Marco Averbeck

Hyaluronan fragments induce cytokine and metalloprotease upregulation in human melanoma cells in part by signalling via TLR4

Abstract:  Small fragments of the extracellular matrix component hyaluronic acid (sHA) are typically produced at sites of inflammation and tissue injury and have been shown to be associated with tumor invasiveness and metastasis. Here we report that exposure of human melanoma cells to sHA leads to nuclear factor kB (NFk‐B) activation followed by enhanced expression of matrix metalloprotease (MMP) 2 and interleukin (IL)‐8, factors that can contribute to melanoma progression. At the receptor level, we found that Toll‐like receptor (TLR) 4 is involved in this signalling pathway, similar to the case in dendritic and endothelial cells. Specifically, we found that melanoma cells expressed TLR4 on their surface in vivo and in vitro, and using specific siRNA, we could clearly demonstrate the functional importance of TLR4 in sHA‐triggered activation of IL‐8 expression in melanoma cells. Furthermore, we also found that sHA treatment enhanced the motility of melanoma cells, an effect that could again be blocked by TLR4‐specific siRNA. Together, our results suggest that sHA in melanoma might promote tumor invasiveness by inducing MMP‐ and cytokine‐expression, in part in a TLR4‐dependent manner, providing new insights into the relationship between cancer and innate immunity.

ADAM10 is the constitutive functional sheddase of CD44 in human melanoma cells.

CD44 proteins are cell surface receptors for hyaluronic acid (HA), a component of the extracellular matrix that has multiple effects on cell behavior. CD44 can be shed from the cell surface by proteolytic cleavage. The resulting soluble form can interfere with the interaction between HA and membrane-bound CD44. Soluble CD44 can abolish the cell proliferation-promoting effect of HA on melanoma cell lines, suggesting that a better understanding of the shedding process might identify ways of blocking tumor cell proliferation. ADAM10, ADAM17, and MMP14 have previously been implicated in the shedding of CD44 from various tumor cells. Using immunohistochemistry we demonstrate that ADAM10 and ADAM17 but not MMP14 are significantly expressed on melanoma cells in histological sections. In human melanoma cell lines expression of these proteases could be blocked by transfection with appropriate siRNAs. However, only blocking of ADAM10 expression led to decreased shedding of CD44. In parallel, cell proliferation was promoted. Confocal microscopy demonstrated that ADAM10 and CD44 colocalize on the cell surface. We conclude that ADAM10 is the predominant protease involved in the constitutive shedding of endogenous CD44 from melanoma cells, and that enhancement of ADAM10 activity could be an approach to decrease the proliferation of melanoma cells.

In situ profiling and quantification of cytokines released during ultraviolet B-induced inflammation by combining dermal microdialysis and protein microarrays

Abstract:  In skin, an evolving inflammatory or immune response is triggered by early release of a cytokine cascade into the extracellular space. Investigation of extracellular cytokine secretion in situ has been limited by low cut‐off filtering membranes and sample volume size and the inability to monitor changes in cytokine protein levels in real‐time in situ. Here, we combine for the first time the methods of intradermal microdialysis and antibody protein arraying to profile the early cascade of multiple cytokines in a complex inflammatory response exemplified by ultraviolet B (UVB)‐induced inflammation. We observed significant differences of the cytokine and growth factor responses after tissue injury by catheter placement and UVB‐induced inflammation. UVB irradiation initiates a rapid proinflammatory response followed by a mixed TH1/TH2 response in which ultimately TH2 cytokines IL‐4 and IL10 predominated after 24 h. This most likely indicates the termination and self limitation of the inflammatory response. We conclude that the combination of dermal microdialysis and protein microarray offers a powerful tool to analyze in real‐time the complex and rapidly changing interstitial protein milieu during cutaneous inflammatory responses.

Dermal Fibroblasts Induce Maturation of Dendritic Cells

To trigger an effective T cell-mediated immune response in the skin, cutaneous dendritic cells (DC) migrate into locally draining lymph nodes, where they present Ag to naive T cells. Little is known about the interaction of DC with the various cellular microenvironments they encounter during their migration from the skin to lymphoid tissues. In this study, we show that human DC generated from peripheral blood monocytes specifically interact with human dermal fibroblasts via the interaction of β2 integrins on DC with Thy-1 (CD90) and ICAM-1 on fibroblasts. This induced the phenotypic maturation of DC reflected by expression of CD83, CD86, CD80, and HLA-DR in a TNF-α- and ICAM-1-dependent manner. Moreover, fibroblast-matured DC potently induced T cell activation reflected by CD25 expression and enhanced T cell proliferation. Together these data demonstrate that dermal fibroblasts that DC can encounter during their trafficking from skin to lymph node can act as potent regulators of DC differentiation and function, and thus may actively participate in the regulation and outcome of DC-driven cutaneous immune responses.

Immunologic Principles of Allergic Disease

Allergy either results from a pathological excessive immune reaction, or from the defective induction of tolerance to otherwise harmless antigens. Allergic reactions are mounted by mechanisms of innate and adaptive immunity. The development of an allergic response can be divided in sensitization and elicitation phases. Immediate type allergic reactions (e.g. anaphylaxis, urticaria, rhinoconjunctivitis allergica, allergic asthma) are mediated by IgE antibodies which are produced by B cells stimulated by allergen‐specific Th2 cells. Crosslinking of allergen‐specific IgE on membrane surfaces of mast cells and basophilic granulocytes leads to release of soluble mediators which may cause systemic symptoms within minutes to hours. The following infiltration of eosinophilic granulocytes and Th2 cells directs chronic inflammation.

Dermal Hyaluronan Is Rapidly Reduced by Topical Treatment with Glucocorticoids

Skin atrophy is part of the normal ageing process, but is accelerated by topical glucocorticoid (GC) treatments that are widely used in dermatology. Hyaluronan (HA) is one of the most abundant components of the cutaneous extracellular matrix and is involved in tissue homeostasis, hydration, and repair processes, but little is known about the effects of GCs on HA synthesis and stability. Here we examined the regulation of HA metabolism in human skin during GC therapy. Expression of the HA synthesizing enzymes hyaluronan synthase (HAS)-2 and HAS-3 and the HA degrading enzymes HYAL-1, HYAL-2, and HYAL-3 in response to GC treatment was evaluated. HAS-2 expression was markedly suppressed by dexamethasone treatment of cultured fibroblasts and HaCaT keratinocyte cells, and in human skin biopsies taken from volunteers treated with dexamethasone ointment. Consistently, the HA content of cell culture supernatants and in human skin was reduced after dexamethasone treatment. Hyaluronidase expression and activity, on the other hand, was not altered by dexamethasone treatment. These data show that the levels of skin HA rapidly decrease after short-term GC treatment due to a reduction in HA synthesis, while HA degradation is not changed. This may reflect an initiation of skin atrophy in response to topically applied GCs.

Mature monocyte‐derived dendritic cells respond more strongly to CCL19 than to CXCL12: consequences for directional migration

The chemokine receptor CCR7 is crucial for migration of mature dendritic cells (DC) directed toward secondary lymphoid organs; however, there is little knowledge about the function of the homeostatic chemokine receptor CXCR4 in DC and its contribution to directional migration of DC during inflammation. By comparing the impact of chemokine receptor engagement on mature DC we found that the CCR7 ligand CCL19 holds a stronger chemotactic potency than the CXCR4 ligand CXCL12. Moreover, CCL19 elicited rapid, steep and long‐lasting mobilization of intracellular calcium in individual cells and induced intense phosphorylation of extracellular signal‐regulated kinase 1/2 and protein kinase B, while the intracellular signals elicited by CXCL12 were in part distinct and significantly weaker. Analysis of chemokine receptor expression revealed that although CCR7 and CXCR4 were expressed by a similar percentage of DC, the mean fluorescence intensity of CCR7 was up to six times higher, suggesting a higher receptor density. Based on these correlations we propose that the type of chemokine signal in conjunction with the expression and functional activity of the respective chemokine receptor is also determining the migration rate and potency of a chemotactic response in mature DC. In conclusion, our data support the fundamental role of CCR7 for rapidly guiding DC toward secondary lymphoid organs at an extra‐ and intracellular molecular level and on the contrary render CXCR4 a weaker contributor to directional migration of DC during inflammation.

A Case of Cutaneous Rosai-Dorfman Disease Refractory to Imatinib Therapy

BACKGROUND Rosai-Dorfman disease is a non-Langerhans cell histiocytosis that recently has been treated successfully with imatinib mesylate in a patient with a systemic variant of the disease. OBSERVATIONS We describe a 69-year-old man with cutaneous Rosai-Dorfman disease manifesting as progressive, deeply infiltrated skin lesions. Histopathologic examination of the lesions demonstrated dense dermal infiltrate positive for CD68, stabilin-1, and S-100, but not for CD1a. The histiocytes were positive for platelet-derived growth factor receptor alpha, the target molecule for imatinib. During the 5-year course of the disease, multiple therapeutic approaches (tuberculostatic drugs, topical and systemic glucocorticoids, thalidomide, isotretinoin, and methotrexate) did not result in significant improvement. Imatinib mesylate therapy (600 mg/d for 2(1/2) weeks and then 400 mg/d for 10 weeks) had no effect, despite the expression of platelet-derived growth factor receptor alpha on the histiocytes. CONCLUSIONS Failure of imatinib therapy in our patient may be due to a lack of functioning target molecules, the therapy protocol, or the course of the disease. Cutaneous and systemic variants of Rosai-Dorfman disease may be different clinical entities or at least may respond differently to tyrosine kinase inhibitors.

Vaccinia virus impairs directional migration and chemokine receptor switch of human dendritic cells

A crucial event for the induction of an anti‐viral immune response is the coordinated, phenotype‐dependent migration of dendritic cells (DC) to sites of infection and secondary lymphoid organs. Here we show that the vaccinia virus (VV) strains Western Reserve (WR) and modified virus Ankara (MVA) inhibit directional migration of mature DC toward the lymphoid chemokines CCL19 and CXCL12 without affecting surface expression of the respective chemokine receptors or impairing undirected cellular locomotion. Instead, infection with VV results in a deficiency of extracellular signal‐regulated kinase‐1 and a disturbance of intracellular calcium mobilization, indicating a viral interference with signaling events downstream of the surface chemokine receptors. In immature DC, apart from inhibiting chemokine‐induced migration of infected DC, infection with both VV strains increases expression of the inflammatory chemokine receptors CCR1 and CXCR1 on non‐infected bystander DC, which depends on the activity of IFN‐α. Although functional, these chemokine receptors are resistant to lipopolysaccharide‐induced down‐regulation. In addition, VV‐infected and non‐infected bystander DC fail to up‐regulate the lymphoid chemokine receptor CCR7 upon activation, together pointing to a disability to undergo the chemokine receptor switch. This study shows that VV targets directional migration of professional antigen‐presenting cells at multiple functional levels, revealing a potent viral strategy of immune escape.

More than just a filler - the role of hyaluronan for skin homeostasis

In recent years, hyaluronan (HA) has become an increasingly attractive substance as a non‐immunogenic filler and scaffolding material in cosmetic dermatology. Despite its wide use for skin augmentation and rejuvenation, relatively little is known about the molecular structures and interacting proteins of HA in normal and diseased skin. However, a comprehensive understanding of cutaneous HA homeostasis is required for future the development of HA‐based applications for skin regeneration. This review provides an update on HA‐based structures, expression, metabolism and its regulation, function and pharmacological targeting of HA in skin.