31st European Society for Dermatological Research (ESDR) Meeting 2001

2001 Journal of Investigative Dermatology  
Pemphigus Vulagaris (PV) is a life threatening skin disease mediated by IgG antibodies to desmoglein-3 (Dsg3), a member of cadherin supergene family of cell-cell adhesion molecules. Development of an animal model of PV is crucial for development of novel approaches in antigen-speci®c immunotherapy of the disease. We here present our data on successful development of PVlike lesions in a speci®c strain of mouse by DNA immunization. cDNA coding for the full length mouse Dsg3 was conjugated with
more » ... g sequence and subcloned into a mammalian expression vector under a CMV promoter. The resulting construct, using in vitro transfection studies, was shown to express the full length Dsg3 as con®rmed by Western blot using anti-Flag and anti-Dsg3 antibodies. The endotoxin free cDNA was then delivered by particle bomardement. The mice developed anti-Dsg3 autoantibodies as detected by immuno¯uorescence microscopy, Elisa and Western blot. Clinically, the mice developed hair loss, and mucosal lesions. Adoptive transfer of splenocytes from affected mice into SCID mice further demonstrated the ability of autoreactive lymphocytes in development of PV-like lesions. This is the ®rst demonstration of an active mouse model of PV by DNA immunization. This model would provide an invaluable tool for exploring the immunopathology of PV and development of novel immunotherapeutical approaches. VOL. 117, NO. 3 SEPTEMBER 2001 ABSTRACTS 767 013 Ribozyme Gene Therapy for Keratin Disorders A. Terron and W.H.I. McLean Dundee, UK There are now 17 keratin genes that are known to be involved in autosomal dominant genodermatoses and other inherited epithelial disorders. Due to the dominant-negative effect of mutant keratins, conventional gene replacement therapy is not appropriate for these diseases. Ribozymes are small catalytic RNA molecules which can be designed to cleave mRNA molecules at speci®c sites. A total of 19 potential ribozyme sites in the K14 mRNA were evaluated and constructs were made for the ®ve sites showing best predicted RNA folding characteristics. Ribozymes were protected from RNAse degradation in vivo by addition of hairpin sequences derived from the U1 snRNA, a naturally occurring small RNA molecule that escapes nuclease attack. Ribozymes directed against the ®ve chosen sites were constructed as mini-genes driven by the U1 snRNA promoter in a modi®ed plasmid vector. The ability of these ribozymes to cleave K14 RNA was investigated under a wide range of conditions both in vitro and in cultured cells. Three ribozymes were capable of cleaving K14 mRNA in vitro. Furthermore, two were capable of rapidly cleaving more than 90% of K14 mRNA following transfection of the ribozyme plasmids into cultured keratinocytes. These experiments show (a) that ribozymes are capable of speci®cally degrading even high-abundance mRNA species such as keratins; and (b) pave the way for gene therapy where all endogenous keratin mRNA might be ablated and replaced by a modi®ed'r ibozyme-immune" keratin gene. In addition, it should be possible to develop ribozymes that target speci®c keratin mutations. Psoriasis is a T-cell mediated skin disease characterized by the in®ltration of activated leukocytes and the presence of a distinct in¯ammatory pattern. There is increasing evidence that bacterial superantigen-mediated activation of T-cells plays a pivotal role in the induction of psoriasis. NF-kB is a transcription factor which is involved in the induction of many pro-in¯ammatory molecules known to be up-regulated in psoriasis. We therefore addressed the question whether attenuation of NF-kB activation by the proteasome inhibitor PS-519 may suppress T-cell mediated responses and may be effective in the treatment of psoriasis. PBMCs of four healthy volunteers were stimulated with the superantigen TSST-1 (100 ng per mL) in the absence or presence of nontoxic concentrations of PS-519 (1±10 mg per mL). We found a dose-dependent inhibition of lymphocyte proliferation of up to 95% compared to TSST-1 treated cells. Additionally, the expression of T-cell activation markers such as CD69, CD25 and HLA-DR was markedly reduced in PS-519 treated, TSST-1 stimulated cultures. Cytokine production by superantigen-stimulated PBMCs was dramatically and signi®cantly reduced in PS519 treated cultures (IL-1b, TNF-a, IFNg). The effects of PS-519 on the severity of psoriasis was assessed in a xenogenic SCID-hu transplantation model. Lesional psoriatic skin obtained from different donors was transplanted onto SCID mice. After 28 days, SCID mice were treated once daily either with PS-519 intraperitoneally (20 mg per mouse; n = 8) or with vehicle for 28 days (n = 4). Thereafter, 20S proteasome activity was determined in the blood and the human transplanted skin examined by immunohistochemistry. As expected, 20S proteasome activity was markedly reduced in PS-519 treated mice (0.22 T 0.01 [pmol AMC per s per mg protein]) compared to untreated mice (1.53 T 0.05; mean T SEM). In PS-519 treated grafts, normalization of epidermal architecture including loss of papillomatosis and marked reduction of akanthosis was observed. We conclude that the proteasome inhibitor PS-519 reduces superantigen mediated T-cell activation in vitro and shows antipsoriatic ef®cacy in the SCID-hu model. ; ²University of Oulu, Finland; ³Scripps Research Institute, USA; §Karolinska Institute, Sweden Collagen XVII is a structural component of the hemidesmosomes and exists in two forms, as 180 kDa type II transmembrane protein and as soluble 120 kDa ectodomain, which corresponds to the extracellular domain of collagen XVII. We have previously shown that the ectodomain was generated by proteases, which were subjected to a furin-mediated process. In this work we identi®ed the enzymes, which release the ectodomain from the cell surface. Time chase experiments with biotinylated cultured keratinocytes showed that the soluble ectodomain was stable in the medium for more than 48 h. The use of domain speci®c antibodies demonstrates that the authentic shedding product contains at least a part of the NC16A domain and the C-terminus of the collagen XVII molecule. Collagen XVII-shedding of cultured keratinocytes was enhanced by phorbol esters and inhibited by phenanthroline, MMP-and sheddase-targeting hydroxamates and TIMP-3, but not by TIMP-1, TIMP-2, a selective gelatinase inhibitor and serine protease inhibitors. The candidate enzymes MMP-2, MMP-9 and MT1-MMP, which are involved in proteolytic cascades on keratinocyte surfaces, were excluded, since they cleaved puri®ed collagen XVII to a nonphysiological fragment and MMP-2 and MT1-MMP-de®cient cells showed normal collagen XVII-shedding. Promising candidate enzymes are members of the ADAMs, like the prototype sheddase TACE, ADAM-10 and ADAM-9. All these sheddases contain a putative furinactivation site in their molecule. RT-PCR analysis and immunoblotting demonstrate that TACE, ADAM-10 and ADAM-9 were expressed and activated in human keratinocytes. Immunohistological analysis of human skin revealed that especially TACE-and ADAM-9 were mainly restricted to basal keratinocytes and therefore showed a localization comparable with collagen XVII. To verify the role of TACE in collagen XVII-shedding, HaCaT cells were transfected with full-length murine TACE-cDNA. A dose dependent increase of ectodomainshedding, with a concomitant decrease of full-length collagen XVII were shown. In addition, TACE-de®cient murine keratinocytes showed signi®cantly reduced collagen XVII-shedding. The results support the conclusion that TACE contributes to collagen XVII-shedding from the keratinocyte surface. Further studies will determine the identity of the other sheddases, which are also involved in this process. The bullous pemphigoid antigen 1 (eBPAG1) is a component of hemidesmosomes (HD), junctional adhesion complexes in strati®ed epithelia. While its COOH-terminus interacts with intermediate ®laments, its NH 2 -terminus is important for its localization into HD. To identify proteins which interact with the NH 2 -terminus of human eBPAG1, we performed a yeast twohybrid screen, which uncovered a novel protein belonging to the LAP/LERP (for LRR and PDZ domain) protein family with 16 NH 2 -terminal leucine rich repeats (LRR) and a COOH-terminal PDZ domain. The gene for this LAP/LERP protein comprises at least 26 exons located on the long arm of chromosome 5. In most human tissues, several transcripts were detected differing in the coding region situated upstream of or within the PDZ domain. One of the encoded variants was found to correspond to the recently described protein ERBIN. In yeast and in in vitro binding experiments ERBIN was shown to interact not only with eBPAG1, but also with the COOHterminal region of the cytoplasmic domain of the integrin b4 subunit, another component of HD. Antibodies raised against the COOH-terminus showed that ERBIN is expressed in keratinocytes. In transfected epithelial cells the protein, however, was not localized in HD but either diffusely distributed over the cytoplasm or concentrated at the basolateral plasma membrane. Since ERBIN has previously been shown to interact with the transmembrane tyrosine kinase receptor Erb-B2, which in turn associates with the integrin b4 subunit, we suggest that ERBIN provides a link between HD assembly and Erb-B2 receptor signaling. The ability to sense the presence of pathogenic organisms is part of the active role of human keratinocytes in mounting an innate immune response resulting in the release of antimicrobial peptides which kill invading microorganisms. To explain our ®ndings of epithelial peptide antibiotic b-defensins 2 and 3 (hBD-2, hBD-3) induction after contact with microorganisms, we speculated about a role of keratinocyte-derived pathogen pattern recognition receptors. Using semiquantitative and real-time RT-PCR with gene speci®c primers we detected mRNA expression of human Toll-like receptors (hTLRs) in cultured primary human keratinocytes (NHK) and the keratinocyte cell line HaCaT. While hTLR 1 and 7 mRNA species seem to be expressed in HaCaT keratinocytes at a low level (after stimulation with heat inactivated Pseudomonas aeruginosa), stronger RT-PCR signals were found for hTLR 2±6. Interestingly, upon incubation with heat inactivated Pseudomonas aeruginosa or Staphylococcus aureus, we found up-regulated expression of mRNA encoding Toll-like receptors hTLR2 and hTLR4 in NHK and HaCaT cells. Relative induction of hTLR4 was found most prominent in NHK and after keratinocyte contact with heat killed Gram-negative Pseudomonas microorganisms. Increased expression of and signalling by hTLR2 (with lipoteichoic acids derived from Gram-positive bacteria as ligands) and/or hTLR4 (with lipopolysaccharides from Gram-negative bacteria as ligands), and perhaps other Toll-receptor homologues, may contribute to the activation and maintenance of innate immunity in infected skin. Langerhans cell (LC) migration from the epidermis to regional lymph nodes is a tightly regulated process, and current data suggest that interleukin (IL)-1b and tumour necrosis factor-a (TNF-a) are involved. In the present investigations we have assessed the role of IL-18, a cytokine structurally similar to IL-1b, in the regulation of LC migration and contact hypersensitivity (CHS) utilising IL-18 knock-out (KO) mice. To determine whether IL-18 is required for optimal contact sensitisation, IL-18 KO and wild type (WT) mice were sensitised on abdominal skin with 1% oxazolone (OX) and challenged on the dorsum of one ear 5 days later with 0.5% OX. Vigorous ear swelling responses were observed in WT mice, but this response was inhibited by 36% (n = 3; p < 0.05) in IL-18 KO mice. This attenuated response was completely restored in IL-18 KO mice by local intradermal injection of IL-18 (50 ng) immediately prior to sensitisation with OX (n = 3) suggesting that the defect in IL-18 KO mice was in the afferent phase of the CHS response. Identical administration of IL-18 to WT mice did not affect the CHS response. To examine the effect of IL-18 on allergen-induced LC migration, epidermal LC density was determined following topical application of 1% OX. OX treatment caused a signi®cant decline in MHC class II + epidermal LC density 4 h after application in WT mice (26%, n = 3; p < 0.05) but this was absent in IL-18 KO mice. Intradermal injection of exogenous IL-1b, TNF-a or IL-18 (50 ng each, n = 3) lead to equivalent LC migration in both IL-18 KO and WT mice, indicating that, given an appropriate signal, IL-18 KO LC are able to migrate normally. Finally, intradermal injection of IL-18 prior to topical sensitisation with 1% OX in caspase-1 de®cient mice (which have impaired release of both IL-1b and IL-18) failed to restore the attenuated CHS response, whereas IL-1b pretreatment resulted in complete restoration (n = 3). These results indicate that IL-18 is a key proximal mediator of LC migration and CHS, acting upstream of IL-1b, and that this cytokine may play a central role in the regulation of cutaneous immune responses. Dendritic cells (DCs) are crucial for the initiation of allergic contact dermatitis (ACD) to haptens. However, the expression of ACD is largely DC-independent, and the mechanisms responsible for T lymphocyte activation at the site of hapten challenge have not been fully elucidated. We have investigated the APC requirement of nickel (Ni)-speci®c CD4 + lymphocytes isolated from the blood of 6 allergic individuals. 42 out of 121 (35%) T cell clones (Tccs) proliferated in vitro to Ni in the absence of professional APCs, suggesting a direct T-T Ni-presentation. Ni recognition by APC-independent clones was MHC class II restricted, was not in¯uenced by CD28 triggering, and occurred independently from the activation state of the Tccs. Additionally, the epitope recognized by these Tccs did not require processing, as indicated by experiments performed with ®xed APCs. APC-independent and -dependent Tccs expressed similar levels of MHC class II and B7 molecules, and did not differ in their antigen presenting capacity, since they both induced comparable activation of APC-independent, but not -dependent Tccs. T-T presentation induced prominent CD3/TCR down-regulation, CD25 up-regulation and IFN-g release, although to a lesser extent compared to those induced by DCs. Finally, T-T presentation was not followed by T cell anergy, as indicated by the capacity of T cells to respond to the hapten after multiple cycles of activation with Ni in the absence of professional APCs. Our data suggest that APC-independent T cell activation could represent an important mechanism for the initiation and ampli®cation of ACD. The oral antidiabetics tolbutamide, glibenclamide, and glipizide, and the diuretics bendroumethiazide, butizide, furosemide, hydrochlorothiazide, and trichlormethiazide were investigated for their potential to cause phototoxicity in the HaCaT cell line. The cells were incubated with different concentrations of the drugs and then exposed to UVA1 irradiation. Cell survival was evaluated in a clonogenic assay and phototoxic DNA damage was investigated by the single cell gel elctrophoresis (comet assay). The effects of the antioxidants L-ascorbic acid, and a-tocopherol on oxidative DNA damage were also assessed. Bendro¯umethiazide, furosemide, hydrochlorothiazide, trichlormethiazide, or tolbutamide induced dose-dependent phototoxicity in the clonogenic assay. Cells incubated with bendro¯umethiazide, tolbutamide, and glibenclamide, and irradiated with UVA1 demonstrated an increased oxidative DNA damage revealed as alkali-labile sites in the comet assay. Pre-treatment with L-ascorbic acid, or a-tocopherol, suppressed the UVA-induced DNA damage in cells incubated with 1 mM of bendro¯umethiazide, furosemide, glibenclamide, glipizide, tolbutamide, and trichloromethiazide, further implying the involvement of reactive oxygen species in the phototoxic DNA damage. These results indicate a link between phototoxic and photocancerogenic potential of the sulfonamide-derived oral antidiabetic and diuretic drugs, as it has previously been recognized for psoralen, chlorpromazine, and¯uoroquinolones. Excessive exposure to UV light may be deleterious for patients treated with oral antidiabetic and diuretic drugs. VOL. 117, NO. 3 SEPTEMBER 2001 ABSTRACTS 785
doi:10.1046/j.1523-1747.2001.00117-3.x fatcat:6d465i4jovb3xie6bf2wqbgw2a