Role of Th17 Cells in Skin Inflammation of Allergic Contact Dermatits

Extending the classical concept considering an imbalance exclusively of T helper(h) 1 and Th2 cells on the bottom of many inflammatory diseases, Th17 cells were recently described. Today, there is sufficient experimental evidence to classify psoriasis and allergic contact dermatitis (ACD) amongst other inflammatory skin disorders as IL-17 associated diseases. In several human studies, T-cell-clones could be isolated from eczema biopsies, and high IL-17 levels were observed after challenge with allergen. In the last years, the phenotype of these IL-17 releasing T cells was in the focus of discussion. It has been suggested that Th17 could be identified by expression of retinoic acid receptor-related orphan receptor (ROR)C (humans) or RORγt (mice) and IL-17, accompanied by the absence of IFN-γ and IL-22. In cells from skin biopsies, contact allergens elevate IL-17A, IL-23, and RORC within the subset of Th cells. The indications for a participation of Th17 in the development of ACD are supported by data from IL-17 deficient mice with reduced contact hypersensitivity (CHS) reactions that could be restored after transplantation of wild type CD4+ T cells. In addition to Th17 cells, subpopulations of CD8+ T cells and regulatory T cells are further sources of IL-17 that play important roles in ACD as well. Finally, the results from Th17 cell research allow today identification of different skin diseases by a specific profile of signature cytokines from Th cells that can be used as a future diagnostic tool

Nickel Challenge In Vitro Affects CD38 and HLA-DR Expression in T Cell Subpopulations from the Blood of Patients with Nickel Allergy

Nickel allergy is a major health problem and shows clinical manifestation of contact eczema. The response of specific lymphocyte subpopulations in sensitized patients after new challenge to nickel has until now not been studied in detail. To evaluate if nickel-based elicitation reaction could be objectively identified by multi-parametric flow cytometry, immunophenotyping of specific T cells was applied. White blood cells from 7 patients (4 positive in patch test, 3 negative) were challenged by nickel and in vitro short-term culture. Standardized antibody-dye combinations, specific for T helper(h)1, Th17 and cytotoxic T cell activation, were selected according to the recommendations of Stanford Human Immune Monitoring Center. In cytotoxic CD8+CCR7+CD45RA+ T cells from patients suffering from nickel allergy, CD38 and HLA-DR were elevated comparing to healthy donors. After challenge to nickel in vitro both markers decreased in CD8+CCR7+CD45RA+ T cells but found up-regulated in CD4+CCR7+CD45RA+CCR6−CXCR3+Th1 cells. Intracellular expression of T-bet and RORγt further indicated Th1 and Th17 cells. Finally, CD4+CD25+CCR4− T cells increased after challenge with nickel in PBMCs of patients with nickel allergy. Flow cytometry based quantification of T cell markers might be used as a specific and reliable method to detect chemical induced skin sensitization and confirm diagnostic patch testing in the clinics

Efficient material-induced activation of monocyte-derived dendritic cells releasing surface molecules, matrix metalloproteinases, and growth factors needed for regenerative tissue remodeling

New experimental approaches for tissue repair have recently been proposed and include the application of natural or synthetic biomaterials and immune cells. Herein, fully synthetic poly(glycidyl ether) (PGE) copolymer coatings are evaluated as bioinstructive materials for the in vitro culture and intrinsic activation of human immune cells. Immature monocyte-derived dendritic cells (moDCs) are exposed to PGE brush and gel coatings of varying copolymer composition, wettability, and deformability immobilized on polystyrene culture dishes. Compared to moDCs cultured on standard tissue culture-treated polystyrene, activation marker levels on the cell surface are strongly enhanced on PGE substrates. Thereby, moDCs undergo a distinct morphological change and reach levels of activation comparable to those achieved by toll-like receptor (TLR) ligand liposaccharide (LPS), specifically for the expression of costimulatory molecules CD86 and CD40 as well as human leukocyte antigen (HLA)-DR. In addition, PGE coatings induce a significantly enhanced level of programmed cell death ligands 1 and 2 (PD-L1/-L2) on the moDC surface, two molecules crucially involved in maintaining immune tolerance. In addition, an increased release of matrix metalloproteinases MMP-1 and MMP-7, as well as transforming growth factor (TGF)-β1 and epidermal growth factor (EGF) was observed in moDCs cultured on PGE substrates. As fully synthetic biomaterials, PGE coatings demonstrate intrinsic functional competence in instructing immature human moDCs for phenotypic activation in vitro, accompanied by the secretion of bioactive molecules, which are known to be crucial for tissue regeneration. Hence, PGE coatings hold strong potential for immune-modulating implant coatings, while PGE-activated moDCs are promising candidates for future clinical cell-based immunoengineering therapies

Efficient material-induced activation of monocyte-derived dendritic cells releasing surface molecules, matrix metalloproteinases, and growth factors needed for regenerative tissue remodeling

New experimental approaches for tissue repair have recently been proposed and include the application of natural or synthetic biomaterials and immune cells. Herein, fully synthetic poly(glycidyl ether) (PGE) copolymer coatings are evaluated as bioinstructive materials for the in vitro culture and intrinsic activation of human immune cells. Immature monocyte-derived dendritic cells (moDCs) are exposed to PGE brush and gel coatings of varying copolymer composition, wettability, and deformability immobilized on polystyrene culture dishes. Compared to moDCs cultured on standard tissue culture-treated polystyrene, activation marker levels on the cell surface are strongly enhanced on PGE substrates. Thereby, moDCs undergo a distinct morphological change and reach levels of activation comparable to those achieved by toll-like receptor (TLR) ligand liposaccharide (LPS), specifically for the expression of costimulatory molecules CD86 and CD40 as well as human leukocyte antigen (HLA)-DR. In addition, PGE coatings induce a significantly enhanced level of programmed cell death ligands 1 and 2 (PD-L1/-L2) on the moDC surface, two molecules crucially involved in maintaining immune tolerance. In addition, an increased release of matrix metalloproteinases MMP-1 and MMP-7, as well as transforming growth factor (TGF)-β1 and epidermal growth factor (EGF) was observed in moDCs cultured on PGE substrates. As fully synthetic biomaterials, PGE coatings demonstrate intrinsic functional competence in instructing immature human moDCs for phenotypic activation in vitro, accompanied by the secretion of bioactive molecules, which are known to be crucial for tissue regeneration. Hence, PGE coatings hold strong potential for immune-modulating implant coatings, while PGE-activated moDCs are promising candidates for future clinical cell-based immunoengineering therapies

T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays

Chemicals can elicit T-cell-mediated diseases such as allergic contact dermatitis and adverse drug reactions. Therefore, testing of chemicals, drugs and protein allergens for hazard identification and risk assessment is essential in regulatory toxicology. The seventh amendment of the EU Cosmetics Directive now prohibits the testing of cosmetic ingredients in mice, guinea pigs and other animal species to assess their sensitizing potential. In addition, the EU Chemicals Directive REACh requires the retesting of more than 30,000 chemicals for different toxicological endpoints, including sensitization, requiring vast numbers of animals. Therefore, alternative methods are urgently needed to eventually replace animal testing. Here, we summarize the outcome of an expert meeting in Rome on 7 November 2009 on the development of T-cell-based in vitro assays as tools in immunotoxicology to identify hazardous chemicals and drugs. In addition, we provide an overview of the development of the field over the last two decades

Stimulation of isolated Langerhans cells from human epidermis by T cell and danger signals

Titelblatt, Danksagungen Inhaltsverzeichnis Abkürzungsverzeichnis Zusammenfassung, Summary Einleitung Material und Methoden Ergebnisse Diskussion Literaturverzeichnis LebenslaufDendritische Zellen sind die Initiatoren spezifischer Immunantworten. Die Langerhans-Zelle (LC) aus der Epidermis gilt als der Prototyp einer dendritischen Zelle im unreifen Zustand. Von allen dendritischen Zellen wurde die LC zuerst beschrieben und viele grundlegende Erkenntnisse über dendritische Zellen wurden aus Versuchen mit LC gewonnen. Diese seltenen Zellen lassen sich nur mit hohem präparativem Aufwand aus der Epidermis isolieren. Alternativ wird der Großteil der Experimente in der DC-Forschung heute mit einer in der Kultur generierten, also artifiziellen dendritischen Zelle betrieben. Es häufen sich jedoch die Hinweise, dass sich diese Zellen in Phänotyp und funktionellen Eigenschaften von LC aus der Haut unterscheiden. LC sind in ihrer epidermalen Umgebung dem Angriff pathogener Mikroorganismen ausgesetzt. Über die Stimulation von Toll-Rezeptoren, die spezifische molekulare Muster verschiedener Erregerklassen erkennen, ist bei LC bis heute nichts bekannt. Ebenfalls nicht untersucht ist die Expression neu beschriebener inflammatorischer Zytokine. Auch Mikrochip-Analysen von LC- ähnlichen Zellen werden in dieser Arbeit erstmals publiziert. Die Ergebnisse meiner Arbeit lassen sich in folgenden Punkten zusammenfassen: 1. Über magnetische Zellsortierung mit CD1c konnten viable epidermale LC in bislang nicht erreichter Reinheit isoliert werden. Die angereicherten Zellen reiften nicht spontan aus und erwarben nach Stimulation die funktionelle Kapazität, T-Zellen in die Proliferation und zur Sekretion von IFN-γ zu bringen. 2. Elektronenmikroskopische Analysen zeigten direkten Membrankontakt von LC und CD40L+Zellen. Die LC zeigten morphologisch deutliche Anzeichen von Zellaktivierung. 3. Eine Kostimulation mit CD40-Ligand war die Voraussetzung, dass TLR-Liganden das Zytokin IL-12p70 in LC induzieren konnten. Dabei produzierten epidermale LC geringere Mengen dieses TH1-Zytokins als LC, die aus Monozyten in Kultur generiert wurden (MoLC). 4. Bei TLR-Stimulation setzten LC mit IL-12 und IL-10 inflammatorische und anti-inflammatorische Zytokine frei. 5. Erstmals wurde eine Expression der neu beschriebenen Zytokine IL-27 und IL-18 in LC gefunden. Das Gen von GM-CSF konnte in epidermalen LC aktiviert werden. TGF-β wurde konstitutiv exprimiert. 6. Das Antigen CD1c eignete sich ebenfalls, um aus der Gesamtpopulation der MoLC die Subpopulation anzureichern, die im stimulierten Zustand das kostimulatorische Molekül CD80 exprimierte. Die CD1c-negativen MoLC ließen sich am wenigsten stimulieren. In vitro generierte dendritische Zellen, die in aktuellen klinischen Studien eingesetzt werden, stellen also eine Mischpopulation dar. Über CD1c-Sortierung konnten optimale T-Zell-reaktive APC gewonnen werden. 7. Stimulierte LC und CD1c+MoLC verstärkten in ähnlichem Ausmaß die Expression von CD83 und CD86. Die Profile von CD80, CD54 und HLA-DR unterschieden sich dagegen deutlich. 8. Mikroarray-Experimente und RT-PCR detektierten Expressionen von TNF-α, IL-1β, GM-CSF, IL-6 und IL-8 in CD40L- und LPS-stimulierten CD1c+MoLC. Über die Expression von NF-κB, RelB, STAT5 und BclX und Bcl-A1 wurden Gene der Zellaktivierung und des Anti-Apoptose-Programms induziert. Das Expressionsprofil repräsentierte deutlich eine Entzündungsreaktion. Die Vergleichsstudien von MoLC mit LC als Modell einer ex vivo-isolierten dendritischen Zelle ergaben neue Erkenntnisse über die funktionelle Kapazität von LC. Dendritische Zellen, generiert aus Monozyten oder Vorläufer-Zellen, werden bereits bei Therapien von Tumor- und Autoimmunerkrankungen eingesetzt (Steinmann, 2003). Die Befunde über natürliche dendritische Zellen ermöglichen Fortschritte in der Immuntherapie mit dendritischen Zellen.Dendritic cells are the initiators of specific immune responses. The epidermal Langerhans cell (LC) is considered to be the prototype of a dendritic cell in an immature state. The LC was the first described dendritic cell and a lot of fundamental findings about dendritic cells were gained by experiments with LCs. These rare cells can only be isolated from the epidermis by high preparative effort. Today, the majority of experiments in DC-research is done with artificial dendritic cells generated in culture. However, there are many indications for phenotypical and functional differences of these cells and LCs derived from skin. In their epidermal environment LCs are exposed to the attack of pathogenic microorganisms. Up to this day, in LCs nothing is known about stimulation of toll-like receptors recognizing specific molecular patterns of different pathogens. The expression of newly identified inflammatory cytokines is also not explored. In this study, microchip analyses of LC-like cells are published for the first time as well. The results of my work can be summarized as follows: 1. Viable epidermal LCs could be isolated via CD1c by magnetic cell sorting at a purity that was not achieved before. The purified cells did not mature spontaneously but gained the functional capacity to induce T cell proliferation and IFN-γ secretion only after stimulation. 2. Electron microscopical analyses revealed direct membrane contact between LCs and CD40L+cells. LCs exhibited obvious morphological marks of cell activation. 3. Costimulation with CD40 ligand was the basic requirement for the induction of the cytokine IL-12p70 in LC by TLR ligands. Epidermal LCs produced lower amounts of this TH1 cytokine than did LCs generated in culture from monocytes (MoLCs). 4. After TLR stimulation, LCs secreted both inflammatory IL-12 and anti-inflammatory IL-10. 5. An expression of the newly identified cytokines IL-27 and IL-18 was found for the first time in LCs. LC activation induced the transcription of GM-CSF. TGF-β was constitutively expressed 6. The antigen CD1c was also suited for the enrichment of a subpopulation of MoLCs which expressed the costimulatory molecule CD80 after stimulation. The CD1c-negative MoLCs were resistant to stimulation. In vitro generated dendritic cells that are used in current clinical trials therefore represent a mixed cell population. Optimal T cell-reactive APCs could be obtained by CD1c sorting. 7. The expression of CD83 and CD86 was equally enhanced in stimulated LCs and CD1c+MoLCs. However the profiles of CD80, CD54, and HLA-DR showed significant differences. 8. Microarray experiments and RT-PCR revealed the expression of TNF-α, IL-1β, GM-CSF, IL-6, and IL-8 in CD40L- and LPS-stimulated CD1c+MoLCs. Genes of cell activation and of the anti-apoptosis program were induced by the expression of NF-κB, RelB, STAT5 and BclX and Bcl-A1. The expression profile obviously represents an inflammatory reaction. The comparative studies of MoLCs and LCs as a model for an ex vivo-isolated dendritic cell gave new insights on the functional capacity of LCs. At this time, dendritic cells generated from monocytes or precursor cells are used in therapies of tumour- and autoimmune diseases (Steinmann, 2003). The findings on natural dendritic cells will enable proceedings in immunotherapy with dendritic cell

In Vitro Induction of T Helper 17 Cells by Synergistic Activation of Human Monocyte-Derived Langerhans Cell-Like Cells with Bacterial Agonists

In the case of epidermal barrier disruption, pathogens encounter skin-resident Langerhans cells (LCs) and are recognized by pathogen recognition receptors such as Toll-like receptors (TLRs). As the majority of microorganisms exhibit more than one TLR ligand, the mechanisms of subsequent T cell differentiation are complex and far from clear. In this study, we investigated combinatory effects on Th cell polarization by bacterial cell wall compounds peptidoglycan (PGN) and lipopolysaccharide (LPS) and by bacterial nucleic acid (DNA). Expression of maturation markers CD40, CD80, HLA-DR and CCR7 and the release of IL-1β, IL-6 and IL-23 was strongly enhanced by simultaneous exposure to PGN, LPS and DNA in LCs. As all these factors were potential Th17 driving cytokines, we investigated the potency of combinatory TLR stimuli to induce Th17 cells via LC activation. High amounts of IL-17A and IL-22, key cytokines of Th17 cells, were detected. By intracellular costaining of IL-17+T cells, IL-22− (Th17) and IL-22+ (immature Th17) cells were identified. Interestingly, one population of LPS stimulated cells skewed into IL-9+Th cells, and LPS synergized with PGN while inducing high IL-22. In conclusion, our data indicates that when mediated by a fine-tuned signal integration via LCs, bacterial TLR agonists synergize and induce Th17 differentiation

Efficient material-induced activation of monocyte-derived dendritic cells releasing surface molecules, matrix metalloproteinases, and growth factors needed for regenerative tissue remodeling

New experimental approaches for tissue repair have recently been proposed and include the application of natural or synthetic biomaterials and immune cells. Herein, fully synthetic poly(glycidyl ether) (PGE) copolymer coatings are evaluated as bioinstructive materials for the in vitro culture and intrinsic activation of human immune cells. Immature monocyte-derived dendritic cells (moDCs) are exposed to PGE brush and gel coatings of varying copolymer composition, wettability, and deformability immobilized on polystyrene culture dishes. Compared to moDCs cultured on standard tissue culture-treated polystyrene, activation marker levels on the cell surface are strongly enhanced on PGE substrates. Thereby, moDCs undergo a distinct morphological change and reach levels of activation comparable to those achieved by toll-like receptor (TLR) ligand liposaccharide (LPS), specifically for the expression of costimulatory molecules CD86 and CD40 as well as human leukocyte antigen (HLA)-DR. In addition, PGE coatings induce a significantly enhanced level of programmed cell death ligands 1 and 2 (PD-L1/-L2) on the moDC surface, two molecules crucially involved in maintaining immune tolerance. In addition, an increased release of matrix metalloproteinases MMP-1 and MMP-7, as well as transforming growth factor (TGF)-β1 and epidermal growth factor (EGF) was observed in moDCs cultured on PGE substrates. As fully synthetic biomaterials, PGE coatings demonstrate intrinsic functional competence in instructing immature human moDCs for phenotypic activation in vitro, accompanied by the secretion of bioactive molecules, which are known to be crucial for tissue regeneration. Hence, PGE coatings hold strong potential for immune-modulating implant coatings, while PGE-activated moDCs are promising candidates for future clinical cell-based immunoengineering therapies