Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4.

Low molecular weight fragmentation products of the polysaccharide of Hyaluronic acid (sHA) produced during inflammation have been shown to be potent activators of immunocompetent cells such as dendritic cells (DCs) and macrophages. Here we report that sHA induces maturation of DCs via the Toll-like receptor (TLR)-4, a receptor complex associated with innate immunity and host defense against bacterial infection. Bone marrow-derived DCs from C3H/HeJ and C57BL/10ScCr mice carrying mutant TLR-4 alleles were nonresponsive to sHA-induced phenotypic and functional maturation. Conversely, DCs from TLR-2-deficient mice were still susceptible to sHA. In accordance, addition of an anti-TLR-4 mAb to human monocyte-derived DCs blocked sHA-induced tumor necrosis factor alpha production. Western blot analysis revealed that sHA treatment resulted in distinct phosphorylation of p38/p42/44 MAP-kinases and nuclear translocation of nuclear factor (NF)-kappa B, all components of the TLR-4 signaling pathway. Blockade of this pathway by specific inhibitors completely abrogated the sHA-induced DC maturation. Finally, intravenous injection of sHA-induced DC emigration from the skin and their phenotypic and functional maturation in the spleen, again depending on the expression of TLR-4. In conclusion, this is the first report that polysaccharide degradation products of the extracellular matrix produced during inflammation might serve as an endogenous ligand for the TLR-4 complex on DCs.

Plasmin plays a role in the in vitro generation of the linear IgA dermatosis antigen LADB97.

Collagen XVII (BP180) and its shed ectodomain represent major autoantigens in dermatoses of the pemphigoid group. The 120 kDa ectodomain is constitutively shed from the cell surface by disintegrin-metalloproteinases (ADAMs). Part of it is further processed to a 97 kDa fragment (LABD97), an autoantigen in linear IgA dermatosis (LAD), but the responsible proteinases remain elusive. In this study, we identified the 120 and the 97 kDa ectodomain in blister fluids of bullous pemphigoid patients using new mAbs. As blister fluids contain significant plasmin-like serine protease activity, HaCaT keratinocytes or purified 120 kDa ectodomain were incubated with several human serine proteases. In vitro, only plasmin generated a stable 97 kDa fragment that was also targeted by LAD sera. Characterization of the plasmin-derived 97 kDa fragment with domain-specific collagen XVII antibodies, heparin binding and N-glycosylation studies indicates that the N-terminus is located approximately at AA 515 and the C-terminus N-terminally from AA 1,421. Interestingly, plasmin-derived LABD97 was also generated in the presence of ADAM inhibitors and remained stable over more than 12 hours incubation at 37 degrees C, indicating that this disease relevant collagen XVII fragment can also arise in an ADAM-independent manner through direct action by plasmin.

The autoantigen in anti-p200 pemphigoid is synthesized by keratinocytes and fibroblasts and is distinct from nidogen-2.

Anti-p200 pemphigoid is a subepidermal immunobullous disorder associated with tissue-bound and circulating autoantibodies reactive with a 200 kDa protein on the dermal side of salt-split-skin. The autoantigen, named p200, is a non-collagenous glycoprotein located at the lamina lucida-lamina densa border of the epidermal basement membrane. However, its identity and cellular origin remain elusive. Here, we used biochemical and genetic approaches to characterize the autoantibody reactivity in three new patients with anti-p200 pemphigoid. We show that the target antigen p200 is synthesized by both keratinocytes and fibroblasts, is disulfide-bonded, and participates in calcium-dependent molecular interactions. Lack of collagen XVII (BP 180), collagen VII, or laminin 332 (laminin 5) from the dermal-epidermal junction does not destabilize p200. Colocalization within the basement membrane zone and an identical molecular weight suggested nidogen-2 as candidate autoantigen in anti-p200 pemphigoid, but biochemical analysis demonstrated that p200 is distinct from nidogen-2. In conclusion, the results define further the biochemical characteristics of p200 and demonstrate its in vitro-synthesis by keratinocytes and fibroblasts, thus providing a basis for identification and further characterization of this autoantigen.

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

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.