Hyaluronan (HA), a major component of the cutaneous extracellular-matrix, is involved in tissue repair. Human skin is exposed to and damaged by UVB-irradiation. Here, we investigate the regulation of HA metabolism in human skin during acute UVB-induced inflammation. Expression of HA synthesizing (HAS) and degrading enzymes hyaluronidase (HYAL) as evaluated by quantitative reverse transcribed PCR in response to UVB differed when fibroblasts and HaCaT-keratinocytes, representative cell types in dermis and epidermis, respectively, were compared. Both demonstrated temporally different expression patterns of these genes 3- and 24-hours post-irradiation. This resulted 24-hours post-irradiation in an increase in HAS gene expression in both fibroblasts and HaCaT-keratinocytes, and an increase in HYAL expression only in fibroblasts. HA-production as analyzed by the HA content of conditioned medium was reduced in HaCaT and fibroblast cultures 3-hours post-irradiation, whereas HA increased in HaCaT-cultures 24-hours post-irradiation but remained suppressed in fibroblasts-cultures. Consistently, immunohistochemical staining for HA in human skin 24-hours post-irradiation demonstrated an increased epidermal HA, but a decrease in the dermal compartment. Moreover, analysis of the HA content of dermal microdialysis-fluid revealed increased accumulation of HA degradation products 24-hours post-irradiation. These data demonstrate that there is a complex temporal and spatial regulation of HA-metabolism in skin in response to UVB irradiation.

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 MMPand cytokine-expression, in part in a TLR4-dependent manner, providing new insights into the relationship between cancer and innate immunity.Key words: hyaluronan -cytokine -metalloprotease -melanoma cells -TLR4Please cite this paper as: Hyaluronan fragments induce cytokine and metalloprotease upregulation in human melanoma cells in part by signalling via TLR4.

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Leukocyte recruitment in response to inflammatory signals is governed, in part, by binding to Thy-1 (CD90) on activated endothelial cells (EC). In this study, we characterized the adhesion G-protein coupled receptor CD97, present on peripheral myeloid cells, as a novel interacting partner for Thy-1. CD97 was upregulated on polymorphonuclear cells (PMNC) of patients with psoriasis. In psoriatic skin lesions, CD97+ myeloid cells colocalized with Thy-1+ EC of small vessels in microabscesses, suggesting an interaction between CD97 and Thy-1 that was further examined by adhesion and protein-binding assays. PMNC and cell lines stably overexpressing CD97 adhered specifically to Thy-1+–activated human dermal EC, Thy-1+ CHO cells, and immobilized Thy-1 protein. Binding of the CD97+ CHO clones correlated with their CD97 expression level. Soluble CD97 bound specifically to immobilized Thy-1 protein, as well as Thy-1+–activated EC and CHO cells. In all assays, cellular adhesion or protein binding was blocked partially by CD97 and Thy-1–blocking mAb. Our data suggested that CD97 interacts via its stalk with Thy-1 because mAb directed to the stalk of CD97 showed stronger blocking compared with mAb to its epidermal growth factor-like domains, and binding was calcium independent. Moreover, soluble CD97 without the stalk and soluble EMR2, containing highly homologous epidermal growth factor-like domains but a different stalk, failed to bind. In summary, binding of leukocytes to activated endothelium mediated by the interaction of CD97 with Thy-1 is involved in firm adhesion of PMNC during inflammation and may play a role in the regulation of leukocyte trafficking to inflammatory sites.

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.

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.

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.

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.