Platelet, not endothelial, P-selectin expression contributes to generation of immunity in cutaneous contact hypersensitivity.

Leukocyte extravasation is a prerequisite for host defense and autoimmunity alike. Detailed understanding of the tightly controlled and overlapping sequences of leukocyte extravasation might aid development of novel therapeutic strategies. Leukocyte extravasation is initiated by interaction of selectins with appropriate carbohydrate ligands. Lack of P-selectin expression leads to decreased contact hypersensitivity responses. Yet, it remains unclear if this is due to inhibition of leukocyte extravasation to the skin or due to interference with initial immune activation in lymph nodes. In line with previous data, we here report a decreased contact hypersensitivity response, induced by 2,4,-dinitrofluorobenzene (DNFB), in P-selectin-deficient mice. Eliciting an immune reaction towards DNFB in wild-type mice, followed by adoptive transfer to P-selectin-deficient mice, had no impact on inflammatory response in recipients. This was significantly reduced in wild-type recipient mice adoptively transferred with DNFB immunity generated in P-selectin-deficient mice. To investigate if platelet or endothelial P-selectin was involved, mice solely lacking platelet P-selectin expression generated by bone marrow transplantation were used. Adoptive transfer of immunity from wild-type mice reconstituted with P-selectin-deficient bone marrow led to a decrease of inflammatory response. Comparing this decrease to the one observed using P-selectin-deficient mice, no differences were observed. Our observations indicate that platelet, not endothelial, P-selectin contributes to generation of immunity in DNFB-induced contact hypersensitivity.

Endogenous µ-opioid peptides modulate immune response towards malignant melanoma.

Opioids exert major effects not only in the central nervous system but also in immune responses. We investigated the effects of µ-opioid peptides, secreted by tumor cells, on anti-tumor immune responses. For this purpose, tumor growth was studied in wild-type and µ-opioid receptor-deficient (MOR-/-) mice injected with B16 melanoma cells. The ability of these cells to produce opioids was studied by Western blots in vitro. Finally, biopsy material from human melanomas was investigated by immunohistochemistry for ß endorphin expression. Injection of B16 melanoma cells, producing endogenous ß endorphin, in the flank of MOR-/- mice revealed a profound reduction in tumor growth, paralleled by a significantly higher infiltration of immune cells into the tumors, when compared to tumor growth after injection of B16 melanoma cells into wild-type mice. Opioids present in B16 cell supernatant significantly reduced the proliferation of normal but not MOR-/- leucocytes. Immunohistochemical analyses of biopsies from human melanoma tissues showed a positive correlation between expression of ß endorphin and tumor progression. Our data provide evidence that µ-opioid peptides may play a major role in cancer progression by modulating immune response. This finding may have implications for the future optimization of immunointerventions for cancer.

ADAM15 expression is downregulated in melanoma metastasis compared to primary melanoma.

In a mouse melanoma metastasis model it has been recently shown that ADAM15 overexpression in melanoma cells significantly reduced the number of metastatic nodules on the lung. Unfortunately, the expression of ADAM15 in human melanoma tissue has not been determined so far. In our study, we characterized the expression of ADAM15 in tissue micro-arrays of patients with primary melanoma with melanoma metastasis. ADAM15 was expressed in melanocytes and endothelial cells of benign nevi and melanoma tissue. Importantly, ADAM15 was significantly downregulated in melanoma metastasis compared to primary melanoma. We further demonstrate that IFN-γ and TGF-ß downregulate ADAM15 protein levels in melanoma cells. To investigate the role of ADAM15 in melanoma progression, we overexpressed ADAM15 in melanoma cells. Importantly, overexpression of ADAM15 in melanoma cells reduced the migration, invasion and the anchorage dependent and independent cell growth of melanoma cells. In summary, the downregulation of ADAM15 plays an important role in melanoma progression and ADAM15 act as a tumorsuppressor in melanoma.

Junctional adhesion molecule (JAM)-B supports lymphocyte rolling and adhesion through interaction with alpha4beta1 integrin.

Junctional adhesion molecule-A (JAM-A), JAM-B and JAM-C have been implicated in leucocyte transmigration. As JAM-B binds to very late activation antigen (VLA)-4, a leucocyte integrin that contributes to rolling and firm adhesion of lymphocytes to endothelial cells through binding to vascular cell adhesion molecule (VCAM)-1, we hypothesized that JAM-B is also involved in leucocyte rolling and firm adhesion. To test this hypothesis, intravital microscopy of murine skin microvasculature was performed. Rolling interactions of murine leucocytes were significantly affected by blockade of JAM-B [which reduced rolling interactions from 9.1 +/- 2.6% to 3.2 +/- 1.2% (mean +/- standard deviation)]. To identify putative ligands, T lymphocytes were perfused over JAM-B-coated slides in a dynamic flow chamber system. JAM-B-dependent rolling and sticking interactions were observed at low shear stress [0.3 dyn/cm(2): 220 +/- 71 (mean +/- standard deviation) versus 165 +/- 88 rolling (P < 0.001; Mann-Whitney rank sum test) and 2.6 +/- 1.3 versus 1.0 +/- 0.7 sticking cells/mm(2)/min (P = 0.026; Mann-Whitney rank sum test) on JAM-B- compared with baseline], but not at higher shear forces (1.0 dyn/cm(2)). As demonstrated by antibody blocking experiments, JAM-B-mediated rolling and sticking of T lymphocytes was dependent on alpha4 and beta1 integrin, but not JAM-C expression. To investigate whether JAM-B-mediated leucocyte-endothelium interactions are involved in a disease-relevant in vivo model, adoptive transfer experiments in 2,4,-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity reactions were performed in mice in the absence or in the presence of a function-blocking JAM-B antibody. In this model, JAM-B blockade during the sensitization phase impaired the generation of the immune response to DNFB, which was assessed as the increase in ear swelling in untreated, DNFB-challenged mice, by close to 40% [P = 0.037; analysis of variance (anova)]. Overall, JAM-B appears to contribute to leucocyte extravasation by facilitating not only transmigration but also rolling and adhesion.

Computer-aided analysis of cell interactions under dynamic flow conditions.

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.

Junctional adhesion molecules (JAM)-B and -C contribute to leukocyte extravasation to the skin and mediate cutaneous inflammation.

Leukocyte extravasation is a finely tuned process, in which transmigration is the final step. Transmigration depends on molecules located at borders of endothelial cells; e.g., junctional adhesion molecules (JAM-A, -B and -C). In vivo blockade of JAM-A lead to decreased migration of monocytes into the skin. In contrast, the role of JAM-B and -C in development of cutaneous inflammation is unknown. We therefore elicited an allergic contact dermatitis in mice using 2,4-dinitro-1-fluorobenzene. RT-PCR and immunofluorescent staining of healthy skin revealed a constitutive JAM-B (66.4%+/-6.7% of all vessels) and -C expression (88.6+/-13.2%), which remained constant after induction of contact dermatitis. Functional studies, in which either JAM-B or -C neutralizing antibodies were injected into sensitized mice prior to allergen challenge showed a concentration-dependent reduction of the contact dermatitis. Decreased ear swelling was accompanied by reduction of leukocyte infiltration as analyzed by hematoxylin and eosin (H&E) histology and enzyme activity. Combined antibody treatment at doses of 1.25 mg per kg bodyweight lead to additive inhibition of allergic contact dermatitis, indicating that JAM-B and -C may have distinct functions. In conclusion, interactions with JAM-B and -C are essential for development of cutaneous inflammation.

Interleukin-1ß interferes with epidermal homeostasis through induction of insulin resistance: implications for psoriasis pathogenesis.

Response pathways of the metabolic and the immune system have been evolutionary conserved, resulting in a high degree of integrated regulation. Insulin is a central player in the metabolic system and potentially also in the homeostasis of the skin. Psoriasis is a frequent and often severe autoimmune skin disease, clinically characterized by altered epidermal homeostasis, of which the molecular pathomechanisms are only little understood. In this study, we have examined a potential role for insulin signaling in the pathogenesis of this disease. We show that IL-1ß is present in high quantities in tissue fluid collected via microdialysis from patients with psoriasis; these levels are reduced under successful anti-psoriatic therapy. Our results suggest that IL-1ß contributes to the disease by dual effects. First, it induces insulin resistance through p38MAPK (mitogen-activated protein kinase), which blocks insulin-dependent differentiation of keratinocytes, and at the same time IL-1ß drives proliferation of keratinocytes, both being hallmarks of psoriasis. Taken together, our findings point toward insulin resistance as a contributing mechanism to the development of psoriasis; this not only drives cardiovascular comorbidities, but also its cutaneous phenotype. Key cytokines inducing insulin resistance in keratinocytes and kinases mediating their effects may represent attractive targets for novel anti-psoriatic therapies.

Systemic therapy of plaque-type psoriasis ameliorates endothelial cell function: results of a prospective longitudinal pilot trial.

Severe psoriasis is associated with significant cardiovascular mortality. We therefore investigated the effects of systemic therapy on the cardiovascular risk of psoriasis patients. Thirteen consecutive patients receiving fumaric acid esters were included and followed for 24 weeks both clinically and by means of laboratory monitoring, 10 completed the study. Eight of ten patients showed a PASI-50 response. Two of three patients with clinical insulin resistance (Homeostasis Model Assessment of insulin resistance >2.5) showed normal insulin responsiveness at the end of the study. Clinical improvement was paralleled by a reduction of high-sensitive CRP serum levels (median -25%). There was a trend toward reduced serum levels for the vascular endothelial growth factor (median -10%) and resistin (median -4%), while the potentially cardio-protective adiponectin showed a trend toward increased serum levels under therapy (median +19%). Systemic endothelial function assessed by venous occlusion plethysmography revealed an improvement of endothelial vasodilator function after 24 weeks of treatment (p < 0.02). This is the first prospective study documenting an amelioration of endothelial cell function in patients with moderate-to-severe plaque-type psoriasis under effective continuous systemic therapy. Future studies need to compare the cardioprotective effects of different treatment modalities, based on hard end points such as the rate of myocardial infarction.

PAX2 regulates ADAM10 expression and mediates anchorage-independent cell growth of melanoma cells.

PAX transcription factors play an important role during development and carcinogenesis. In this study, we investigated PAX2 protein levels in melanocytes and melanoma cells by Western Blot and immunofluorescence analysis and characterized the role of PAX2 in the pathogenesis of melanoma. In vitro we found weak PAX2 protein expression in keratinocytes and melanocytes. Compared to melanocytes increased PAX2 protein levels were detectable in melanoma cell lines. Interestingly, in tissue sections of melanoma patients nuclear PAX2 expression strongly correlated with nuclear atypia and the degree of prominent nucleoli, indicating an association of PAX2 with a more atypical cellular phenotype. In addition, with chromatin immunoprecipitation assay, PAX2 overexpression and PAX2 siRNA we present compelling evidence that PAX2 can regulate ADAM10 expression, a metalloproteinase known to play important roles in melanoma metastasis. In human tissue samples we found co-expression of PAX2 and ADAM10 in melanocytes of benign nevi and in melanoma cells of patients with malignant melanoma. Importantly, the downregulation of PAX2 by specific siRNA inhibited the anchorage independent cell growth and decreased the migratory and invasive capacity of melanoma cells. Furthermore, the downregulation of PAX2 abrogated the chemoresistance of melanoma cells against cisplatin, indicating that PAX2 expression mediates cell survival and plays important roles during melanoma progression.

Dimethylfumarate reduces leukocyte rolling in vivo through modulation of adhesion molecule expression.

Esters of fumaric acid have a long tradition in the treatment of psoriasis. Dimethylfumarate (DMF) is perceived as the main active substance. However, the molecular mechanisms of DMF action are not completely understood. Here, we investigate the effects of DMF on lymphocyte adhesion molecule expression in vitro and interactions with endothelial cells in vivo. DMF dose-dependently reduced superantigen-induced expression of CD25, human leukocyte antigen-DR, and cutaneous lymphocyte antigen by 27, 22, and 48% on CD3-positive cells, respectively. No change was observed for CD54, VLA-4, and P-selectin glycoprotein ligand-1. An enhancement of CD69 expression was noted (22%). DMF led to a significant reduction in binding of human peripheral blood mononuclear cells (PBMCs) to E-selectin (72%), P-selectin (36%), and vascular cell adhesion molecule-1 (33%) in vitro. Intravital microscopy of PBMCs in ear vasculature of wild-type and knockout mice showed that rolling was mainly P-selectin-dependent and could be reduced by 61% through DMF incubation. We provide early evidence that DMF affects adhesion molecule expression on human leukocytes and their rolling behavior in vivo, indicating that DMF directly affects the initial step of leukocyte extravasation.

Pharmacological and histopathological characterization of a hyperalgesia model induced by freeze lesion.

Induction of a freeze lesion in human skin is an experimental model of hyperalgesia that allows assessing the antihyperalgesic effects of traditional non-steroidal anti-inflammatory drugs (NSAIDs). We have investigated whether this model is also sensitive to selective cyclooxygenase (COX)-2 inhibitors and have characterized morphological substrates of the generated hyperalgesia in the skin. In eight healthy subjects, a freeze lesion was induced and mechanical pain thresholds (MPT) were tested for 5h following administration of the non-selective COX inhibitor diclofenac (75mg), the COX-2-selective inhibitor parecoxib (40mg) or placebo in a randomized, double-blind cross-over study. In five additional healthy subjects, biopsies were taken from normal skin and the area of freezing injury. Induction of the freeze lesion resulted in hyperalgesia expressed by a decrease of MPT after 24h. Diclofenac and parecoxib, but not placebo, statistically significantly elevated MPT. Histochemical and Western blot analyses of skin biopsies revealed a strong upregulation of COX-2, a slight decrease of COX-1 and activation of nuclear factor kappa B (NF-kappaB) in the area of the freezing injury. These findings indicate that the freeze lesion model is sensitive to NSAIDs including selective COX-2 inhibitors, and that NF-kappaB-dependent COX-2 upregulation contributes to the hyperalgesia in this model.

Oligonucleotides suppress IL-8 in skin keratinocytes in vitro and offer anti-inflammatory properties in vivo.

DNA codes for genetic information. Furthermore, recent findings suggest that DNA offers additional function, particularly in the recognition of microorganisms. In this study, we investigated two classes of oligodeoxynucleotides (ODN) in skin keratinocytes; namely, an ODN comprising two cytidine-phosphate-guanosine (CpG) motifs (CpG-1-phosphorothioate (PTO)) and a poly-cytidine (Non-CpG-5-PTO) as control. Both fluorescence-tagged ODN were rapidly taken up by cells and accumulated already after 5 minutes in perinuclear compartments. In order to test whether ODN convey immunological effects in keratinocytes, secretion of IL-8 was measured. Interestingly, both CpG-1-PTO and Non-CpG-5-PTO suppressed basal and tumor necrosis factor alpha-induced IL-8 levels measured in cell culture supernatants. Experiments using deletion mutant revealed a critical length of approximately 16 nucleotides conveying IL-8 suppression. Studies regarding the ODN backbone offered that PTO bondings are critical for significant IL-8 suppression. In order to substantiate the anti-inflammatory response, a contact hypersensitivity mouse model was utilized. Topical application of Non-CpG-5-PTO-containing ointments reduced ear thickness in sensitized mice. Taken together, these findings suggest an anti-inflammatory effect of ODN in epithelial cells in vitro and in vivo, indicating that DNA molecules offer distinct biological activities restricted to the physiological compartment applied. This effect seems to be independent from Toll-like receptor 9.

Eosinophilic cellulitis (Wells' syndrome) associated with colon carcinoma.

Eosinophilic cellulitis (Wells' syndrome) is an inflammatory dermatosis characterized by marked eosinophilic infiltrates. Drugs and various infections are recognized causes of eosinophilic cellulitis. Eosinophilic cellulitis has been reported in non-hematological malignancies in two patients with squamous cell carcinoma and one with nasopharyngeal carcinoma. We report the association of eosinophilic cellulitis with adenocarcinoma of the colon. Curative hemicolectomy led to a complete remission, suggesting that underlying malignancies can trigger eosinophilic cellulitis.