Miriam Wittmann

Evidence for a pathogenetic role of interleukin-18 in cutaneous lupus erythematosus.

OBJECTIVEnCutaneous manifestations are the most common clinical features of lupus erythematosus (LE). The aim of this study was to analyze differences in the inflammatory response of keratinocytes from patients with cutaneous LE (CLE) compared with healthy controls.nnnMETHODSnKeratinocytes from LE patients and controls were cultured from epidermal stem cells of the hair follicle of anagen head hairs. Functional responses of keratinocytes to cytokine stimulation were determined by flow cytometry and enzyme-linked immunosorbent assay. Biopsy samples of lesional skin were analyzed by immunohistochemistry.nnnRESULTSnKeratinocytes from CLE patients expressed higher levels of IL-18 receptor on their cell surface in response to tumor necrosis factor alpha (TNFalpha) or interferon-gamma stimulation. In response to IL-18 stimulation, these cells produced large amounts of TNFalpha. Of note, in the presence of IL-18, CLE keratinocytes failed to express IL-12. IL-12 has previously been shown to protect keratinocytes from ultraviolet irradiation-induced apoptosis. Keratinocytes from LE patients were more prone to die upon exposure to IL-18, and this increased apoptosis was abrogated by blockade of endogenously produced TNFalpha as well as by the addition of exogenous IL-12. IL-18 was highly expressed in biopsy samples of lesional skin from CLE patients.nnnCONCLUSIONnOur results demonstrate an intrinsic difference in the inflammatory response of keratinocytes and indicate an autocrine feedback loop involving TNFalpha, IL-18, and IL-12 family members. Our results suggest that IL-18 may occupy an important position in the cytokine hierarchy in CLE, indicating the potential benefit of a local agent that blocks IL-18 activity in the treatment of the manifestations of CLE.

IL-27 is expressed in chronic human eczematous skin lesions and stimulates human keratinocytes

BACKGROUNDnIL-27 is produced by antigen-presenting cells early during immune responses. IL-27 has been described to support T-cell polarization along the T(H)1 lineage but also to exert important anti-inflammatory responses in later phases of inflammation in murine models.nnnOBJECTIVEnIt was the aim of this study to analyze the potential role of IL-27 in epidermal inflammatory skin responses in human subjects.nnnMETHODSnSurface receptor expression and apoptosis of human primary keratinocytes were analyzed by means of flow cytometry. Supernatants of stimulated keratinocytes were either analyzed by means of ELISA or submitted to chemotaxis assays. RT-PCR from lesional skin and phospho-specific Western blotting were performed.nnnRESULTSnBoth subunits of IL-27 were expressed in chronic lesional allergic eczematous skin, whereas the IL-27 subunit EBV-induced gene 3 was not detectable in the acute phase of eczema. Human primary keratinocytes responded to IL-27. Stimulation of keratinocytes with IL-27 resulted in activation of the signal transducer and activator of transcription 1 and 3 pathways. Major effects found for IL-27 include CXCL10 production and MHC class I upregulation. Importantly, we could demonstrate that IL-27 acts as a priming signal on keratinocytes able to amplify chemokine production and surface molecule expression when used before a second signal, such as TNF-alpha. The effects of IL-27 could not be mimicked by IL-6, IL-12, or IL-23.nnnCONCLUSIONnThese results support the notion that IL-27 might act in an inflammatory, disease-maintaining manner in the epidermal compartment of patients with eczema.

Histamine Upregulates Keratinocyte MMP-9 Production via the Histamine H1 Receptor

Skin inflammation and the migration of cells at the site of the immune response play an important role in allergic skin diseases. It has already been described that matrix metalloproteinase 9 (MMP-9) influences tissue remodeling and facilitates cell migration by proteolytic degradation of basal membrane components. The aim of this study was to investigate MMP-9 expression on human primary keratinocytes (KCs) upon stimulation with histamine, a potent mediator in allergic responses. With ELISA and zymography, we could show that histamine induced dose-dependent upregulation of MMP-9 in cultured KCs and in punch biopsies of human skin. The histamine H(1) receptor (H(1)R) agonist beta-histine-but not agonists for H(2)R, H(3)R, and H(4)R-induced MMP-9, whereas the H(1)R antagonist clemastine blocked the effect in a dose-dependent manner. Immunohistological staining showed that histamine-induced MMP-9 led to destruction of type IV collagen at the basement membrane in healthy skin. In a coculture system of KCs and T cells, migration of T cells through an artificial basement membrane was increased after histamine stimulation of KCs. Our findings demonstrate enhanced MMP-9 production and cell migration after histamine stimulation and may represent a new mechanism by which KCs contribute to the pathology of skin diseases.

Intracutaneous injection of the macrophage‐activating lipopeptide‐2 (MALP‐2) which accelerates wound healing in mice – a phase I trial in 12 patients

Abstract:u2002 Chronic skin ulcers, such as leg ulcers, pressure sores and diabetic foot ulcers, are a challenge to physicians and medical personnel and a cause of tremendous discomfort and ensuing loss of quality of life to the patients. Wound healing involves production and action of various growth factors. A novel approach, distinct from the application of single growth factors, is the administration of the macrophage stimulator macrophage‐activating lipopeptide‐2 (MALP‐2). The rationale is based on the finding that macrophages are the main source of several growth factors required for wound healing, which are sequentially released during this process. MALP‐2 has previously been shown to be effective in an established animal model with diabetic mice. The purpose of the present phase I study was to establish tolerability of MALP‐2 when applied into small cutaneous wounds in human beings. Twelve patients (six females and six males; mean age 66.8u2003years; range 52–87u2003years) with different diagnoses were enrolled into the study. An artificial wound was created with a 2‐mm diameter skin biopsy punch and a volume of 30u2003μl MALP‐2 (0.125–1u2003μg) or vehicle control, respectively, was injected intracutaneously into the wound and closed with a water‐resistant transparent adhesive. Photos were taken daily from every patient up to 6u2003days, and skin biopsies were performed after 1u2003week from six patients. We could show in the present study for the first time that MALP‐2 caused a transient erythema and was tolerated without any systemic side effects up to a dose of 1u2003μg per wound in human beings. In healthy as well as in diabetic patients, MALP‐2 induced local inflammation that faded after 48u2003h. The effectiveness of MALP‐2 in the healing of chronic wounds in humans, e.g. in chronic skin ulcers, such as leg ulcers, pressure sores and diabetic foot ulcers, could now be addressed in further studies.

Critical involvement of IL-12 in IFN-γ induction by calcineurin antagonists in activated human lymphocytes

Calcineurin antagonists are known as potent immunosuppressants working particularly on T cells by virtue of their capacity to block nuclear factor of activated T cell (NFAT) activation and translocation to the nucleus. In addition to interleukin (IL)‐2 suppression, T helper cell type 1 (Th1) as well as Th2 cytokine transcription is blocked by calcineurin antagonists. Here, we show that calcineurin antagonists such as cyclosporin A (CsA) or tacrolimus can markedly enhance the production of interferon‐γ (IFN‐γ) by human T cells. This increased IFN‐γ production is dependent on T cell receptor (TCR) and CD28 signaling as well as on the presence of IL‐12. IL‐27, which could mimic the effect of IL‐12, was however less potent in inducing IFN‐γ production in the presence of CsA and TCR stimulation. Other cytokines such as IL‐23, IL‐18, IL‐2, or the Th2‐related cytokine IL‐4 are not able to support a calcineurin antagonist‐dependent up‐regulation of IFN‐γ. CsA‐dependent IFN‐γ production is observable in therapeutic concentrations. The effect is independent of IL‐10 or IL‐4, as addition of these cytokines could not inhibit the CsA‐induced IFN‐γ production. The effect of calcineurin antagonists is associated with an increased c‐fos expression and DNA‐binding activity of the transcription factor activated protein‐1 but not with increased DNA‐binding activity of T‐bet. Our study further supports the relevance of known calcineurin activities other than NFAT activation. The presented data may help to explain why concomitant infections (resulting in increased IL‐12 expression) under therapy with calcineurin antagonists often have a negative impact on the activity of the underlying disease (e.g., autoimmune disease).

Regulatory Role of T Lymphocytes in Atopic Dermatitis

Eczema does not occur in the absence of T cells. Here we provide an overview on the regulatory impact which T cells have on the establishment and maintenance of atopic dermatitis. Particularly, we outline the role of different T-helper cell subsets (i.e. Th-1, Th-2, T-regulatory and Th-17 cells) and their distinct influence on the cutaneous inflammatory reaction at different stages of the disease. Eczema is characterized by epidermal inflammation and thus T-cell/ker - atinocyte interactions are of particular relevance in this condition. Alterations in innate and adaptive immunity involving T cells result in susceptibility to skin infections and in hyperreactivity reactions to environmental stimuli which in turn determine the course and severity of atopic dermatitis.

Keratinocytes enriched for epidermal stem cells differ in their response to IFN-γ from other proliferative keratinocytes

Abstract:u2002 The epidermis has a pool of adult stem cells [epidermal stem cells (ESC)]. Although the localization of ESC is well described, we lack a clear understanding of their role in perturbed conditions such as inflammation. One of the most important mediators in inflammatory skin diseases acting on keratinocytes (KCs) is interferon gamma (IFN‐γ). The assumption that ESC might generate a protected niche prompted us to investigate their response to the pro‐inflammatory cytokine IFN‐γ. In this study, we isolated two populations of KCs according to their adherence ability. ESC enriched by adherence showed a higher CD29 and CD49f expression compared with other KCs. Surprisingly, surface expression of CD54 was more inducible upon IFN‐γ stimulation in short‐term cultures of the ESC subpopulation. In contrary to that, a markedly lower induction of IL‐18 and reduced basal production of CCL2 were observable in ESC. No differences in IFN‐γ‐induced interleukin (IL)‐10, CXCL10, CCL22 or transforming growth factor (TGF)β1 secretion were detectable between the two keratinocyte subpopulations. These results suggest that ESC respond to IFN‐γ with a ‘restricted’ pattern of pro‐inflammatory cytokines, and do not build up an anti‐inflammatory microenvironment by means of TGF‐β or IL‐10. Activated ESC possess the capability to interact with infiltrating lymphocytes via CD54. In conclusion, the ESC compartment might actively contribute to the immunological properties of the skin organ.

Heat Shock Protein 70 (HSP70) induces cytotoxicity of T-helper cells

Stress-inducible heat shock protein 70 (HSP70) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific CD8+ cytotoxic T-lymphocyte (CTL) and CD4+ T-helper cell (Th1) responses. In this study, we investigated the behavior of T-cell subsets stimulated with endotoxin-free recombinant HSP70 with respect to proliferation, cytokine expression, cytotoxicity against allogeneic B-lymphoblastoid cell line (B-LCL) and K562 cells as well as target-independent cytotoxicity. n nCD4+ cells exhibited a strong increase in proliferation after stimulation with HSP70, with rates of up to 29%. In the presence of target cells, a 35-fold up-regulation of granzyme B mRNA was observed after stimulation of CD4+ T-helper cells with HSP70 in combination with IL-7, -12 and -15. The target cell-independent secretion of granzyme B by CD4+ cells was greatly augmented after stimulation with HSP70 plus IL-2 or IL-7, -12 and -15. n nIn this study, we have shown that HSP70 is capable of inducing a cytotoxic response of T-helper cells in the absence of LPS or any other PAMPs. The granzyme B secretion and the cytolytic activity of CD4+ T cells is induced in a target-independent way, whereas the cytotoxic activity of CD3+ and CD8+ T cells can be further enhanced in the presence of the target cells. Our data provide novel insights into the role of extracellular HSP70 on T-cell immune response concerning the induction of target-independent T-helper cell cytotoxicity.

Human calreticulin can act as adjuvant in the maturation of antigen presenting cells

Calreticulin (CRT), an endoplasmic reticulum (ER) resident protein, is involved in critical cellular functions, such as protein folding and antigenic peptide cross presentation. Furthermore, this chaperone has been proposed to act as an adjuvant during the activation of dendritic cells (DCs) in vivo. We assessed human eukaryotically expressed CRT for its potential to induce NF-kappa B regulated maturation of monocyte-derived DCs. In order to facilitate eukaryotic expression procedures, we established and compared three different methods to express recombinant endotoxin-free CRT to be secreted in the supernatant of HEK 293 cells: (1.) the complete, unmodified CRT coding sequence was cloned into the pcDNA3.1V5/His vector (euCRT), (2.) the C-terminal ER-retrieval KDEL amino acid sequence was mutated into KDQL in order to disturb the endoplasmic retention and support the protein secretion (euCRT_KDQL) and (3.) a shRNA was designed to knock down the expression of aminoacyl-tRNA synthetase-interacting multifunctional protein-1 (AIMP-1), which is known to regulate protein retention in the ER. An efficient shRNA sequence specific to AIMP-1 transcripts was delivered to HEK 293 cells, which were afterwards transfected with the CRT-expressing vector (euCRT). No relevant differences between these different approaches were observed in regard to mRNA levels of the transfected CRT determined by Real Time RT-PCR as well as protein expression levels of CRT determined by V5/HIS ELISA in the cell culture supernatants. Thus, for large scale expression of CRT the first strategy with the unmodified CRT sequence (euCRT) was chosen. The functional capability of the expressed calreticulin to induce maturation of DCs was tested. By flow cytometry the translocation of NF-kappa B into the nuclei of the monocytes after stimulation with the recombinant CRT could be demonstrated. Using low-dose CRT (10 μg/ml) the phenoptype of the immature DCs changed to a more matured one, as indicated by an increased surface expression of CD40, CD86, CD83. In summary, our first data indicate, that this recombinant CRT can act as an adjuvant for in vitro maturation of DCs and therefore has the potential to assist in T cell stimulation and expansion protocols.