Search

Heat shock protein 90 (Hsp90) and Hsp70 are chaperones implicated in different inflammatory disorders, given their property to impact innate and adaptive immune responses. Here, we determined the so far unknown role of extracellular Hsp70 in epidermolysis bullosa acquisita (EBA), an anti-type VII collagen autoantibody-mediated blistering dermatosis. The in vivo pathophysiological relevance of extracellular Hsp70 was demonstrated in an anti-type VII collagen antibody transfer-induced EBA mouse model in which elevated blood levels of this chaperone were recorded. We found that Hsp70-treated mice had a more intense clinical disease severity compared to controls that were paralleled by increased levels of cutaneous matrix metalloproteinase 9 and plasma hydrogen peroxide. The latter finding was confirmed in an independent reactive oxygen species release assay using EBA-specific immune complexes combined with recombinant Hsp70. Finally, cell culture experiments using human naive peripheral blood mononuclear cells (PBMC) revealed that extracellular Hsp70 stimulated the secretion of the T cell-derived pro-inflammatory cytokines IL-6 and IL-8. This work extends knowledge about the role of Hsps in autoimmune bullous diseases, suggesting that extracellular Hsp70 represents a pathophysiological factor and potential treatment target in EBA.

Pemphigoid diseases (PDs) are chronic and life-threatening autoimmune diseases of the skin and mucous membranes. PDs are characterized and caused by autoantibodies targeting components of the basement membrane. In the PD epidermolysis bullosa acquisita (EBA), the target autoantigen is type VII collagen. Current treatment options of PD, especially EBA, are limited and are mostly based on systemic immunosuppression. Animal models of PD have greatly advanced our understanding of PD pathogenesis. This has led to the identification of several novel therapeutic targets, including signalling molecules. Herein, the contribution of signalling molecules in the pathogenesis of the PD EBA and the effects of pharmacological targeting of these pathways are reviewed in detail. The p38 MAPK, ERK1/2, AKT, PI3Kβ, Hsp90, RORα, PDE4, Src kinases and CARD9 have been demonstrated to be critically involved in EBA pathogenesis. With the advent of new signal transduction inhibitors, we expect that the so far poor prognosis of EBA and other PD will improve significantly in the future.

The proteins secreted by parasitic nematodes are evolutionarily optimized molecules with unique capabilities of suppressing the immune response of the host organism. Neutrophil inhibitory factor (NIF), which is secreted by the dog hookworm Ancylostoma caninum, binds to the β2 integrin CD11b/CD18, which is expressed on human neutrophils, eosinophils, monocytes and macrophages and inhibits neutrophil-dependent lung injury and neutrophil invasion of ischaemic brain tissue. Neutrophils are key players in the pathogenesis of subepidermal autoimmune blistering diseases (sAIBDs), and their pathogenic activities are crucially dependent on β2 integrin functionality. Based on the template of single-stranded, dimerizing antibody derivatives, which are already used in cancer treatment, we designed a novel biologic, NIF-IGHE-CH4, comprising NIF and the dimerizing but otherwise inert constant heavy subdomain 4 (CH4) of human IgE (IGHE). This molecule was evaluated in a variety of in vitro assays, demonstrating its ability to inhibit pathogenically relevant neutrophil functions such as migration, adhesion and spreading, and release of reactive oxygen species. Finally, we confirmed that NIF-IGHE-CH4 inhibits blister formation in an ex vivo assay of sAIBD. These results suggest that NIF-IGHE-CH4 is a novel potential anti-inflammatory drug for the treatment of neutrophil-mediated diseases such as sAIBDs. This study promotes the drugs from bugs concept and encourages further research and development focused on turning parasite proteins into useful anti-inflammatory biologics.

Previous reports have demonstrated that cell-derived nanoparticles (CDNPs) composed of bovine or porcine protein complexes exerted therapeutic effects against viral infections and cancer in mice and humans. Based on these observations, we asked whether CDNPs would improve inflammatory skin disorders. To address this, we utilized two distinct mouse models of cutaneous inflammation: the autoimmune skin-blistering disease epidermolysis bullosa acquisita (EBA) as an example of an autoantibody-induced cutaneous inflammation, and Leishmania major (L. major) infection as an example of a pathogen-induced cutaneous inflammation. In both models, we observed that CDNPs increased mRNA expression of the Th2 cytokine IL-4. Clinically, CDNPs decreased inflammation due to EBA and increased L. major-specific IgG1 levels without major effects on infected skin lesions. In addition, CDNPs supported the growth of keratinocytes in human skin cultures. In vitro studies revealed that CDNPs were taken up predominantly by macrophages, leading to a shift towards the expression of anti-inflammatory cytokine genes. Altogether, our data demonstrate that treatment with porcine CDNPs may be a new therapeutic option for the control of autoimmune-mediated inflammatory skin disorders.

Autoimmune diseases affect a large fraction of the population in Western countries. To elucidate the underlying causes, autoantibody transfer-induced mouse models have been established that greatly contributed to the understanding of the pathophysiology of these diseases. However, the role of a potentially co-occurring murine xenogeneic immune response to commonly utilized rabbit anti-mouse IgG remains poorly understood. Using the established rabbit anti-mouse type VII collagen (COL7) IgG-induced mouse model of the mucocutaneous blistering disorder epidermolysis bullosa acquisita (EBA), we found in this study a profound T- and B-cell response along with an altered cytokine expression profile in draining lymph nodes of mice injected with the xenogeneic IgG. This was associated with the formation of circulating and skin-bound mouse anti-rabbit IgG in wild-type but not CD154-deficient or B-cell-deficient JHT mice challenged with pathogenic rabbit IgG. Development of EBA skin lesions was attenuated in the two mouse strains lacking a B-cell response at later observation time points, but was not affected in mice treated with the T-cell trafficking blocker FTY720. Collectively, our results implicate a host's xenoreactive immune response to rabbit anti-mouse COL7 IgG, a confounding effect that may contribute to immune complex-driven inflammation and tissue damage in this antibody transfer-induced EBA mouse model, especially at later time points. In this regard, it may be recommended to finish the evaluation of results obtained by experiments employing antibody-transferred mouse models within the first 2 weeks after the pathogenic antibody injection.

Experimental models of bullous pemphigoid (BP), the most frequent subepidermal autoimmune bullous disease, revealed that the immune response leading to blister formation represents an incompletely understood complex process involving different inflammatory cells. In contrast to previous reports commonly focusing on limited molecular and cellular phenotypes of the disease, the aim of this study was to investigate a broad spectrum of markers of cellular immune activation in patients with BP. We found that serum levels of soluble CD4, myeloperoxidase, S100A12, eosinophil cationic protein and soluble P-selectin were significantly elevated in patients with active BP compared with normal controls. Mast cell tryptase and neopterin serum levels significantly decreased at the time of clinical remission of the patients. Additionally, serum concentrations of soluble IL-2 receptor, mast cell tryptase and soluble P-selectin were significantly associated with levels of circulating anti-BP180 autoantibodies. Our findings confirm and extend previous reports suggesting some concomitant involvement of a panel of molecules representative for a wide spectrum of cellular players (T cells, mast cells, neutrophils, eosinophils, macrophages and platelets) orchestrating the inflammatory reaction in BP. These data may favour the employment of broad-spectrum or combined immunosuppressants, potentially together with an anticoagulant treatment, over cell- or molecule-specific targeted therapy in patients with this disorder.

Heat shock protein 90 (Hsp90) inhibition blocks T-cell-linked inflammatory disease pathways and exhibits therapeutic activity in autoimmune disease mouse models, including the blistering disease epidermolysis bullosa acquisita. Although we previously showed that preformed autoreactive plasma cells do not seem to be directly affected by anti-Hsp90 treatment, immunomodulatory effects of Hsp90 inhibition on (auto-)antibody responses are not yet fully understood. In this study, the Hsp90 blocker 17-DMAG inhibited proliferation of activated total B cells and their IgG secretion in cultures of human peripheral B cells from healthy subjects, but IgG production was no longer affected when these activated B cells were allowed to differentiate prior to a deferred application of the inhibitor. 17-DMAG treatment was associated with induction of nuclear and cytoplasmic heat shock factor 1 and Hsp70 in stimulated human B cells, respectively. Type VII collagen (epidermolysis bullosa acquisita)-immunized mice early treated with 17-DMAG had reduced total B cells in spleens, a relative increase in splenic regulatory B cell fractions, higher serum IL-10 concentrations, and lower levels of circulating autoantibodies (paralleled by less pronounced disease induction) compared with vehicle-treated immunized mice. Autoantibody production was blunted in isolated and autoantigen-restimulated lymph node cells from immunized mice by either 17-DMAG or purified autologous splenic regulatory B cells. Thus, in addition to the previously described T cell inhibitory effects of Hsp90 blockade, this treatment potently modulates humoral immune responses at the B cell level, further supporting the introduction of Hsp90 inhibitors into the clinical setting for treatment of autoantibody-mediated disorders.

A broad range of immunosuppressive and immunomodulatory effects of heat shock protein 90 (Hsp90) blockade has been described in models of autoimmune bullous diseases, but the direct contribution of this chaperone to neutrophil effector pathways in the context of autoantibody-driven blistering is generally unknown. Therefore, this has been addressed in the current study on the basis of the subepidermal blistering disease epidermolysis bullosa acquisita (EBA) characterized by autoantibodies against type VII collagen, in which a crucial role of neutrophils and both their reactive oxygen species and matrix metalloproteinases in mediating tissue injury has been established. First, the Hsp90 antagonist 17-DMAG dose-dependently inhibited dermal-epidermal separation ex vivo in cryosections of human skin induced by co-incubation of EBA patient autoantibodies with neutrophils from healthy blood donors. Next, 17-DMAG dose-dependently suppressed production and release of reactive oxygen species by human neutrophils induced by both fMLP ± LPS and EBA-specific immune complexes. In addition, co-immunoprecipitation studies revealed that extracellular Hsp90 interacted with secreted matrix metalloproteinases 2 and 12 in sera of EBA patients, suggesting that these basement membrane-degrading proteolytic enzymes are client proteins of Hsp90 and dependent on its chaperone function. Our findings add to the knowledge of the multimodal anti-inflammatory effects of Hsp90 blockade and implicate that Hsp90 is closely involved in the effector mechanisms of neutrophil-driven autoantibody-induced tissue damage, thus being a relevant therapeutic target in patients with neutrophil-mediated autoimmune diseases such as inflammatory types of EBA.

Although vibrissae hair follicles (VHFs) have long been a key research model in the life sciences, their immune system (IS) is essentially unknown. Therefore, we have characterized basic parameters of the VHF-IS of C57BL/6J mice by quantitative (immuno-)histomorphometry. Murine anagen VHF harbour few CD4+ and CD8+ T cells in the distal mesenchyme and sinuses but hardly any gamma-delta T cells in their distal epithelium. MHC class II+ Langerhans cells are seeded in the VHF infundibulum, which is also surrounded by MHC class II+ and CD11b+ cells (macrophages). The number of Langerhans cells then declines sharply in the VHF bulge, and the VHF bulb lacks MHC class II+ cells. Mast cells densely populate the VHF connective tissue sheath, where they strikingly cluster around the bulge. Both the bulge and the bulb of VHF display signs of immune privilege, that is, low MHC class I and MHC class II expression and local immunoinhibitor expression (CD200, TGFβ1). This immunophenotyping study fills an important gap in the immunobiology of murine skin and identifies differences between the IS of VHF, mouse pelage and human terminal HFs. This facilitates utilizing murine VHF as a versatile organ culture model for general immunology and immune privilege research in situ.

IgG antibodies are potent inducers of proinflammatory responses by cross-linking Fc receptors on innate immune effector cells resulting in tissue injury. The recently discovered enzymes endoglycosidase S (EndoS) and IgG-degrading enzyme (IdeS) of Streptococcus pyogenes are able to modulate the interaction between IgG antibodies and the Fc receptors, by hydrolysis of the glycan associated with the heavy chain of the IgG molecule (EndoS), or cleavage in the hinge region of the heavy IgG chain (IdeS). In this work, we investigated their ability to inhibit damage mediated by skin-bound antibodies in vivo in two different experimental models, the Arthus reaction, and epidermolysis bullosa acquisita, an autoimmune blistering skin disease associated with autoantibodies against type VII collagen. We demonstrate that both enzymes efficiently interfere with IgG-mediated proinflammatory processes, offering a great asset to specifically target pathological IgG antibodies in the skin and holding great promise for future applications in human therapy.

Autoimmune bullous dermatoses are a group of severe diseases, which are clinically characterized by blisters and erosions of skin and/or mucous membranes. In order to investigate the pathogenesis of these potentially life-threatening diseases and to develop more specific therapeutic approaches, animal models have been developed that aim to reproduce the clinical, histological and immunopathological findings. We here review established and novel animal models of autoimmune skin blistering diseases and discuss their applications and limitations.

Haemostasis-maintaining platelets are also recognized as important modulators in the regulation of immune response. Activated platelets expressing P-selectin (CD62P) are involved in the extravasation of leucocytes. This study evaluated platelet P-selectin expression as a biomarker for cutaneous inflammation. P-selectin expression was assessed by flow cytometry in 147 successive patients suffering from an inflammatory or infectious skin condition at the day of admission for in-patient treatment as well as a day prior to demission. Forty-one patients admitted for allergy testing served as controls. A commercially available ELISA was used in 17 patients to determine soluble P-selectin in the plasma. In patients with psoriasis, the Psoriasis Area and Severity Index (PASI) was documented as a measure for disease severity. We observed a significant increase in platelet P-selectin expression in patients with inflammatory or infectious disorders, when compared to the control group (3,01% vs. 1,46%; P < 0.000001). Successful treatment resulted in a significant decrease in P-selectin expression to the level of the control group. In the case of psoriasis (n = 47), we found highly significant correlation between P-selectin and PASI (r = 0.51; P < 0.000001), as well as between the change in the PASI and the change in P-selectin expression (r = 0.4; P = 0.006). Platelet P-selectin expression as determined by flow cytometry correlated well with the results of soluble P-selectin, determined by ELISA (r = 0.63; P < 0.01). Thus, platelet P-selectin expression may be used as an efficacy biomarker to monitor treatment success in psoriasis. As platelet P-selectin correlates with soluble P-selectin in patient plasma, which can be measured by ELISA, the latter is feasible also for routine use.

Experimentally, initial steps of leucocyte extravasation, including tethering and rolling, are analysed in endothelial cell flow chambers. Given the complexity and speed of endothelial-immune cell interaction, computer-aided advances of this analysis are highly desirable. Herein, we compared two established methods, hand counting and tracking software, with novel analysis software using defined movies recorded at standard conditions of endothelial-leucocyte interactions. As a first validation, cell counts and velocity parameters determined by seven experienced experts revealed no statistic differences to both semi-automated tracking and fully computerized analyses. Nevertheless, interindividual variations were substantial for hand counting. In additional experiments, velocity distributions between 1 and 800 microm/s picked up by the fully computerized analysis matched well with the tracking software as indicated by speed vector histograms. With respect to the time consumed for a defined set of movies, hand counting took 3.6 +/- 1.6 h, tracking software 4.5 +/- 1.2 h, whereas fully automated analysis consumed less than 15 min, reaching real-time mode. Thus, a validated and fully computerized method yielded functional flow chamber data unbiased, independent from an examiner, and reaching high-throughput level, which in turn will allow a substantial progress in understanding this process central for skin inflammation.

While the transition from basic life science research to clinical applications is often much more cumbersome than promised, Gostynski et al. took the opposite approach and demonstrated how a scholarly, biology-guided perspective on human skin disease can reveal basic principles of human biology. Hereditary blistering diseases represent not only a disastrous fate for affected patients, but also an opportunity for understanding human molecular physiology and pathophysiology. On the basis of their clinical expertise, Gostynski et al. have elegantly used differences between lesional and non-lesional as a unique opportunity to dissect the role of structural skin proteins – namely type XVII collagen, laminin β3 and type VII collagen – in melanocyte biology, thus elucidating a new concept in melanocyte biology by transferring knowledge in reverse direction from bedside to bench.

Autoimmune diseases are rare, but their incidence has increased over the past decades. Interestingly, the co-occurrence of autoimmune diseases is well documented; however, data on the presence of more than one specific autoantibody in healthy individuals are not available. Here, we investigated the prevalence of several autoantibodies in a cohort of over 6000 healthy persons. While individual autoantibodies were rarely detected (i.e. ranging from 0.3% for ANCA to 4.6% for anti-TPO), the cumulative prevalence of the tested autoantibodies was as high as 10%. Furthermore, our results demonstrate co-occurrence of ANA with specific autoantibodies that target TPO, CCP and Dsg1/3, while ANCA and autoantibodies to PCA and BP180/BP230 were not more frequent in ANA-positive compared to ANA-negative samples. This indicates that shared and independent mechanisms influence loss of tolerance to distinct sets of self-antigens.

Psoriasis is a common chronic inflammatory skin disease, associated with significant comorbidity, for example, metabolic syndrome (MetS) and coronary heart disease (CHD). This association implies that the risk to develop these diseases is commonly controlled or that the presence of one disease favours manifestation of the other. Therefore, we assessed the catalogue of genome-wide association studies (GWAS) to analyse whether psoriasis, MetS and CHD share susceptibility loci. Interestingly, genetic control of psoriasis is almost completely independent from both MetS and CHD. In contrast, MetS and CHD share 10 common loci. Like by GWAS analysis, psoriasis susceptibility genes showed close clustering in Ingenuity Pathway Analysis, while genes conferring susceptibility to MetS and CHD were interlinked separately. These findings lead to the hypothesis that the clinically observed co-occurrence of psoriasis with MetS and CHD may be due to a common environmental factor, for example, diet, which is known as a risk factor for all of these diseases.

Pro-inflammatory chemokines and their receptors exhibit elementary functions in cell migration and in Th1-driven inflammatory conditions. One therapeutic strategy to prevent accumulation of pro-inflammatory immune cells is the use of specific chemokine receptor antagonists. An interesting and promising candidate in this context is the viral antagonist MIP-II (vMIP-II) that acts on a broad spectrum of chemokine receptors. To study the in vitro and in vivo effects of vMIP-II on pro-inflammatory chemokine receptor function, we further characterized an ovalbumin-specific murine central memory Th1IF12 clone by using RT-PCR, cDNA array and cytometry. Using in vitro chemotaxis assays we show that eukaryotically generated vMIP-II strongly inhibited migration of CCL2- or CCL5-stimulated Th1 IF12 cells. Using intravital microscopy, we observed that CCL5 induced rolling of Th1 cells in the ear vasculature of C57Bl/6 mice. Pre-treatment with vMIP-II significantly reduced CCL5-induced rolling of Th1 cells to basal levels, indicating, that vMIP-II is also active in vivo (proportion of rolling cells: 19.4 +/- 3.8%, 39.8 +/- 2.9% and 26.1 +/- 3.2%). In addition, investigating the anti-inflammatory action of vMIP-II in adoptive transfer of immunity and dinitrofluorobenzene-induced cutaneous hypersensitivity reaction using C57Bl/6 mice, we show a direct inhibitory effect of vMIP-II on the sensitization phase [Delta ear swelling 62 and 37 cm x 10(-3) for controls and vMIP-II treated mice (2.5 mg/kg), respectively] and effector phase (Delta ear swelling 14.8 and 3.6 cm x 10(-3) for controls and vMIP-II treated mice (2.5 mg/kg), respectively) of cutaneous hypersensitivity. These data indicate that vMIP-II is a promising agent to interfere with chronic inflammatory (skin) diseases.

Lymphocyte trafficking through the dermal compartment is part of the physiological surveillance process of the adaptive immune system. On the other hand, persistent or recurrent lymphocyte infiltrates are hallmarks of both types of chronic inflammatory skin diseases, Th1-type such as psoriasis or Th2/allergic-type like atopic dermatitis. A better understanding of the mechanisms underlying lymphocyte movements is one of the key prerequisites for developing more effective therapies. In this review, we introduce a range of simple-to-sophisticated experimental in vitro and in vivo approaches to analyze lymphocyte migration. These methods start from static in vitro adhesion and chemotaxis assays, include dynamic endothelial flow chamber, intravital dual photon, and transcutaneous live-video microscopy, and finally encompass specific genetically deficient or engineered animal models. Discussing pros and cons of these assay systems hopefully generates both state-of-the-art knowledge about the factors involved in most common chronic skin diseases as well as an improved understanding of the limitations and chances of new biologic pharmaceuticals that are currently introduced into clinical practice.