ABSTRACT
Immune sensing of DNA is critical for antiviral immunity but can also trigger autoimmune diseases such as lupus erythematosus (LE). Here we have provided evidence for the involvement of a damage-associated DNA modification in the detection of cytosolic DNA. The oxidized base 8-hydroxyguanosine (8-OHG), a marker of oxidative damage in DNA, potentiated cytosolic immune recognition by decreasing its susceptibility to 3' repair exonuclease 1 (TREX1)-mediated degradation. Oxidizative modifications arose physiologically in pathogen DNA during lysosomal reactive oxygen species (ROS) exposure, as well as in neutrophil extracellular trap (NET) DNA during the oxidative burst. 8-OHG was also abundant in UV-exposed skin lesions of LE patients and colocalized with type I interferon (IFN). Injection of oxidized DNA in the skin of lupus-prone mice induced lesions that closely matched respective lesions in patients. Thus, oxidized DNA represents a prototypic damage-associated molecular pattern (DAMP) with important implications for infection, sterile inflammation, and autoimmunity.
Subject(s)
DNA Damage , DNA/metabolism , Exodeoxyribonucleases/metabolism , Membrane Proteins/metabolism , Phosphoproteins/metabolism , Animals , Cells, Cultured , DNA Repair , Guanosine/analogs & derivatives , Guanosine/metabolism , Humans , Interferon Type I , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/metabolism , Mice , Mice, Inbred C57BL , Neutrophils/metabolism , Oxidation-Reduction , Oxidative Stress , Reactive Oxygen Species/metabolismABSTRACT
Cutaneous lupus erythematosus (CLE) is an autoimmune skin disorder that is characterized by an anti-epidermal lymphocytic infiltrate invading the dermo-epidermal junction, causing an interface dermatitis (ID). Pathogenesis of CLE has been linked to activation of innate immunity. NKG2D is an innate immune receptor on NK cells and distinct T-cell populations. The NKG2D ligands MHC class I polypeptide-related sequence A and B (MICA, MICB) have been associated to CLE susceptibility. Our gene microarray analyses of chronic discoid lupus erythematosus (CDLE) skin lesions, separated in epidermal, junctional and dermal skin areas via laser microdissection, revealed a high expression of NKG2D in the lymphocytic infiltrate and led us to further investigate the role of NKG2D in CLE. Pathway analyses showed a strong "interferon (IFN) signature" and vast activation of innate immune response pathways (TLR, RIG-I, cytosolic DNA sensing, JAK/STAT) in CDLE, that expressed the high NKG2D signal. Immunohistochemistry (IHC) confirmed the presence of NKG2D and its ligand MICB in CDLE and subacute cutaneous lupus erythematosus (SCLE) lesions. Finally, HaCaT cells were stimulated with nucleic acids and extracted RNA was sequenced with Illumina HiSeq and showed that stressed keratinocytes express typical NKG2D ligands MICA/B and ULBP2. This study provides first evidence that NKG2D is present in CDLE and SCLE skin lesions and could be relevant for cytotoxicity in IFN-driven skin lesions with upregulated innate immune response pathways present in CLE. It could furthermore play a role in CLE inflammation promoted by keratinocytes under cell stress.
Subject(s)
Cytotoxins/metabolism , Keratinocytes/metabolism , Lupus Erythematosus, Cutaneous/metabolism , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Humans , Immunity, Innate/genetics , Ligands , NK Cell Lectin-Like Receptor Subfamily K/genetics , Up-RegulationABSTRACT
Irradiation with ultraviolet (UV) light is an important exacerbating factor in cutaneous lupus erythematosus (CLE) and induces various effects in the skin of patients with the disease, such as cell death and inflammation. Recently, we demonstrated the ability of a broad-spectrum sunscreen to prevent UV-induced damage both in patients with CLE and healthy controls (HCs). The aim of this study was to evaluate whether the UV-dependent activation of interferon (IFN)-driven inflammation in CLE can also be prevented by application of the sunscreen. In 20 patients with different subtypes of CLE and 10 HCs, defined areas on the upper back were treated with a broad-spectrum liposomal sunscreen 20 min prior to a combined standardized UVA/UVB irradiation. Immunohistological analyses using antibodies directed against MxA, CD11c, CD123 and CD68 were performed from skin biopsies taken from areas before UV irradiation as well as from sunscreen-treated and sunscreen-untreated areas 24 and 72 h after UV irradiation. The expression of MxA was completely prevented by the sunscreen applied prior to UV irradiation in CLE patients and HCs. Additionally, sunscreen protection significantly diminished the number of the CD11c- and CD123-positive dendritic cells, which are suggested to be a major source of type I/III IFNs, in UV-irradiated skin of patients with CLE. Moreover, the application of the sunscreen prevented the increase in CD68-positive macrophages in both groups 72 h after UV irradiation. The data of this study demonstrate that UV protection reduces lesional tissue damage and inhibits the typical IFN-driven inflammatory response in CLE.
Subject(s)
Inflammation/metabolism , Interferons/metabolism , Lupus Erythematosus, Cutaneous/immunology , Skin/pathology , Sunscreening Agents/administration & dosage , Ultraviolet Rays , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , CD11c Antigen/metabolism , Case-Control Studies , Humans , Interleukin-3 Receptor alpha Subunit/metabolism , Liposomes/chemistry , Lupus Erythematosus, Cutaneous/metabolism , Macrophages/metabolism , Macrophages/radiation effects , Myxovirus Resistance Proteins/metabolism , Time Factors , Ultraviolet Rays/adverse effectsABSTRACT
Actinic cheilitis (AC) is an early keratocyte neoplasia with inflammation that occurs in the lip vermillion with the potential to develop into invasive squamous cell carcinoma (SCC). The expression of the intracellular enzyme indoleamine 2,3-dioxygenase (IDO) by antigen-presenting cells and/or tumor cells has been described to arrest T cell proliferation by degrading the essential amino acid tryptophan from the environment. The expression of IDO in AC may support cancer progression by inhibiting T cell-mediated rejection responses. The aim of this study was to identify the cellular nature and extent of IDO expression in early keratocye neoplasia of the lower lip (n=25), and to correlate IDO expression to the severity of epithelial atypia (KIN I°- KIN III°) and to the extent of actinic inflammation. The expression of IDO was analyzed together with expression markers for T-cells (CD3), myeloid DCs (S100, CD11c), macrophages (CD68, CD11c), and Langerhans cells (CD1a) by immunohistochemistry and immunofluorescence analysis. Analyses showed that IDO was expressed in myeloid S100(+) CD11c(+) DCs. The expression of IDO correlated significantly with the degree of epithelial atypia (P=0.0005) but not to the extent of inflammation (P=0.4283). Expression of IDO in early atypic skin epithelial conditions might be a predictor to promote carcinogenesis.
Subject(s)
Cheilitis/enzymology , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Lip/enzymology , Skin/enzymology , Cheilitis/pathology , Humans , Inflammation/enzymology , Inflammation/pathology , Lip/pathology , Severity of Illness Index , Skin/pathologySubject(s)
Sunscreening Agents/chemistry , Administration, Cutaneous , Drug Evaluation, Preclinical , Friction , Humans , Hydrophobic and Hydrophilic Interactions , Liposomes , Prospective Studies , Skin , Sunburn/prevention & control , Sunscreening Agents/radiation effects , Ultraviolet Rays , WaterABSTRACT
Immunity against leishmaniasis has primarily been studied in experimental infections of mice. It was shown that infected skin dendritic cells (DC) are critical for the induction of protection against this pathogen, and targeting skin DC in vaccination approaches in mice has proven to be successful. However, little is known about the contribution of human DC subsets from the skin to primary immunity against this pathogen. In this study, we have analysed the interaction between different human DC subsets and Leishmania major. Primary human myeloid and monocyte-derived DC ingested the parasite comparable to that of murine skin DC, and this resulted in DC activation and IL-12 release, a cytokine essential for the induction of Th1/Tc1-dependent protection. Interestingly, both Langerhans cells and plasmacytoid DC did not appear to contribute to protection in humans. Thus, in leishmaniasis, both murine and human data suggest that dermal inflammatory DC appear to be superior in promoting protection.
Subject(s)
Interleukin-12/metabolism , Langerhans Cells/immunology , Langerhans Cells/parasitology , Leishmania major/immunology , Leishmaniasis/immunology , Animals , Humans , Langerhans Cells/metabolism , Leishmania major/growth & development , MiceABSTRACT
Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) represent the 2 most common types of nonmelanoma skin cancer. Both derive from keratinocytes but show a distinct biological behavior. Here we present transcriptional profiling data of a large cohort of tumor patients (SCC, n = 42; BCC, n = 114). Differentially expressed genes reflect known features of SCC and BCC including the typical cytokeratin pattern as well as upregulation of characteristic cell proliferation genes. Additionally, we found increased expression of interferon (IFN)-regulated genes (including IFI27, IFI30, Mx1, IRF1 and CXCL9) in SCC, and to a lower extent in BCC. The expression of IFN-regulated genes correlated with the extent of the lesional immune-cell infiltrate. Immunohistological examinations confirmed the expression of IFN-regulated genes in association with a CXCR3+ cytotoxic inflammatory infiltrate on the protein level. Of note, a small subset of SCC samples with low expression of IFN-regulated genes included most organ transplant recipients receiving immunosuppressive medication. Collectively, our findings support the concept that IFN-associated host responses play an important role in tumor immunosurveillance in the skin.
Subject(s)
Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/immunology , Gene Expression Regulation, Neoplastic/genetics , Interferons/immunology , Skin Neoplasms/genetics , Skin Neoplasms/immunology , Transcription, Genetic/genetics , Aged , Aged, 80 and over , Carcinoma, Squamous Cell/pathology , Cell Differentiation , Cell Proliferation , Female , Gene Expression Profiling , Humans , Immunohistochemistry , Keratins/genetics , Male , Middle Aged , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/immunology , Neoplasm Invasiveness/pathology , Neoplasms, Basal Cell/genetics , Neoplasms, Basal Cell/immunology , Neoplasms, Basal Cell/pathology , Skin Neoplasms/pathologyABSTRACT
BACKGROUND: Lichenoid graft-versus-host disease (liGVHD) histologically shares several common features with other lichenoid dermatoses, such as cutaneous lupus erythematosus and lichen planus (LP), which collectively show a junctional infiltrate of cytotoxic lymphocytes with liquefaction of the basal layer ("interface dermatitis"). Because recent studies have shown a role for type I interferon (IFN)-associated inflammation, including lymphocyte recruitment via CXCR3 <-> ligand interaction in cutaneous lupus erythematosus and LP, we hypothesized that similar mechanisms might also be involved in liGVHD. METHODS: Ten representative lesional skin biopsies taken from patients with different subsets of chronic cutaneous graft versus host disease (GvDH) were recovered from the authors' archives. Eight LP specimens and 5 punch biopsies taken from healthy skin were analyzed for control purposes. Immunohistochemistry was performed to characterize the lesional infiltrate (CD3, CD4, CD8, CD20, CD56, or CD68), to analyze type I IFN signaling (MxA), and to investigate expression of the IFN-inducible chemokines CXCL9 and CXCL10 and their ligand CXCR3. In situ hybridization was performed to visualize IFNalpha expression on the mRNA level. RESULTS: Our analyses revealed striking similarities between the inflammatory pattern seen in LP and liGVHD. Both disorders presented with a predominantly T-cellular inflammation with CD8(+) lymphocytes affecting the basal epidermal layer. The majority of lesional lymphocytes expressed the chemokine receptor CXCR3. The corresponding chemokines CXCL9 and CXCL10 were found in the epidermis and within the inflammatory infiltrate. Analyses of MxA and IFNalpha mRNA expression supported a role for type I IFNs in these conditions. LIMITATIONS: This study was limited by the number of well characterized cases in our archives. In situ hybridization was realizable only in single cases. CONCLUSION: Our results support the hypothesis that CXCR3 <-> ligand-mediated lymphocyte recruitment is involved in cutaneous liGVHD. The fact that CXCL10 was seen in precisely those areas with extensive liquefaction of the basal epidermis supports a role of this chemokine for the development of the typical histologic "interface" pattern.
Subject(s)
Dermatitis/etiology , Graft vs Host Disease/metabolism , Lichenoid Eruptions/metabolism , Receptors, CXCR3/metabolism , Skin Diseases/metabolism , Chemokine CXCL10/metabolism , Chemokine CXCL9/metabolism , Chronic Disease , Dermatitis/pathology , Epidermis/metabolism , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Graft vs Host Disease/complications , Humans , In Situ Hybridization , Interferon Type I/metabolism , Interferon-alpha/genetics , Interferon-alpha/metabolism , Lichen Planus/pathology , Lichenoid Eruptions/complications , Ligands , Lymphocytes/metabolism , Myxovirus Resistance Proteins , RNA, Messenger/metabolism , Skin Diseases/complications , T-Lymphocytes/pathologyABSTRACT
Thought-shape fusion (TSF) is a cognitive distortion associated with eating disorders (ED). A similar distortion, thought-abandonment fusion (TAbF), is assumed to occur in borderline personality disorder (BPD). In this study the specificity of TSF in participants with anorexia nervosa (AN) and TAbF in participants with BPD was examined. 63 patients completed questionnaires assessing the manifestation of trait-TAbF and trait-TSF, as well as relevant psychopathology. Nonparametric conditional inference trees were used to test for cognitive disorder-specificity. Participants with AN exhibited higher trait-TSF-scores than those with BPD, when participants with BPD and a co-occurring AN were removed. Trait-TSF in participants with AN seemed to be disorder-specific. Participants with BPD and a co-occurring AN had the highest TAbF-scores. The specificity hypothesis could only be partially confirmed for trait-TAbF: while participants with BPD and a co-occurring AN tended to have the highest trait-TAbF scores, high mean values could also be found in participants with AN. The results indicate that TAbF is not specific to BPD, but may also play a role in AN. Both distortions seem to play a role in the maintenance of the respective disorders.
Subject(s)
Anorexia Nervosa/physiopathology , Borderline Personality Disorder/physiopathology , Cognitive Dysfunction/physiopathology , Thinking/physiology , Adult , Anorexia Nervosa/complications , Borderline Personality Disorder/complications , Cognitive Dysfunction/etiology , Female , Humans , Young AdultABSTRACT
BACKGROUND: Lupus erythematosus profundus (LEP) is a rare variant lupus erythematosus with unclear etiology characterized by lobular panniculitis. Recently, we observed a case of LEP involving the lower right eyelid. Our immunohistological analyses of lesional skin biopsies revealed a type I IFN signature in the context of cytotoxic lobular panniculitis. OBJECTIVE: Since type I IFNs have been shown to be involved in other cutaneous LE subtypes, especially in chronic discoid LE, we hypothesized that a type I IFN driven immune response might play an important role in the pathogenesis of LEP. METHODS: In addition to the above case, 9 skin biopsies taken from 5 patients with LEP were analyzed for a type I interferon signature by immunohistochemistry. Furthermore, 8 skin biopsies taken from patients with active chronic discoid LE and 5 biopsies of healthy skin were included for control purposes. The inflammatory infiltrate was characterized using monoclonal antibodies specific for CD3, CD4, CD8, CD20, CD68, and CD123. Subsequently, we analyzed the expression the type I IFN Marker MxA, the cytotoxic molecules granzyme B and Tia1, the chemokine receptor CXCR3 and its ligand, the interferon inducible protein IP10/CXCL10. RESULTS: LEP skin lesions were characterized by a lobular panniculitis, dominated by cytotoxic CXCR3(+) lymphocytes. Strong MxA expression indicated extensive type I IFN production within the fat lobules. Numerous plasmacytoid dendritic cells appear to be the major source of type I IFNs. Lesional expression of IP10 links the type I IFN production and recruitment of CXCR3(+) lymphocytes. LIMITATIONS: The study was based on histological and immunohistological analyses in a limited number of patients, due to the rareness of the investigated disease. CONCLUSION: Our results demonstrate a type I IFN driven immune response in active LEP skin lesions. We suggest that this type I IFN driven inflammation is responsible for the recruitment of CXCR3(+) lymphocytes into fat lobules and enhance their cytotoxic capacity.
Subject(s)
Panniculitis, Lupus Erythematosus/pathology , Panniculitis, Lupus Erythematosus/physiopathology , Receptors, Chemokine/immunology , T-Lymphocytes, Cytotoxic/immunology , Biopsy, Needle , Humans , Immunohistochemistry , Panniculitis, Lupus Erythematosus/immunology , Receptors, CXCR3 , Receptors, Chemokine/metabolism , Sensitivity and Specificity , T-Lymphocytes, Cytotoxic/metabolismABSTRACT
Cutaneous lupus erythematosus (CLE) is a photosensitive autoimmune disease characterized by a strong type I IFN-associated inflammation. Keratinocytes are known to determine the interface dermatitis pattern in CLE by production of proinflammatory cytokines in the lower epidermis. These cytokines drive a cytotoxic anti-epithelial immune response resulting in keratinocytic cell death and release of endogenous nucleic acids. We hypothesized that these endogenous nucleic acids (RNA and DNA motifs) have the capacity to activate innate immune pathways in keratinocytes via pathogen recognition receptors. Gene expression analyses showed an excessive activation of innate immune response pathways with strong expression of IFN-regulated cytokines in CLE skin lesions. Cultured keratinocytes produce large amounts of these cytokines in response to stimulation of PRR with endogenous nucleic acids. UV stimulation enhances the immunogenicity of endogenous nucleic acids and induces CLE-like skin lesions in knockout mice lacking the cytosolic DNase TREX1. Our results provide evidence for a pathogenetic role of endogenous nucleic acids in CLE. They are released within the cytotoxic inflammation along the dermo-epidermal junction and have the capacity to drive the CLE-typical inflammation. UV irradiation supports this inflammation by generation of highly immunostimulatory DNA motifs (8-hydroxyguanosine). These findings explain the photosensitivity of patients with lupus and identify pathways of the innate immune system as targets for future therapies.
Subject(s)
Cytokines/metabolism , Immunity, Innate , Inflammation/metabolism , Lupus Erythematosus, Cutaneous/metabolism , Nucleic Acids/metabolism , Animals , Apoptosis , Cells, Cultured , Humans , Inflammation/immunology , Inflammation/pathology , Keratinocytes/metabolism , Lupus Erythematosus, Cutaneous/immunology , Lupus Erythematosus, Cutaneous/pathology , Mice, KnockoutABSTRACT
BACKGROUND: Patients with depression often have limited access to outpatient psychotherapy following inpatient treatment. The objective of the study was to evaluate the long-term effectiveness of a telephone-based aftercare case management (ACM) intervention for patients with depression. METHODS: We performed a prospective randomized controlled trial in four psychotherapeutic inpatient care units with N = 199 patients with major depression or dysthymia (F32.x, F33.x, F34.1, according to the ICD-10). The ACM consisted of six phone contacts at two-week intervals performed by trained and certified psychotherapists. The control group received usual care (UC). The primary outcome was depressive symptom severity (BDI-II) at 9-month follow-up, and secondary outcomes were health-related quality of life (SF-8, EQ-5D), self-efficacy (SWE), and the proportion of patients initiating outpatient psychotherapy. Mixed model analyses were conducted to compare improvements between treatment groups. RESULTS: Regarding the primary outcome of symptom severity, the groups did not significantly differ after 3 months (p = .132; ES = -0.23) or at the 9-month follow-up (p = .284; ES = -0.20). No significant differences in health-related quality of life or self-efficacy were found between groups. Patients receiving ACM were more likely to be in outpatient psychotherapy after 3 months (OR: 3.00[1.12-8.07]; p = .029) and 9 months (OR: 4.78 [1.55-14.74]; p = .006) than those receiving UC. CONCLUSIONS: Although telephone-based ACM did not significantly improve symptom severity, it seems to be a valuable approach for overcoming treatment barriers to the clinical pathways of patients with depression regarding their access to outpatient psychotherapy.
Subject(s)
Aftercare , Case Management , Depressive Disorder/therapy , Telephone , Adult , Female , Humans , Male , Middle Aged , Prospective Studies , Treatment OutcomeABSTRACT
Strong contact sensitizers are able to induce signal transduction mechanisms such as tyrosine phosphorylation and activation of MAP kinases in antigen-presenting cells. We studied the capacity of different antioxidants (ascorbic acid, alpha-tocopherol, pyrrolidine dithiocarbamate, N-acetylcysteine, and glutathione) to block the increase in tyrosine phosphorylation in human monocytes seen after stimulation with strong contact sensitizers. Human peripheral blood mononuclear cells were stimulated with 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone in the presence or absence of these antioxidants. The total amount of membrane-associated phosphotyrosine in CD14+ cells was quantified using flow cytometric techniques. Complete inhibition of tyrosine phosphorylation was noticed when cells were stimulated in the presence of N-acetylcysteine or glutathione. Using N-acetylcysteine as inhibitor similar results were obtained for cells stimulated with formaldehyde, thimerosal methyldibromoglutaronitrile, diphenylcyclopropenone, p-phenylenediamine, toluene-2,5-diamine, and 2,4-dinitrofluorobenzene. By use of a trinitrophenyl-specific monoclonal antibody it was shown that N-acetylcysteine as well as cysteine prevents the binding of 2,4,6-trinitrochlorobenzene to proteins in monocytes and monocyte-derived mature dendritic cells. Furthermore, the capacity of N-acetylcysteine to block the activation of p38 and ERK1/2 MAP kinases by 2,4,6-trinitrochlorobenzene was demonstrated. The radical scavengers ascorbic acid and alpha-tocopherol as well as the nuclear factor kappaB inhibitor pyrrolidine dithiocarbamate failed to prevent the increase in tyrosine phosphorylation. Our data present evidence that reactive oxygen species as well as transcription factor nuclear factor kappaB seem to be unimportant for the induction of tyrosine phosphorylation by contact sensitizers. On the other hand, protection of thiol groups using compounds with free sulfhydryl groups is very effective to block this process. This finding may have implications for prevention of occupational sensitization to strong contact allergens.
Subject(s)
Antigen-Presenting Cells/drug effects , Antioxidants/physiology , Dermatitis, Contact/prevention & control , Sulfhydryl Compounds/pharmacology , Acetylcysteine/pharmacology , Cells, Cultured , Cysteine/pharmacology , Glutathione/pharmacology , Humans , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/physiology , Phosphorylation , Picryl Chloride/metabolism , Picryl Chloride/toxicity , Tyrosine/metabolismABSTRACT
Strong contact sensitizers are able to induce distinct signal transduction mechanisms in antigen-presenting cells by coupling to cell proteins. The predominant target structures of haptens are thought to be thiol and amino groups in cysteine and lysine residues. We studied whether coupling of small reactive chemicals to thiol or amino groups might be responsible for the activation of monocytes and mature monocyte-derived dendritic cells. Human peripheral blood mononuclear cells were stimulated in vitro with subtoxic concentrations of the strong haptens 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone and 2, 4, 6-trinitrochlorobenzene, the thiol-reactive reagents N-hydroxymaleimide and N-ethylmaleimide, as well as the amino-reactive compounds sulfosuccinimidyl acetate and 2-iminothiolane. Flow cytometric quantification of tyrosine phosphorylation in CD14+ monocytes showed that 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone, 2, 4, 6-trinitrochlorobenzene, N-hydroxymaleimide, and N-ethylmaleimide but not sulfosuccinimidyl acetate and 2-iminothiolane strongly induced this process. Tyrosine phosphorylation induced by 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone and 2, 4, 6-trinitrochlorobenzene was completely prevented in the presence of cysteine but not lysine, suggesting a competitive mechanism between cysteine and sulfhydryl groups of cell proteins. Using the mouse ear swelling test N-hydroxymaleimide could be classified as a significant contact allergen in comparison to 2, 4, 6-trinitrochlorobenzene, whereas no sensitizing potential became apparent for sulfosuccinimidyl acetate and 2-iminothiolane. Western blot analysis on monocytes and mature monocyte-derived dendritic cells confirmed the flow cytometric data for tyrosine phosphorylation and demonstrated a selective capacity of haptens and thiol-reactive compounds to activate ERK1/2 mitogen-activated protein kinase. Our data show that strong affinity of a small reactive chemical toward thiol groups is important for the activation of monocytes and monocyte-derived dendritic cells and can support the process of sensitization.
Subject(s)
Dendritic Cells/immunology , Monocytes/immunology , Sulfhydryl Compounds/pharmacology , Acetates/pharmacology , Anti-Infective Agents/pharmacology , Antioxidants/pharmacology , Cysteine/pharmacology , Dendritic Cells/drug effects , Ethylmaleimide/pharmacology , Humans , Interleukin-1/metabolism , Lysine/pharmacology , MAP Kinase Signaling System/drug effects , Maleimides/pharmacology , Monocytes/drug effects , Monocytes/metabolism , Phosphorylation/drug effects , Picryl Chloride/pharmacology , Succinimides/pharmacology , Sulfhydryl Reagents/pharmacology , Thiazoles/pharmacology , Tyrosine/metabolismABSTRACT
Recently we described the induction of tyrosine phosphorylation by contact sensitizers as an early molecular event during the activation of antigen- presenting cells. In this study, the role of the p38 mitogen-activated protein kinase for the activation of human monocytes after exposure to four structurally unrelated contact sensitizers was analyzed in comparison with the irritant benzalkonium chloride and an inductor of oxidative stress (H2O2) using immunofluorescence, Western blotting, and enzyme-linked immunosorbent assay techniques. Bio chemical analysis revealed a translocation of p38 from the cytoplasm to the detergent-resistant cell fraction only upon stimulation with contact sensitizers. The activity of p38 was studied by quantification of its phosphorylated active form with a specific antibody and by kinase assay. Although all stimulants used in this study led to the activation of p38, a translocation to the detergent-resistant fraction as well phosphorylation of the mitogen-activated protein kinase dependent transcription factor Elk-1 was induced only by contact sensitizers. Evidence for a functional relevance of mitogen-activated protein kinase activation was provided by measurement of the hapten-induced production of the proinflammatory cytokine interleukin-1beta. Its release was inhibited by blocking p38-mediated signaling using the imidazole compounds SB203580 and SB202190. These data show that contact sensitizers are strong activators of the p38 mitogen-activated protein kinase. Although activation of this stress-associated pathway has been reported for many other stimuli, a unique translocation of p38 from the cytoplasm to the detergent-resistant fraction seems to be a specific event during hapten-induced activation of antigen-presenting cells.
Subject(s)
DNA-Binding Proteins , Haptens/pharmacology , Irritants/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Monocytes/enzymology , Transcription Factors , Benzalkonium Compounds/pharmacology , Biological Transport/drug effects , Cytoplasm/enzymology , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Humans , Imidazoles/pharmacology , In Vitro Techniques , Interleukin-1/metabolism , MAP Kinase Signaling System/drug effects , Phosphorylation , Proto-Oncogene Proteins/metabolism , Pyridines/pharmacology , ets-Domain Protein Elk-1 , p38 Mitogen-Activated Protein KinasesABSTRACT
JAK/STAT pathways are described as the major mechanisms by which cytokine receptors transduce intracellular signals. The signalling mechanisms in antigen-presenting cells (APC) in the sensitization phase of contact hypersensitivity are poorly understood. The aim of this study was to clarify whether well-established JAK/STAT signalling pathways might be activated directly by contact sensitizers as described previously for tyrosine kinases and some MAP kinases. As a model of epidermal APC, human monocytes and human monocyte-derived dendritic cells were stimulated with the structurally unrelated contact sensitizers MCI/MI, thimerosal, TNCB and formaldehyde. The phosphorylation states of the transcription factors STAT1, STAT3, STAT4, STAT5 and STAT6 were determined by Western blot analysis using phosphospecific antibodies. In contrast to the positive controls performed with the cytokines IFN-gamma, IL-10, IFN-alpha, GM-CSF and IL-4, no significant increase in the phosphorylation of STAT molecules was recognized in hapten-treated cells. These results suggest that contact allergens do not directly activate common JAK/STAT pathways. Therefore the activation of APC in the early sensitization phase of contact hypersensitivity by haptens does not involve signals normally delivered by JAK-associated cytokine receptors.
Subject(s)
Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/metabolism , DNA-Binding Proteins/metabolism , Haptens/toxicity , Milk Proteins , Mitogen-Activated Protein Kinases/metabolism , Trans-Activators/metabolism , Antigen-Presenting Cells/immunology , Cytokines/pharmacology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/metabolism , Dermatitis, Contact/etiology , Dermatitis, Contact/immunology , Dermatitis, Contact/metabolism , Epidermis/drug effects , Epidermis/immunology , Epidermis/metabolism , Humans , In Vitro Techniques , JNK Mitogen-Activated Protein Kinases , Monocytes/drug effects , Monocytes/immunology , Monocytes/metabolism , STAT1 Transcription Factor , STAT3 Transcription Factor , STAT4 Transcription Factor , STAT5 Transcription Factor , STAT6 Transcription Factor , Signal TransductionABSTRACT
Type I IFNs (IFNα/ß) have been shown to have a central role in the pathophysiology of lupus erythematosus (LE). The recently discovered type III IFNs (IFNλ1/IL29, IFNλ2/IL28a, IFNλ3/IL28b) share several functional similarities with type I IFNs, particularly in antiviral immunity. As IFNλs act primarily on epithelial cells, we investigated whether type III IFNs might also have a role in the pathogenesis of cutaneous LE (CLE). Our investigations demonstrate that IFNλ and the IFNλ receptor were strongly expressed in the epidermis of CLE skin lesions and related autoimmune diseases (lichen planus and dermatomyositis). Significantly enhanced IFNλ1 could be measured in the serum of CLE patients with active skin lesions. Functional analyses revealed that human keratinocytes are able to produce high levels of IFNλ1 but only low amounts of IFNα/ß/γ in response to immunostimulatory nuclear acids, suggesting that IFNλ is a major IFN produced by these cells. Exposure of human keratinocytes to IFNλ1 induced the expression of several proinflammatory cytokines, including CXCL9 (CXC-motiv ligand 9), which drive the recruitment of immune cells and are associated with the formation of CLE skin lesions. Our results provide evidence for a role of type III IFNs in not only antiviral immunity but also autoimmune diseases of the skin.
Subject(s)
Interferon-gamma/immunology , Keratinocytes/immunology , Lupus Erythematosus, Cutaneous/immunology , Lupus Erythematosus, Cutaneous/physiopathology , Biopsy , Cells, Cultured , Chemokine CXCL9/immunology , Chemokine CXCL9/metabolism , Epidermis/immunology , Epidermis/pathology , GTP-Binding Proteins/immunology , GTP-Binding Proteins/metabolism , Humans , Immunohistochemistry , Interferon-gamma/blood , Interferon-gamma/pharmacology , Keratinocytes/drug effects , Keratinocytes/pathology , Lupus Erythematosus, Cutaneous/pathology , Membrane Proteins/immunology , Membrane Proteins/metabolism , Myxovirus Resistance Proteins , Receptors, Interferon/immunology , Receptors, Interferon/metabolism , Toll-Like Receptors/immunology , Toll-Like Receptors/metabolism , Interferon gamma ReceptorABSTRACT
Label-free biosensor technology based on dynamic mass redistribution (DMR) of cellular constituents promises to translate GPCR signaling into complex optical 'fingerprints' in real time in living cells. Here we present a strategy to map cellular mechanisms that define label-free responses, and we compare DMR technology with traditional second-messenger assays that are currently the state of the art in GPCR drug discovery. The holistic nature of DMR measurements enabled us to (i) probe GPCR functionality along all four G-protein signaling pathways, something presently beyond reach of most other assay platforms; (ii) dissect complex GPCR signaling patterns even in primary human cells with unprecedented accuracy; (iii) define heterotrimeric G proteins as triggers for the complex optical fingerprints; and (iv) disclose previously undetected features of GPCR behavior. Our results suggest that DMR technology will have a substantial impact on systems biology and systems pharmacology as well as for the discovery of drugs with novel mechanisms.
Subject(s)
Biosensing Techniques/methods , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Adenylyl Cyclases/metabolism , Animals , CHO Cells , Cell Survival , Cricetinae , Cricetulus , Enzyme Activation , GTP-Binding Protein alpha Subunits, G12-G13/metabolism , HEK293 Cells , Humans , Keratinocytes/metabolism , Organ SpecificityABSTRACT
Inappropriate activation of innate immune mechanisms, in particular of the type I interferon (IFN) system, is regarded to play an important role in the pathogenesis of lupus erythematosus (LE). Type I IFN serum levels have been shown to correlate with the disease activity in systemic LE and additionally play a proinflammatory role in the development of LE skin lesions. Recent studies demonstrated a close morphological association between the expression pattern of IFN-inducible chemokines (MxA, CXCL10) and typical histological features of cutaneous LE. These and other studies suggest that a complex network of IFN-associated cytokines, chemokines and adhesion molecules orchestrates and promotes tissue injury observed in LE skin.