, provided further characterization of these cells. Dermal sheets were obtained from normal human skin and irradiated, or not, with UVA at 2 or 12 J per cm 2 . After a 2 d incubation, the phenotype of emigrant cells was analyzed by double immunostaining and flow cytometry. Results showed that migratory DDC were best characterized by CD1c expression and that only few cells co-expressed the Langerhans cell marker Langerin. Whereas the DC extracted from the dermis displayed an immature phenotype, emigrant DDC showed increased expression of HLA-DR and acquired co-stimulation and maturation markers. We showed here that UVA significantly decreased the number of viable emigrant DDC, a process related to increased apoptosis. Furthermore, UVA irradiation impaired the phenotypic and functional maturation of migrating DDC into potent antigen-presenting cells, in a concentration-dependent manner. The results provide further evidence that UVA are immunosuppressive and suggest an additional mechanism by which solar radiation impairs immune response.

Migration and maturation of human dendritic cells derived from CD34+ progenitor cells (DC) infected by Toxoplasma gondii were studied in an in vitro model. We demonstrated that infection with virulent type I strains RH and ENT or type II low virulent strains PRU and CAL induced DC migration towards MIP-3β. However, type II strains induced a higher percentage of migrating cells than that induced by type I strains or positive controls (chemical allergen or lipopolysaccharides). Type II strains produced soluble factors responsible of the high migration whereas heat killed tachyzoites did not induced a migration higher than positive controls. We also demonstrated that infection by virulent strains and not by type II stains or heat killed tachyzoites triggers DC maturation. A soluble factor released by type II strains was responsible of the absence of DC maturation. Taken together, these results demonstrated that the interference of T. gondii in the behaviour of DC functions is related to the strain types and can be supported by secretion of soluble factors by the parasite.

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.

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.

SUMMARY Aspergillus fumigatus is the most common aetiological fungus responsible for human pulmonary aspergilloses. This study investigated the primary contact between Langerhans cells (LC), corresponding to dendritic cells present in pulmonary mucosa and live conidia of A. fumigatus. LC play a key role in antigen presentation for initiation of the primary T cell response. In vitro-generated LC (iLC) were differentiated from cultured human cord blood CD34+ cells and incubated at 4°C or 37°C with fluorescein-isothiocyanate (FITC)-stained conidia or control latex beads. In vitro, conidia were shown by microscopy and cytometry to adhere to iLC in a dose- and time-dependent manner. This adhesion was not limited to iLC because interstitial dendritic and other cells also fluoresced in the presence of conidia-FITC. A lectin other than mannose receptor-type lectin was demonstrated to be responsible of conidial binding. Inhibition of binding was observed with heterologous galactomannan and EDTA, indicating a C-lectin-like receptor with galactomannan structure specificity. After binding only a few conidia were internalized in acidic vesicles, as indicated by the cessation of conidial fluorescence. Conidial binding was followed by activation and maturation of iLC, suggesting that LC present in the lung may play a role in cellular host defence against aspergilloses.

This study evaluated the potential of the skin as a non invasive route for RSV vaccination using two G protein-derived molecules, G2Na and G5 in mice. G2Na contains T and B-cell epitopes whether G5 is a pure B-cell epitope. In contrast to G5, G2Na coadministered with CT three times at 1 month interval onto 1cm of square area shaved skin, elicited a consistent serum anti-G2Na and anti-CT IgG response. The anti-G2Na IgG response was dominated by IgG1 isotype, an indirect marker of a Th2 type of response. Dramatic reduction and decrease of RSV titers in lung tissues and in the nasal tract, respectively, following intranasal virus challenge revealed biological relevance of the transcutaneous immunization in the context of RSV vaccine. These results suggest that the transcutaneous route may offer a promising potential for novel RSV vaccine strategy, simple, painless and economical.

To better characterize human dendritic cells (DCs) that originate from lymphoid progenitors, the authors examined the DC differentiation pathways from a novel CD7(+)CD45RA(+) progenitor population found among cord blood CD34(+) cells. Unlike CD7(-)CD45RA(+) and CD7(+)CD45RA(-) 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)-alpha (standard condition), CD7(+)CD45RA(+) progenitors expanded less (37- vs 155-fold) but yielded 2-fold higher CD1a(+) DC percentages than CD7(-)CD45RA(+) or CD7(+)CD45RA(-) progenitors. As reported for CD34(+)CD1a(-) thymocytes, cloning experiments demonstrated that CD7(+)CD45RA(+) cells comprised bipotent NK/DC progenitors. DCs differentiated from CD7(-)CD45RA(+) and CD7(+)CD45RA(+) progenitors differed as to E-cadherin CD123, CD116, and CD127 expression, but none of these was really discriminant. Only CD7(+)CD45RA(+) or thymic progenitors differentiated into Lag(+)S100(+) Langerhans cells in the absence of exogenous transforming growth factor (TGF)-beta 1. Analysis of the DC differentiation pathways showed that CD7(+)CD45RA(+) progenitors generated CD1a(+)CD14(-) precursors that were macrophage-colony stimulating factor (M-CSF) resistant and CD1a(-)CD14(+) precursors that readily differentiated into DCs under the standard condition. Accordingly, CD7(+)CD45RA(+) progenitor-derived mature DCs produced 2- to 4-fold more IL-6, IL-12, and TNF-alpha on CD40 ligation and elicited 3- to 6-fold higher allogeneic T-lymphocyte reactivity than CD7(-)CD45RA(+) progenitor-derived DCs. Altogether, these findings provide evidence that the DCs that differentiate from cord blood CD34(+)CD7(+)CD45RA(+) progenitors represent an original population for their developmental pathways and function. (Blood. 2000;96:3748-3756)

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 degrees 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/m2 from 14 experiments with values ranging from 500 to 3200 J/m2). 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.

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. (J Allergy Clin Immunol 2000;105:1194-1201.)

Contact hypersensitivity is a major public health concern in most industrial countries, which is why predictive tests which could identify potential allergens are needed. We have established an in vitro approach for the detection of primary immune response. This model uses Langerhans-like dendritic cells (LLDC) derived from cord blood progenitors and autologous T lymphocytes, isolated from the same blood sample. Treatment of day 12-14 LLDC, with strong haptens trinitrobenzene sulfonic acid (TNP), fluorescein isothiocyanate (FITC) or Bandrowski's base (BB), results in the proliferation of T lymphocytes, whereas weak allergens and irritants, such as sodium dodecyl sulfate (SDS) are ineffective. The use of immature (day 8) LLDC and the addition of a 48 h stage of incubation after hapten contact, result in phenotypic maturation of LLDC in addition to lymphocyte activation in all the cultures with strong haptens. The 48 h stage of incubation, results in sensitization and in some cases the induction of T cell proliferation to citronellal (1/8), coumarine (1/8) and to a prohapten p-phenylenediamine (pPDA; 2/8). The phenotype of DC after 48 h of contact with a strong hapten, becomes that of mature DC (CD83(+), CD86(+) and HLA-DR(++)). With fragrance molecules, weak haptens and prohaptens, a comparable phenotype is observed only when T lymphocytes are activated. These data suggest that the unresponsiveness observed with weak haptens, may be the consequence on an incomplete maturation of LLDC.

We have identified a type II Ca2+-dependent lectin displaying mannose-binding specificity, exclusively expressed by Langerhans cells (LC), and named Langerin. LC are uniquely characterized by Birbeck granules (BG), which are organelles consisting of superimposed and zippered membranes. Here, we have shown that Langerin is constitutively associated with BG and that antibody to Langerin is internalized into these structures. Remarkably, transfection of Langerin cDNA into fibroblasts created a compact network of membrane structures with typical features of BG. Langerin is thus a potent inducer of membrane superimposition and zippering leading to BG formation. Our data suggest that induction of BG is a consequence of the antigen-capture function of Langerin, allowing routing into these organelles and providing access to a nonclassical antigen-processing pathway.

In vivo, dendritic cells (DC) form a network comprising different populations. In particular, Langerhans cells (LC) appear as a unique population of cells dependent on transforming growth factor β (TGF-β) for its development. In this study, we show that endogenous TGF-β is required for the development of both LC and non-LC DC from CD34+ hematopoietic progenitor cells (HPC) through induction of DC progenitor proliferation and of CD1a+ and CD14+ DC precursor differentiation. We further demonstrate that addition of exogenous TGF-β polarized the differentiation of CD34+ HPC toward LC through induction of differentiation of CD14+ DC precursors into E-cadherin+, Lag+CD68−, and Factor XIIIa−LC, displaying typical Birbeck granules. LC generated from CD34+ HPC in the presence of exogenous TGF-β displayed overlapping functions with CD1a+ precursor-derived DC. In particular, unlike CD14+-derived DC obtained in the absence of TGF-β, they neither secreted interleukin-10 (IL-10) on CD40 triggering nor stimulated the differentiation of CD40-activated naive B cells. Finally, IL-4, when combined with granulocyte-macrophage colony-stimulating factor (GM-CSF), induced TGF-β-independent development of non-LC DC from CD34+ HPC. Similarly, the development of DC from monocytes with GM-CSF and IL-4 was TGF-β independent. Collectively these results show that TGF-β polarized CD34+ HPC differentiation toward LC, whereas IL-4 induced non-LC DC development independently of TGF-β. J. Leukoc. Biol. 66: 781–791; 1999.

Recently, sustance P receptors (SPR) have been detected in neonatal foreskin. Our purpose was to determine the expression of SPR in other localizations than neonatal foreskin. As SP has been implicated in the pathogenesis of inflammatory cutaneous lesions, we wondered whether SPR localization was modified in psoriatic lesions. In normal skin, SP binding sites were detected using biotinylated SP and abrogated by a specific NK1 antagonist (spantide) on blood vessels, sweat glands and hair follicles. In the normal epidermis, SPR were usually observed on granular layers but may also be observed on other cell layers. The SP binding processed on cultured keratinocytes demonstrated that SPR were expressed in the epidermis, except basal cell layers, confirming that keratinocytes constitutively express SPR. In skin lesions of psoriatic patients, SP binding sites were expressed on the uppermost keratinocytes which are not granular cells, and seem to be overexpressed. Our results raise the question of the role of SPR on psoriatic keratinocytes.

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.

Langerhans cells play a key role in contact hypersensitivity reactions. The application of haptens on the skin leads to many modifications of these cells, including the increase of major histocompatibility complex II expression, allogeneic stimulation potency, and migration towards lymph nodes to activate T cells. Moreover, it has been shown that Langerhans cells cultured in vitro are able to prime naive T cells in response to hapten contact. From CD34+ progenitors present in cord blood, we generated dendritic cells of which some presented the phenotypic markers of Langerhans cells. We show that these cells are able to sensitize syngeneic naive (CD45RA+) T cells to haptens such as trinitrophenyl conjugate of trinitrobenzene sulfonic acid (TNP) and fluoroscein isothiocyanate. The response to TNP is higher than to fluoroscein isothiocyanate, whereas sodium dodecyl sulfate, an irritant molecule used as a control, never caused this effect. Phenotypic analysis of cellular suspensions and experiments of cell sorting lead to the conclusion that only CD1a+ cells are able to induce a primary response of syngeneic T cells to TNP or fluoroscein isothiocyanate. Furthermore, we have shown a close relationship between the differentiation state of dendritic cells and their ability to prime T lymphocytes. Dendritic cells are able to present haptens in an efficient manner between day 10 and 14 of culturing CD34+ progenitors, whereas they were efficient in presenting alloantigens from day 6 until after day 20. This dissociation suggests the need of an active metabolic process for hapten presentation in the direct treatment of dendritic cells with haptens. This model of hapten presentation was used for a panel of fragrance molecules and other molecules considered as weaker haptens than TNP and fluoroscein isothiocyanate.

Summary Dendritic cells (DC) are the most effective cells for antigen presentation in primary immune responses. Human cord blood CD34 + progenitors cultured in the presence of GM-CSF and TNF a generate α heterogeneous population of DC including Langerhans-like DC (LLDC) and monocytes. We describe here that IL-4 exerts different effects in such culture according to the cells considered. Thus, IL-4 favors DC components at the expense of monocytic development, and permits long-time persistence of DC which can be maintained up to one month in culture. These results show an IL-4-dependent inhibition of proliferation and emergence of CD14 + cells. Notably, however IL-4 also acts on the DC precursors. Thus IL-4 enhances survival and delays maturation of LLDC from CD1a + CD14 - precursors. In addition IL-4 also favors orientation of CD14 + CD1a - DC/monocyte precursors towards dermal-type CD1a + DC. DC recovered from IL-4 treated cultures display reduced allostimulatory capacity, but this function is restored upon IL-4 weaning. Finally, a short. (48 h) IL-4 Pulse is sufficient to favor DC development. The present study demonstrates that IL-4 positively regulates DC development at several levels on distinct precursor cells.

To carry out the characterization of feline Langerhans cells (LC), first described in 1994, we used a panel of monoclonal antibodies (MAb) known to react with human, canine and feline leukocyte membrane antigens (Ag). The immunolabeling was performed, at light microscope level, on frozen sections of feline skin and labial mucosa using an avidin-biotin-peroxidase technique, and at electron microscope level on epidermal cell suspensions using an immunogold technique. Out of the 52 MAb tested, six labeled basal or suprabasal DC cells in the frozen sections, either in epidermis or lip epithelium: MHM23 (anti-human CD18), CVS20 and vpg3 (respectively anti-canine and feline-major histocompatibility complex class II molecules), vpg5 (anti-feline leukocytes), vpg39 (anti-feline CD4) and Fel5F4 (anti-feline CD1a). These six MAb were used on suspensions, and labeled cells which showed no desmosomes or melanosomes, but contained 'zipper-like' structures similar to Birbeck granules (BG) in their cytoplasm, revealing they were LC. Consequently, feline LC are CD18-positive (CD18+), major histocompatibility complex class II-positive (Class II+), CD1a-positive (CD1a+), vpg5-positive (vg5+) and CD4-positive (CD4+). This immunophenotypic and ultrastructural characterization demonstrates that feline LC share many characteristics with their human counterparts, a fact that will allow us to study the role of feline LC in certain feline diseases such as Feline Immunodeficiency Virus (FIV) infection, since it has been shown that human LC cells are HIV-permissive, and to establish an animal model for human AIDS.

The infection of macaque monkeys by attenuated simian immunodeficiency virus can vaccinate against pathogenic molecular clones and isolates of the same virus. The correlates of this potent protective immunity are not fully understood but may be the key to an effective AIDS vaccine for humans. Aiming to determine whether host immune responses to envelope glycoprotein are an essential component of the immunity to primate lentiviruses, we have tried to superinfect SIVmac-infected macaque monkeys with SHIVsbg, a chimeric primate lentivirus constructed from the SIVmac239 genome with the env, rev, tat, and vpu genes from HIV-1 Lai. After inoculation of a large dose of SHIVsbg, the chimeric virus was isolated by coculture of mononuclear blood cells from four of five SIV-infected monkeys, but three animals were protected from extracellular SHIV viremia and did not seroconvert to HIV-1 glycoproteins. In the two SIV-infected monkeys that did develop SHIV viremia, cell-associated viral load was reduced at least 100-fold. These data indicate that an antiviral response capable of effectively controlling primate lentivirus replication might not necessarily involve the envelope glycoprotein.

The effect on human peripheral blood mononuclear cells (PBMCs) of neutral glycosphingolipids extracted from metacestodes of the parasite Echinococcus multilocularis was investigated. Neutral glycosphingolipids inhibited [3H]thymidine uptake by human PBMCs upon stimulation by mitogens such as phytohemagglutinin A and pokeweed mitogen or by allogeneic Burkitt B cells. This effect was dose dependent and was related to a decrease in interleukin 2 (IL-2) synthesis, the expression of IL-2 receptors (CD25) being unmodified. Addition of exogenous recombinant IL-2 restored the cell proliferation. Partial inhibition of immunoglobulin G (IgG), IgA, and IgM synthesis was observed in the supernatant of cell culture in association with the inhibitory effect. Identification of active subfractions contained in the neutral glycosphingolipid fraction was also studied in relation to cell viability. The free ceramide fraction had an inhibitory effect, in part related to cell lysis, particularly at high concentration, while the monogalactosylceramides had a paradoxical effect: as an activator at low concentrations and as an inhibitor at high concentrations, with limited cell survival. The immunogenic neutral glycosphingolipids containing at least two carbohydrate residues, all having a structure based on Gal beta 1-->6Gal, were inhibitors of PBMC proliferation and showed good cell survival. These results suggest that parasite neutral glycosphingolipids may play an immunologically relevant role in alveolar hydatid disease.

Human dendritic cells (DC) can now be generated in vitro in large numbers by culturing CD34+ hematopoietic progenitors in presence of GM-CSF+TNF alpha for 12 d. The present study demonstrates that cord blood CD34+ HPC indeed differentiate along two independent DC pathways. At early time points (day 5-7) during the culture, two subsets of DC precursors identified by the exclusive expression of CD1a and CD14 emerge independently. Both precursor subsets mature at day 12-14 into DC with typical morphology and phenotype (CD80, CD83, CD86, CD58, high HLA class II). CD1a+ precursors give rise to cells characterized by the expression of Birbeck granules, the Lag antigen and E-cadherin, three markers specifically expressed on Langerhans cells in the epidermis. In contrast, the CD14+ progenitors mature into CD1a+ DC lacking Birbeck granules, E-cadherin, and Lag antigen but expressing CD2, CD9, CD68, and the coagulation factor XIIIa described in dermal dendritic cells. The two mature DC were equally potent in stimulating allogeneic CD45RA+ naive T cells. Interestingly, the CD14+ precursors, but not the CD1a+ precursors, represent bipotent cells that can be induced to differentiate, in response to M-CSF, into macrophage-like cells, lacking accessory function for T cells. Altogether, these results demonstrate that different pathways of DC development exist: the Langerhans cells and the CD14(+)-derived DC related to dermal DC or circulating blood DC. The physiological relevance of these two pathways of DC development is discussed with regard to their potential in vivo counterparts.

Summary Background The need to develop predictive tests which could identify potential allergens has been recognized for many years. There is as yet no accepted in vitro method for the assessment of contact sensitizers. Objective We have tested the ability of a range of contact allergens to induce in vitro primary sensitization of autologous T cells. Method T-cell proliferation induced by haptens using 2-day cultured human Langerhans cells as antigen-presenting cell was assessed by 3H thymidine incorporation. Antigen specific stimulation was calculated as stimulation indexes. Results Strong allergens induced in vitro a primary T-cell response in all (trinitrophenyl, TNP: 13/13) or in the majority (fluorescein isothiocyanate, FITC: 7/10) of experiments. An irritant, sodium dodecyi sulfate (SDS). failed to generate a significant T-cell proliferation in any of the experiments (0/10). We obtained a significant lymphoproliferative response to weak sensitizers only in a limited number of experiments: (coumarin: 1/12, citronellal: 0/10, hydroxycitronellal: 2/8). p-Phenylenediamine (PPDA), a prohapten and highly sensitizing chemical in vivo, generated primary sensitization in vitro in only one of six experiments, while Bandrowski's base (BB), a metabolization product of PPDA induced a significant T-cell response in all six experiments. Conclusion The present in vitro model allows discrimination between two groups of substances: strong contact sensitizers (TNP, FITC. BB) on the one hand and weak sensitizers (coumarin, citronellal and hydroxycitronellal) and irritants (SDS) on the other hand. It could be used as a screening in vitro assay to eliminate strong contact allergens before further predictive animal tests have to be performed.

In addition to T cell receptor triggering, activation of T cells requires co-stimulatory 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 allogeneic, 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 on B7-1 expression.

To study the migration capacity of Langerhans cells in vitro, a reconstituted basement membrane invasion assay (Matrigel assay) was performed. Partially enriched human epidermal Langerhans cell suspensions (6-20%) were incubated with or without haptens [2,4,6-/trinitrobenzenesulfonic acid (TNBS) and fluorescein isothiocyanate (FITC)] or an irritant sodium lauryl sulfate (SLS) in Hanks' balanced salt solution for 10 min at 37 degrees C, and were then used for the invasion assay. Fibroblast-conditioned medium prepared from human dermal fibroblasts was used as a source of chemoattractants. Non-treated cells showed a gradual increase in migration of HLA-DR-positive cells through the Matrigel-coated filters in a time-dependent manner. In vitro TNBS and FITC treatment resulted in a significant stimulation of this migration. By contrast, SLS treatment did not increase the number of migrating cells. The migration capacity of hapten-treated cells decreased both in the absence of human fibroblast-conditioned medium and in the presence of this medium in the upper compartment of the chamber, indicating that this medium actually functioned as chemoattractant. Our findings suggest that the contact between hapten and Langerhans cells could be one of the triggers to initiate and/or stimulate Langerhans cell migration from the epidermis to the dermis. In addition, some factors derived from the dermal fibroblasts may promote and regulate the directional migration of Langerhans cells.

Murine models for the assessment of the contact sensitizing properties of chemicals rely on mouse ear swelling tests (Mest), which are not sensitive enough to detect weak sensitizers. The aim of the present study was to develop in mice an adjuvant-free Mest appropriate for in vivo detection of any type of sensitizer (weak to strong), and useful for in vitro assessment of contact sensitivity (CS). 3 haptens were tested: dinitrochlorobenzene (DNCB), para-phenylenediamine (pPD) and isoeugenol. We compared various protocols for induction of the CS reaction, differing by the site of induction, the number of applications and the concentrations of the 3 haptens. Comparison of the induction site for optimal CS reaction showed that, in Balb/c mice, the back was a better site of induction than the abdomen. Detection of the sensitizing properties of weak sensitizers (pPD, isoeugenol) was possible using an adjuvant-free protocol, provided that the induction phase comprised hapten applications on 3 consecutive days on the backs of animals. For DNCB, one application was sufficient to obtain optimal CS reaction. For all 3 haptens, a secondary response in vitro was obtained using semi-purified lymph node T cells from animals sensitized 5 days before with the optimized Mest. These results demonstrate that the Mest could be a useful experimental model for the study of all types of contact sensitizers.

In the present study, we analyzed the mechanism by which human Langerhans cells (LC), the dendritic cells (DC) from epidermis, support the induction of a primary allogeneic T cell response. We reported that paraformaldehyde (PF) fixation completely abrogated the stimulatory property of freshly isolated LC, although the level of major histocompatibility complex (MHC) class II antigen (Ag) expression was unaltered by the fixative. Addition of either interleukin (IL)-1 beta and/or IL-6, during the mixed epidermal cell lymphocyte reaction, failed to restore the proliferative response. By contrast, when human LC were incubated for 3 days in culture medium before fixation, they retained a low but significant allostimulatory capacity. Trypsin treatment of incubated LC before fixation did not impair their function, suggesting that stimulatory activity by fixed incubated LC did not merely reflect a repair of LC membrane after trypsin trauma suffered during epidermal cell (EC) isolation. More interestingly, we found that addition of interferon-gamma during LC incubation mediated an enhanced allostimulatory activity by the PF-fixed LC. Acquisition of allostimulatory property by in vitro activated and fixed LC did not correlate with increased MHC class II Ag expression at the cell surface. By contrast, we showed that ICAM-1 Ag expression by human LC is involved in this maturation process. Finally, we found that once human LC have been activated, IL-1 beta, but not IL-6, could serve as a costimulatory factor in the primary allogeneic T cell response. In conclusion, the data suggest that human LC accessory function is not constitutive but requires an activation step which can be provided by interferon-gamma during LC-T cell interaction.

We have performed double immunolabelings for cytofluorometric analysis and electron microscopy to investigate the coexpression of the CD1a (OKT6 and DMC1 monoclonal antibodies) antigen and the promonocyte/monocyte differentiation antigens CD14 (My4) or CD33 (My9) on putative bone marrow and umbilical cord blood precursors of the Langerhans cells (LC) and the epidermal LC. By cytofluorometric analysis, the percentage of CD1a+ cells which coexpressed the CD33 antigen was different from the bone marrow (5% of CD33+ cells are CD1a+), to the cord blood (3% of the CD33+ are CD1a+) and to the epidermis (the whole population of CD33+ LC are CD1a+). The ultrastructural morphology of the CD1a-expressing bone marrow, cord blood cells closely approximated that of a promonocyte/monocyte. Only LC epidermal were specifically recognized by the intracytoplasmic Birbeck granules. These CD1a+/CD33+ or CD14+ subpopulations found in three different locations (epidermis, bone marrow, cord blood) display a similar quantitative expression of the CD14 and CD33 antigens.

Human dendritic cells (DC) obtained in vitro from CD34+ progenitors (CD34-DC) or blood monocytes (mo-DC) are different DC which may be used in a model of T. gondii infection. We compared the survival, infection rate and cell surface receptor expression of both DC types after living T. gondii tachyzoite infection. CD34-DC appeared less resistant to the parasite than mo-DC. At 48 h post-infection, chemokine receptors responsible for DC homing and migration were absent in mo-DC, while down regulation of CCR6 and up regulation of CCR7 was observed in CD34-DC. This result, suggesting migration ability of CD34-DC, was confirmed by in vitro migration experiments against different chemokines. Tachyzoite supernatant, used as chemokine, attracted immature CD34-DC as observed by MIP3α, while MIP3β, as expected, attracted mature CD34-DC. Under similar conditions, no significant difference was noticed between mature or immature mo-DC. These data indicated that CD34-DC represent an alternative model that allows migration assay of infected DC by T. gondii.

Summary We investigated in vitro the properties of soluble factors produced by Toxoplasma gondii on the recruitment, maturation and migration of human dendritic cells (DC) derived from CD34+ progenitor cells. We used soluble factors including excreted secreted antigens (ESA) produced under various conditions by the virulent type I RH strain (ESA-RH) and the less virulent PRU type II strain (ESA-PRU). Soluble factors of both T. gondii strains appeared to possess a chemokine-like activity that attracted immature DC. This recruitment activity required the presence of functional CCR5 molecules on the cell membrane. Incubation of DC for 24 h with ESA triggered the migration of a large percentage of these cells towards the chemokine MIP-3β; ESA-PRU was more efficient than ESA-RH. ESA produced in absence of exogenous protein and crude extract did not induce DC migration but retained recruitment activity. These data indicate that recruitment activity and migration-inducing activity are not governed by the same factors. Moreover, incubation of DC for 48 h with ESA did not modify the expression of costimulation or maturation markers (CD83, CD40, CD80, CD86 or HLA-DR), but induced a decrease in CCR6 expression associated with an increased expression of CCR7. Taken together, these results suggest that T. gondii controls recruitment and migration of immature DC by different soluble factors and may induce a dysfunction in the host-specific immune response.

International audience; 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.

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.

Langerhans cells (LC) are dendritic cells that capture foreign antigens and migrate with them to the regional lymph nodes where they are presented to naive T cells. The possible role of matrix metalloproteinase-9 (MMP-9) in migration was suggested following experiments in a mouse model and in human skin explants. Using in vitro generated LC (iLC) derived from CD34+ cord blood cells and epidermal LC (eLC), we investigated the correlation between MMP-9 and other MMPs production and cell migration. Cells were activated by Bandrowski's base (BB), a chemical allergen, or by recombinant birch pollen allergen 1 (rBetv 1). Contact with allergens triggered migration of these cells, with a maximum rate being reached after 24 h. Migration was preceded by production of MMP-2 and MMP-9; part of the molecules were recovered as pro-MMPs in cell culture supernatant and part were associated with cell membrane proteins. At the cellular level, membrane-type 1 (MT1) and MT3-MMP were also identified. Addition of tumor necrosis factor-alpha (TNF-alpha) initiated pro-MMP-2 and pro-MMP-9 production followed by cell migration in a dose-dependent manner. These data imply that TNF-alpha is a key molecule for MMP production and cell migration. Furthermore, activation of iLC with BB or rBet v 1 induced synthesis of TNF-a and expression of TNF RII on the cell membrane, suggesting an autocrine loop. In conclusion, membrane-associated MMP-2 and-9 rather than soluble MMPs appear to be involved in cell migration.

International audience; We recently demonstrated that dendritic cells (DCs) can be generated from monocytes in the presence of high concentrations of human serum (HS), provided the extra-cellular pH is maintained at plasma values. Because monocyte-derived DCs (Mo-DCs) can also be generated in the presence of fetal calf serum (FCS) or serum-free medium, we have investigated whether these different culture supplements influence DC generation. With this aim, purified monocytes were cultured with GM-CSF plus IL-4 for 6 days and were further exposed to TNF-alpha for 2 additional days, in the presence of HS, autologous plasma (AP), FCS, or X-VIVO 20, a serum-free medium. Our results show that good yields of functionally mature DCs can reproducibly be obtained in the presence of HS or AP, as assessed by CD83 and CD86 up-regulation, dextran-FITC uptake, allogeneic MLR assays and the induction of an autologous response. Interestingly, the effect of serum on DC generation was probably not only quantitative, but also qualitative, since (i) the majority of HS- or AP-cultured DCs expressed CD83 with very weak levels of CD1a, whereas CD83+ DCs cultured in FCS or X-VIVO were mostly CD1a++; (ii) HS- and AP-cultured DCs were much more granular and heterogeneous than FCS- or X-VIVO-cultured DCs, and (iii) the presence of Birbeck-like granules was preferentially observed in HS- or AP-cultured DCs, as assessed by electron microscopy. That these different cells resemble dermal DCs (DDCs) was further supported by the observations that most of the cells displayed intracytoplasmic FXIIIa in the absence of Lag antigen, and expressed E-cadherin at very low levels. Altogether, our results indicate that starting from the same monocytic population, different subsets of DCs can be generated, depending on the culture conditions. Thus, HS or AP favors the generation of fully mature DCs that resemble activated dermal DCs, whereas FCS, or X-VIVO preferentially leads to the generation of less mature CD1a++ dermal-like DCs.

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.

Most monocyte-derived dendritic cells (DC) display CD1a, like Langerhans cells (LC) and some dermal DC, but their relationship with these skin DC remains unclear. To address this issue, we studied the expression of different antigens characteristic of skin DC and of monocyte/macrophages in CD1a+ and CD1a– monocyte-derived DC. Their phenotype indicated that they may be related to dermal DC rather than to LC, i.e., they were all CD11b-positive, and 72% were Factor XIIIa-positive, but they did not express E-cadherin nor VLA-6. It is interesting that CD1a+ and CD1a– cells showed intracytoplasmic granules that were different from LC Birbeck granules. These pheno-typical and ultrastructural features are comparable to those of CD14-derived DC obtained from cord blood precursors [C. Caux et al. J. Exp. Med. 184, 695–706]. These results show a close relationship between these two in vitro models, which are both related to dermal DC. J. Leukoc. Biol. 64: 484–493; 1998.

CD40/CD40 ligand interactions are known to play a key role in the development of immune reactions, especially by enhancing the costimulatory function of professional antigen-presenting cells (APC). Little is known, however, about the role this receptor plays on occasional APC, i.e. cells that are induced to express MHC class II molecules following an inflammatory process. In this study, we used CD40 ligand-transfected cells to analyze the effect of CD40 ligation on the phenotype, as well as accessory function, of human keratinocytes. We found that CD40 ligation enhanced ICAM-1 expression and did not upregulate HLA-DR, CD80 or CD86 expression on IFN-gamma-treated keratinocytes. CD40 triggering was not sufficient to generate primary allogeneic T-cell responses even in the presence of anti-CD28 monoclonal antibody (mAb). Moreover, CD40 ligation, in the presence or not of IFN-gamma, did not alter the accessory function of keratinocytes in PHA- or superantigen-induced T-cell activation. The lack of effect on the T-cell response was confirmed in blocking experiments using anti-CD40 mAbs. Collectively, these results suggest that CD40-CD40 ligand interactions on nonprofessional APC may amplify the inflammatory reaction without providing a mitogenic signal to the T cells.

Cornified cell envelope (CE) is generated during the late stages of epidermal differentiation and is made up of proteins covalently linked together by transglutaminases. To determine whether filaggrin is a component of this structure in humans, we analysed highly purified CE from plantar stratum corneum. An immunoelectron microscopy analysis showed specific binding of four different anti-(pro)filaggrin monoclonal antibodies to the surface of the CE, proved previously to be free of non-covalently linked proteins. Moreover, the anti-filaggrin activity of one of the antibodies was absorbed by preincubation with the plantar CE, as determined by ELISA. Convincingly, fragments of CE produced by proteolytic digestion of the structures were stained by this antibody on immunoblots. These data provide direct evidence that filaggrin is a component of CE purified from human plantar stratum corneum. Cross-linking between CE and the filaggrin-containing fibrous matrix may enhance the structural cohesion of the corneocytes and thus the resistance of the stratum corneum.

ICAM-3 is a newly recognized adhesion molecule, which is a member of the immunoglobulin supergene family of ICAMs, and has been shown to be identical with the CD50 antigen. Recent functional studies have shown that ICAM-3 is a ligand for LFA-1, and plays an important part in immune reactions. To date, very few data exist in the literature concerning its expression in the skin. In the present study, we investigated the expression of ICAM-3 in normal skin and in 98 biopsy specimens of various inflammatory and neoplastic dermatoses. ICAM-3 was found to be expressed by epidermal CD1a+ Langerhans cells, by cells of Langerhans cell histiocytosis, by T and B lymphocytes infiltrating the dermis in cutaneous lymphomas and in a wide spectrum of inflammatory dermatoses. Epidermal keratinocytes were consistently negative; endothelial expression of ICAM-3 was observed in six of the 98 cases. These results show that ICAM-3 is constitutively and widely expressed by cells participating in inflammatory dermatoses (including Langerhans cells and T and B lymphocytes), and that it can be, albeit rarely, induced on endothelial cells and dermal dendrocytes. These results highlight the important part that ICAM-3 may play in cutaneous inflammatory and immune reactions.

The effects of ultraviolet B radiation (UVB) on the immune function of human epidermal Langerhans cells (LC) were studied by using the mixed epidermal cell-lymphocyte reaction (MELR). Exposure of both enriched LC suspensions (eLC, 8-20% LC) and purified LC suspensions (pLC, 70-90% LC) to increasing doses of UVB radiation (25 to 200 J/m 2 ) decreased the proliferative T cell response in a very similar dose-dependent way, suggesting that keratinocytes did not play a major role in the UVB-induced inhibition of MELR. Supernatants from irradiated cultured eLC or pLC failed to inhibit T cell proliferation induced by untreated pLC. Furthermore, addition of irradiated eLC to untreated pLC did not affect the allogeneic T cell response. Taken together, these results provide evidence that in vitro UVB-induced immunosuppression was not mediated by inhibitory soluble factors that could affect either LC allostimulatory property or T cell proliferative response. UVB irradiation of human LC inhibited the capacity of these cells to induce CD4 + as well as CD8 + T cell proliferation. UVB-irradiated LC also induced a decreased T cell response to recall antigen or mitogen. Moreover, addition of exogeneous cytokines such as IL-1β, IL-1α, or IL-2 did not reverse the defective function of UVB-irradiated LC in MELR. The inhibitory effect of UVB radiation on human LC was not related to a decreased HLA-DR expression. Because cultured LC appeared to be less sensitive than freshly isolated LC to UVB-induced suppressive effects, the deleterious effects of UVB radiation on human LC allostimulatory properties may be associated with an impaired development of LC accessory function.

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.

Ultraviolet B (UVB) radiations are known to suppress induction of cutaneous immune responses. Since these radiations are almost completely absorbed within epidermis, epidermal cells were considered as potential targets for their various effects. Among epidermal cells, interest was focused on Langerhans cells (LC) because these cells are the antigen presenting cell of epidermis and play a key role in contact hypersensitivity reactions. In the murine models, UVB-induced immunosuppression has been described to both direct effect on LC function1 and/or to indirect effect through the liberation of keratinocyte-derived factors2. In human being, only few data are available concerning the mechanisms involved in this process. Here, we analysed the effects of narrow-band of UVB radiation (312 nm) on human LC antigen presenting function by using the mixed epidermal cell lymphocyte reaction (MELR).

The skin and mucosa are the first line of defense of the organism against external agents, not only as a physical barrier between the body and the environment but also as the site of initiation of immune reactions. The immunocompetent cells which act as antigen-presenting cells are Langerhans cells (LC). Originating in the bone marrow, LC migrate to the peripheral epithelia (skin, mucous membranes) where they play a primordial role in the induction of an immune response and are especially active in stimulating naive T lymphocytes in the primary response through a specific cooperation with CD4-positive lymphocytes after migration to proximal lymph nodes. Apart from many plasma membrane determinants, LC also express CD4 molecules which make them a susceptible target and reservoir for human immunodeficiency virus type 1 (HIV-1). Once infected, these cells, due to their localization in areas at risk (skin, mucous membranes), to their ability to migrate from the epidermal compartment to lymph nodes and to their ability to support viral replication without major cytopathic effects, could play a role as a vector in the dissemination of virus from the site of inoculation to the lymph nodes, and may thereby contribute to the infection of T lymphocytes. The in vivo consequences resulting from LC infection in the pathobiology of HIV will be discussed.

Langerhans cells are epidermal dendritic cells which take up antigens and transport them to paracortical areas in regional draining lymph nodes, where they induce T-lymphocyte activation. According to Moll1, LC’s play a key role in the capture, transport and presentation of leishmania to T cells, and in the initiation of the specific immune response in the mouse version of leishmaniasis. In order to establish a canine model of this disease, we first examined the ability of canine LC’s to migrate to regional lymph nodes in response to cutaneous stimulation.

The classification of linear IgA bullous dermatosis in the group of subepidermal blistering diseases is still a matter of controversy. This situation is due partly to the considerable clinical heterogeneity of the disease but also results from the difficulties in characterization and localization of the specific basement membrane zone antigen(s) recognized by immunoglobulin (Ig)A antibodies. In the present study, we have combined the Western blot detection of circulating autoantibodies with an ultrastructural immunogold labeling of human skin antigens using the same patients' sera. Our results, obtained with a short series of sera showing exclusive IgA class reactivity with the epidermal portion of salt-split skin, indicate that the antibodies recognizing the 97-kD antigen on immunoblot bind to the hemidesmosomal plaques of basal keratinocytes and the adjacent lamina lucida. These homogeneous laboratory results remain in striking contrast to the heterogeneity of clinical pictures in the patients studied, suggesting a participation of complementary, possibly not humoral, phenomena in the pathogenesis of linear IgA bullous dermatosis.

In this study, we analyzed the effect of interleukin-10 (IL-10) on the primary allogeneic T cell response induced by human Langerhans cells (LC), the dendritic cells from epidermis. We showed that IL-10 strongly inhibited the T cell response, provided it was added at the beginning of the mixed epidermal cell lymphocyte reaction (MELR). Proliferation of both CD4+ and CD8+ T cell subsets was affected by the cytokine. An inhibitory effect of IL-10 on human LC allostimulatory function was evidenced by the fact that IL-10-preincubated LC, but not IL-10-preincubated T cells, can display inhibitory effect. LC treatment with IL-10 partially inhibited the increase of HLA-DR expression on cultured LC as the percentage of highly positive HLA-DR cells was lower than that observed in the absence of the cytokine. IL-10 inhibited T cell alloreaction induced by 2-day-cultured human LC which constitutively display high levels of HLA class II, as well as ICAM-1 and LFA-3 antigens. This suggests that the suppressive effect of the cytokine was not merely related to an impaired up-regulation of these molecules. Addition of IL-1 during the MELR potentiated the allogeneic T cell proliferation and could reverse, at least partly, the inhibitory effect of IL-10. Collectively, these data indicate that IL-10 can prevent the alloreaction induced by human dendritic cells, providing new insights into the potential clinical use of this cytokine.

Introduction Tumor necrosis factor-α (TNFα) has received significant attention as a mediator of lumbar radiculopathy, with interest in TNF antagonism to treat radiculopathy. Prior studies have demonstrated that TNF antagonists can attenuate heightened nociception resulting from lumbar radiculopathy in the preclinical model. Less is known about the potential impact of TNF antagonism on gait compensations, despite being of clinical relevance. In this study, we expand on previous descriptions of gait compensations resulting from lumbar radiculopathy in the rat and describe the ability of local TNF antagonism to prevent the development of gait compensations, altered weight bearing, and heightened nociception. Methods Eighteen male Sprague-Dawley rats were investigated for mechanical sensitivity, weight-bearing, and gait pre- and post-operatively. For surgery, tail nucleus pulposus (NP) tissue was collected and the right L5 dorsal root ganglion (DRG) was exposed (Day 0). In sham animals, NP tissue was discarded (n = 6); for experimental animals, autologous NP was placed on the DRG with or without 20 μg of soluble TNF receptor type II (sTNFRII, n = 6 per group). Spatiotemporal gait characteristics (open arena) and mechanical sensitivity (von Frey filaments) were assessed on post-operative Day 5; gait dynamics (force plate arena) and weight-bearing (incapacitance meter) were assessed on post-operative Day 6. Results High-speed gait characterization revealed animals with NP alone had a 5% decrease in stance time on their affected limbs on Day 5 (P ≤0.032). Ground reaction force analysis on Day 6 aligned with temporal changes observed on Day 5, with vertical impulse reduced in the affected limb of animals with NP alone (area under the vertical force-time curve, P