SummaryCD34 § cells in human cord blood and marrow are known to give rise to dendritic cells (DC), as well as to other myeloid lineages. CD34 § cells are rare in adult blood, however, making it difficult to use CD34 + ceils to ascertain if DC progenitors are present in the circulation and if blood can be a starting point to obtain large numbers of these immunostimulatory antigenpresenting cells for clinical studies. A systematic search for DC progenitors was therefore carried out in several contexts. In each case, we looked initially for the distinctive proliferating aggregates that were described previously in mice. In cord blood, it was only necessary to deplete erythroid progenitors, and add granulocyte/macrophage colony-stimulating factor (GM-CSF) together with tumor necrosis factor (TNF), to observe many aggregates and the production of typical DC progeny. In adult blood from patients receiving CSFs after chemotherapy for malignancy, GM-CSF and TNF likewise generated characteristic DCs from HLA-DR negative precursors. However, in adult blood from healthy donors, the above approaches only generated small DC aggregates which then seemed to become monocytes. When interleukin 4 was used to suppress monocyte development (Jansen, J. H., G.-J. H. M. Wientjens, W. E. Fibbe, K. Willemze, and H. C. Kluin-Nelemans. 1989. J. Exp. Med. 170:577.), the addition of GM-CSF led to the formation of large proliferating DC aggregates and within 5-7 d, many nonproliferating progeny, about 3-8 million cells per 40 ml of blood. The progeny had a characteristic morphology and surface composition (e.g., abundant HLA-DK and accessory molecules for cell-mediated immunity) and were potent stimulators of quiescent T cells. Therefore, large numbers of DCs can be mobilized by specific cytokines from progenitors in the blood stream. These relatively large numbers of DC progeny should facilitate future studies of their FceRI and CD4 receptors, and their use in stimulating T cell-mediated resistance to viruses and tumors.

There is consensus that an optimized cancer vaccine will have to induce not only CD8+ cytotoxic but also CD4+ T helper (Th) cells, particularly interferon (IFN)-γ–producing, type 1 Th cells. The induction of strong, ex vivo detectable type 1 Th cell responses has not been reported to date. We demonstrate now that the subcutaneous injection of cryopreserved, mature, antigen-loaded, monocyte-derived dendritic cells (DCs) rapidly induces unequivocal Th1 responses (ex vivo detectable IFN-γ–producing effectors as well as proliferating precursors) both to the control antigen KLH and to major histocompatibility complex (MHC) class II–restricted tumor peptides (melanoma-antigen [Mage]-3.DP4 and Mage-3.DR13) in the majority of 16 evaluable patients with metastatic melanoma. These Th1 cells recognized not only peptides, but also DCs loaded with Mage-3 protein, and in case of Mage-3DP4–specific Th1 cells IFN-γ was released even after direct recognition of viable, Mage-3–expressing HLA-DP4+ melanoma cells. The capacity of DCs to rapidly induce Th1 cells should be valuable to evaluate whether Th1 cells are instrumental in targeting human cancer and chronic infections.

Although it is widely accepted that filaggrin (FLG) deficiency contributes to an abnormal barrier function in ichthyosis vulgaris and atopic dermatitis, the pathomechanism of how FLG deficiency provokes a barrier abnormality in humans is unknown. We report here that the presence of FLG mutations in Caucasians predicts dose-dependent alterations in epidermal permeability barrier function. Although FLG is an intracellular protein, the barrier abnormality occurred solely via a paracellular route in affected stratum corneum. Abnormal barrier function correlated with alterations in keratin filament organization (perinuclear retraction), impaired loading of lamellar body contents, followed by nonuniform extracellular distribution of secreted organelle contents, and abnormalities in lamellar bilayer architecture. In addition, we observed reductions in corneodesmosome density and tight junction protein expression. Thus, FLG deficiency provokes alterations in keratinocyte architecture that influence epidermal functions localizing to the extracellular matrix.These results clarify how FLG mutations impair epidermal permeability barrier function.

Dendritic cells migrate from the skin to the draining lymph nodes. They transport immunogenic MHC-peptide complexes, present them to Ag-specific T cells in the T areas, and thus generate immunity. Migrating dendritic cells encounter physical obstacles, such as basement membranes and collagen meshwork. Prior work has revealed that matrix metalloproteinase-9 (MMP-9) contributes to mouse Langerhans cell migration. In this study, we use mouse and human skin explant culture models to further study the role of MMPs in the migration and maturation of skin dendritic cells. We found that MMP-2 and MMP-9 are expressed on the surface of dendritic cells from the skin, but not from other sources. They are also expressed in migrating Langerhans cells in situ. The migration of both Langerhans cells and dermal dendritic cells is inhibited by a broad spectrum inhibitor of MMPs (BB-3103), by Abs to MMP-9 and -2, and by the natural tissue inhibitors of metalloproteinases (TIMP), TIMP-1 and TIMP-2. Inhibition by anti-MMP-2 and TIMP-2 define a functional role for MMP-2 in addition to the previously described function of MMP-9. The importance of MMP-9 was emphasized using MMP-9-deficient mice in which Langerhans cell migration from skin explants was strikingly reduced. However, MMP-9 was only required for Langerhans cell migration and not maturation, since nonmigrating Langerhans cells isolated from the epidermis matured normally with regard to morphology, phenotype, and T cell stimulatory function. These data underscore the importance of MMPs, and they may be of relevance for therapeutically regulating dendritic cell migration in clinical vaccination approaches.

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Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) is a rare (occurring in approximately 2 to 3 people/million population/year in Europe and the US), life-threatening, intolerance reaction of the skin. It is most often caused by drugs (most commonly sulfonamides, nonsteroidal anti-inflammatory drugs, antimalarials, anticonvulsants, and allopurinol). SJS/TEN is characterized by a macular exanthema ('atypical targets') which focusses on the face, neck, and the central trunk regions. Lesions show rapid confluence, a positive Nikolsky's sign, and quickly result in widespread detachment of the epidermis and erosions. Mucosal, conjunctival, and anogenital mucous membranes are prominently involved. Histopathology shows satellite cell necrosis in the early stages progressing to full thickness necrosis of the epidermis, contrasting with rather inconspicuous inflammatory infiltrates of the dermis. Damage to the skin is thought to be mediated by cytotoxic T lymphocytes and mononuclear cells which induce apoptosis in keratinocytes expressing drug-derived antigens at their surfaces. No guidelines for the treatment of SJS/TEN exist since no controlled clinical trials have ever been performed. The controversy over whether systemic corticosteroids should be used to curtail progression is still unresolved; while many authors agree that corticosteroids do in fact suppress progression, it is obvious that they also greatly enhance the risk of infection, the complication which most frequently leads to a fatal outcome. It appears reasonable to only administer corticosteroids in the phase of progression and to withdraw them as soon as possible, and to add antibacterials for prophylaxis. Recently, in a small series of patients, intravenous immunoglobulins were presumed to be effective by the blockade of lytic Fas ligand-mediated apoptosis in SJS/TEN. However, these results have to be confirmed by large clinical trials. Supportive treatment and monitoring of vital functions is of utmost importance in SJS/TEN, and out-patient treatment is unacceptable. Recovery is usually slow, depending on the extent and severity and the presence of complications, and may take 3 to 6 weeks. Skin lesions heal without scars as a rule, but scarring of mucosal sites is a frequent late complication, potentially leading to blindness, obliteration of the fornices and anogenital strictures. There is no reliable laboratory test to determine the offending drug; diagnosis rests on the patient's history and the empirical risk of drugs to elicit skin SJS/TEN. Provocation tests are not indicated since re-exposure is likely to elicit a new episode of SJS/TEN of increased severity.

We provide evidence that prophylactic and ongoing use of topical therapy with either topical corticosteroid or a dexpanthenol-containing emollient ameliorates, but does not prevent radiation dermatitis. Our data suggest, but do not prove, a benefit of a topical corticosteroid vs. a dexpanthenol-containing emollient. Further controlled studies with larger cohorts will be needed to determine optimal forms of topical therapy for radiation dermatitis.

Prior studies have established the requirement for enzymatic hydrolysis of glucosylceramides to ceramide for epidermal barrier homeostasis. In this study, we asked whether sphingomyelin-derived ceramide, resulting from acid-sphingomyelinase activity, is also required for normal barrier function. We showed first, that a subset of Niemann-Pick patients with severe acid-sphingomyelinase deficiency (i.e., <2% residual activity) demonstrate abnormal permeability barrier homeostasis, i.e., delayed recovery kinetics following acute barrier disruption by cellophane tape-stripping. To obtain further mechanistic insights into the potential requirement for sphingomyelin-to-ceramide processing for the barrier, we next studied the role of acid-sphingomyelinase in hairless mouse skin. Murine epidermis contains abundant acid-sphingomyelinase activity (optimal pH 5.1-5.6). Two hours following acute barrier disruption by tape-stripping, acid-sphingomyelinase activity increases 1. 44-fold (p<0.008 versus vehicle-treated controls), an increase that is blocked by a single topical application of the acid-sphingomyelinase inhibitor, palmitoyldihydrosphingosine. Furthermore, both palmitoyldihydrosphingosine and desipramine, a chemically and mechanically unrelated acid-sphingomyelinase inhibitor, significantly delay barrier recovery both 2 and 4 h after acute barrier abrogation. Inhibitor application also causes both an increase in sphingomyelin content, and a reduction of normal extracellular lamellar membrane structures, in the stratum corneum. Both of the inhibitor-induced delays in barrier recovery can be overridden by co-applications of topical ceramide, demonstrating that an alteration of the ceramide-sphingomyelin ratio, rather than sphingomyelin accumulation, is likely responsible for the barrier abnormalities that occur with acid-sphingomyelinase deficiency. These studies demonstrate an important role for enzymatic processing of sphingomyelin-to-ceramide by acid-sphingomyelinase as a mechanism for generating a portion of the stratum corneum ceramides for permeability barrier homeostasis in mammalian skin.