Histamine suppresses regulatory T cells mediated by TGF‐β in murine chronic allergic contact dermatitis

Regulatory T cells (Tregs) suppress effector T cells and ameliorate contact hypersensitivity (CH); however, the role of Tregs in chronic allergic contact dermatitis (CACD) has not been assessed. Repeated elicitation of CH has been used to produce CACD models in mice. We previously showed that the presence of histamine facilitates the creation of eczematous lesions in this model using histidine decarboxylase (HDC) (?/?) mice. Therefore, the effects of histamine on Tregs in the CACD model were investigated in this study. CACD was developed by repeated epicutaneous application of 2, 4, 6‐trinitro‐1‐chlorobenzene (TNCB) on HDC (+/+) and HDC (?/?) murine skin to assess the effects of histamine in CACD. Histamine aggravated CACD in the murine model and suppressed the number of Tregs in the skin. Histamine also suppressed the level of TGF‐β1 in this model. Recombinant TGF‐β1 or anti‐TGF‐β1 antibody was injected into the dorsal dermis of HDC (+/+) mice daily just before TNCB challenge to determine the effects of histamine‐regulated TGF‐β on the Treg population in CACD. Recombinant TGF‐β1 injection promoted the infiltration of Tregs in the skin and the production of IL‐10; however, anti‐TGF‐β1 antibody injection suppressed the number of Tregs in the skin and the production of IL‐10. Histamine suppresses the number of Tregs in CACD, and this effect is mediated by TGF‐β.     

Biological clock dysfunction exacerbates contact hypersensitivity in mice

Background Immediate‐type skin allergic reactions, such as passive cutaneous anaphylactic reaction, are associated with circadian rhythm, but the role of circadian mechanisms on delayed‐type skin allergic reactions, such as contact hypersensitivity (CHS), remains uncertain. In mice, CHS, a T‐cell‐mediated immune response, is a classic model of human allergic contact dermatitis. Objectives We investigated whether biological clock dysfunction affects CHS pathogenesis in CLOCK mutant mice compared with wild‐type (WT) mice. Methods Mice were treated with 2,4,6‐trinitro‐1‐chlorobenzene (TNCB) on the abdominal skin on day 0 (sensitization) and then treated with TNCB on the ears on day 5 (challenge). Results We found that biological clock dysfunction resulted in severe inflammation. Ear swelling, serum immunoglobulin E level and mast cell number were significantly increased in CLOCK mutant mice compared with WT mice. These results provide evidence that CLOCK mutation promotes the T‐helper type 2 immune response and exacerbates CHS. Corticosterone has a protective effect on CHS. The serum corticosterone level lost rhythmicity and showed a decreased daily level in CLOCK mutant mice compared with WT mice, supporting the exacerbating effect of CLOCK mutation on CHS. Adrenalectomy markedly worsened TNCB‐induced CHS in WT mice but not in CLOCK mutant mice. In addition, dramatic dexamethasone‐induced protection of CHS was observed in CLOCK mutant mice compared with WT mice. Conclusions The present results suggest that circadian rhythm might be an important factor in the regulation of CHS via corticosterone rhythmicity and/or level.     

Interleukin (IL)-6 modulates transforming growth factor-β expression in skin and dermal fibroblasts from IL-6-deficient mice

Background Interleukin (IL‐6) and transforming growth factor (TGF)‐β have been shown to play a role in skin development and maintenance. Objectives A link between these two cytokines has yet to be identified and therefore in this study we investigated the modulation of TGF‐β1 and TGF‐β type 2 receptor (TGF‐βR2) by IL‐6 in skin. Methods An IL‐6 knockout (IL‐6KO) fibroblast‐populated lattice model and intradermal injections of IL‐6 into unwounded IL‐6KO mice were used to investigate the direct effects of IL‐6 treatment on TGF‐β and TGF‐βR2 expression and to determine the signalling mechanism. In addition, IL‐6KO and C57BL/6 control mice were wounded by a 4‐mm punch biopsy to monitor expression of TGF‐β1 and TGF‐βR2 within a wound over time. The expression of TGF‐β1 and TGF‐βR2 was assessed by real‐time quantitative polymerase chain reaction, enzyme‐linked immunosorbent assay and immunohistology. Results Recombinant IL‐6 treatment of IL‐6KO lattices and intradermal injections of IL‐6 showed a significant induction of TGF‐β1 mRNA and protein, with TGF‐β1 expression localized in the dermis, while TGF‐βR2 expression was primarily in the epidermis in IL‐6KO mice. During healing, the expression of TGF‐β1 and TGF‐βR2 mRNA was significantly greater in unwounded and 7‐day‐old wounds from wild‐type mice; however, protein expression did not differ. Treatment with signal transduction inhibitors indicated that IL‐6 modulates TGF‐β through a mitogen‐activated protein kinase/extracellular signal‐regulated kinase (Mapk/Erk)‐dependent mechanism. Conclusion These studies indicate that IL‐6 has the ability to modulate the expression of TGF‐β and TGF‐βR2 to varying degrees in the skin, which may provide a possible mechanism for defining the role of IL‐6 in skin maintenance and a new association of IL‐6 with TGF‐β in pathologies associated with fibrosis.     

Interleukin (IL)‐6 modulates transforming growth factor‐β receptor I and II (TGF‐βRI and II) function in epidermal keratinocytes

It been shown that IL‐6 modulates TGF‐β1 expression in fibroblasts, however, what role IL‐6 plays concerning TGF‐βR expression and function in skin is unknown. Therefore, the aim of this study was to investigate the mechanism by which IL‐6 might modulates TGF‐β receptors in skin. Skin from WT, IL‐6 over‐expressing mice and IL‐6 treated keratinocyte cultures was analysed for TGF‐βRI and TGF‐βRII expression via histology, PCR and flow cytometry. Receptor function was assessed by cell migration, bromodeoxyuridine (BrdU) proliferation assays, and Smad7 expression and Smad2/3 phosphorylation. Receptor localization within the membrane was determined by co‐immunoprecipitation. IL‐6 overexpression and treatment increased TGF‐βRII expression in the epidermis. IL‐6 treatment of keratinocytes induced TGF‐βRI and II expression and augmented TGF‐β1‐induced function as demonstrated through increased migration and decreased proliferation. Additionally, IL‐6 treatment of keratinocytes altered receptor activity as indicated by altered Smad2/3 phosphorylation and increased Smad7 and membrane localization. These results suggest that IL‐6 regulates keratinocyte function by modulating TGF‐βRI and II expression and signal transduction via trafficking of the receptor to lipid raft pools.     

Impaired contact hypersensitivity in diet-induced obese mice

BACKGROUND: Epidemiological studies suggest that obesity is associated with the impairment of immunity. However, there is no experimental evidence that obesity prejudices immune responses. OBJECTIVE: This study was designed to determine the effects of obesity on contact hypersensitivity (CHS) response using a diet-induced obese (DIO) mouse model. METHODS: The effect of high fat diet (HFD) on CHS response to trinitrochlorobenzene (TNCB) was assessed by ear swelling, cytokine production, functional analysis of epidermal Langerhans cells, and adoptive transfer of immune cells. Immune response to ovalbumin was also analyzed in DIO mice. RESULTS: C57BL/6 mice but not BALB/c mice that fed with HFD for 4 weeks or more became obese and showed impaired CHS response, although both strain of mice showed enhanced irritant response to TNCB. CHS response was slightly impaired when C57BL/6 mice fed with HFD for 1 or 2 weeks. This suggests that diet-induced obesity or the HFD itself impairs the CHS response in the susceptible mice. The adoptive transfer of immune cells from DIO mice sensitized with TNCB to na?ve mice failed to show vigorous CHS, which suggests dysfunction of an afferent phase of CHS in DIO mice. However, the number and allo-stimulating ability of epidermal Langerhans cells were comparable between DIO mice and lean mice. In addition, the immune response to ovalbumin (delayed type hypersensitivity, and antigen-dependent production of antibodies and cytokine) was preserved in DIO mice. CONCLUSION: These results suggest that the diet-induced obesity or the HFD only partially impairs immunity in the susceptible mice.     

Inhibitory effects of dietary soyasaponin on 2,4‐dinitrofluorobenzene‐induced contact hypersensitivity in mice

Soyasaponins (SSs) abundant in soybean have anti‐inflammatory activities; however, their therapeutic effects on allergic contact dermatitis (ACD) remain unknown. To assess the effects of SS‐enriched diets on ACD, we used a mouse model of contact hypersensitivity (CHS). Mice were fed low‐dose or high‐dose SS‐containing diets for 3 weeks prior to CHS induction with 2,4‐dinitrofluorobenzene (DNFB). The low‐dose SS diet attenuated DNFB‐induced ear swelling and tissue oedema, and reduced the number of infiltrating Gr‐1‐positive myeloid cells. Low‐dose, but not high‐dose, SSs decreased chemokine (C‐X‐C motif) ligand 2 (CXCL2) and triggering receptor expressed on myeloid cells (TREM)‐1 production in ear tissues, compared to a control. Taxonomic 16S rRNA analysis revealed significant alterations in faecal microbiota caused by CHS, which were reversed by low‐dose SSs. The low‐dose SS and non‐CHS groups clustered together, while the high‐dose SS group split between CHS and non‐CHS clusters. Our results demonstrated that low‐dose SSs alleviated CHS symptoms by attenuating inflammation and improving the intestinal microbiota composition, suggesting that dietary SSs may have beneficial effects on ACD.     

Altered expression of dermokine in skin disorders

Background Although dermokine‐β, a glycoprotein expressed in epithelial cells, does not have significant homology to other proteins, its carboxyl‐terminal domain shares a high pI value with many cytokines, suggesting similar functions. Objective To better understand the biology of dermokine, we here determined its localization under pathological conditions and examined factors that regulate its expression. Methods We generated an anti‐human dermokine‐β/γ monoclonal antibody cross‐reacting with the mouse protein. Using this antibody, immunohistological staining and Western blotting of dermokine‐β/γ were performed with various tissue samples. Results Although human dermokine‐β/γ was expressed in almost all granular layers, upper spinous layers of the skin were also stained with anti‐dermokine‐β/γ antibody in inflammatory skin disorders. Dermokine‐β/γ was expressed in keratoacanthoma and a part of well‐differentiated squamous cell carcinoma (SCC). However, dermokine‐β/γ was not detected in poorly differentiated SCC or tumours derived from non‐keratinocytes. In mice, dermokine‐β/γ‐expressed keratinocytes were increased in models of contact hypersensitivity, ultraviolet‐irradiated skin injury and wound healing. Consistent with expanded distribution in inflammatory skin diseases, proinflammatory cytokines such as interleukin‐1β, interleukin‐12, and tumour necrosis factor‐α augmented dermokine‐β/γ expression in cultured human keratinocytes. In contrast, growth factors including epidermal growth factor, insulin‐like growth factor‐I, keratinocyte growth factor and transforming growth factor‐α significantly reduced dermokine expression. Conclusion These results provide novel insights into the physiological and pathological significance of dermokine in the epidermis.     

Attenuation of contact hypersensitivity by cell‐permeable heat shock protein 70 in BALB/c mouse model

 In contact hypersensitivity (CHS), multiple cells, inflammatory mediators and cytokines are known to be involved in the regulation of the immune response. Previously, we revealed the reactive oxygen species generation by 2, 4, 6‐trinitrobenzene sulphonic acid (TNBS) in vivo, followed by heat shock protein 70 (Hsp70) carbonylation and the exogenous antioxidant role of cell‐permeable Hsp70. Here, we demonstrate the role of Hsp70 using cell‐permeable Hsp70 in the mouse CHS model. Pretreatment of cell‐permeable Hsp70: (i) suppressed ear swelling; (ii) down‐regulated phosphorylated p38, but up‐regulated phosphorylated extracellular signal‐regulated kinase; (iii) increased population of CD4⁺CD25⁺Foxp3⁺ T cells; (iv) decreased secretion of tumor necrosis factor‐α (TNF‐α), IL‐12, interferon‐γ and IL‐2 and (v) but up‐regulated IL‐4 and transforming growth factor beta (TGF‐β) in the lymph nodes. In conclusion, cell‐permeable Hsp70 attenuates CHS through modulation of MAPK pathway and regulation of Th1, Th2 and regulatory T cells.     

EC-SOD suppresses contact hypersensitivity in mouse skin by impairing Langerhans cell migration

Extracellular superoxide dismutase (EC-SOD) is primarily a tissue enzyme and has been implicated in the modulation of inflammatory response. The biological role of EC-SOD in skin, however, has rarely been investigated. In this study, we aim to explore the effects of EC-SOD on the inflammatory response in skin by evaluating the contact hypersensitivity response (CHS) in EC-SOD transgenic mice. Transgenic mice with skin-specific expression of EC-SOD were sensitized and challenged with 2,4,6-trinitro-1-chlorobenzene (TNCB), followed by measurement of ear swelling. EC-SOD transgenic mice showed significantly reduced CHS responses compared with wild-type mice. Histological evaluation of the challenged ears of EC-SOD transgenic mice revealed diminished infiltration of inflammatory cells with a failure to induce expression of inflammatory cytokines, such as tumor necrosis factor-alpha and IFN-gamma, on sensitization and challenge with TNCB. Furthermore, Langerhans cell migration to lymph nodes was impaired in EC-SOD transgenic mice. These results indicate that EC-SOD downregulates CHS through inhibition of the inflammatory response, suggesting a possible therapeutic regimen in inflammatory skin diseases.     

Increased contact hypersensitivity response in mice by topical application of 1α,25-dihydroxyvitamin D3 to elicitation site

Summary Recent evidence indicates that the biologically active metabolite of vitamin D₃, 1,25-dihydroxy-vitamin D₃ [1,25(OH)₂D₃], has an effect on the regulation of the immune response. We investigated whether topical treatment of mice with 1,25(OH)₂D₃ influences the contact hypersensitivity (CHS) response to trinitrochlorobenzene (TNCB). 1,25(OH)₂D₃ was applied to the dorsal trunk of A/J mice on days 0–3, and on day 4 topical application of 5% TNCB on the 1,25(OH)₂D₃-treated site was performed. The mice were tested for CHS on day 10 by applying 1% TNCB to the ears. No effect on induction of CHS response to TNCB was observed in 1,25(OH)₂D₃-treated mice compared with 24,25-dihydroxyvitamin D₃[24,25-(OH)₂D₃]-treated mice as control. In a second experiment, the dorsal trunk of A/J mice was treated with 5% TNCB on day 0. The topical application of 1,25(OH)₂D₃ on the ears was performed from days 2 to 5. On day 6, the mice were tested for CHS by applying 1% TNCB to the 1,25(OH)₂D₃-treated ears. When 1,25(OH)₂D₃ increased their response to TNCB by 40% compared with 24,25(OH)₂D₃-treated mice as 1,25(OH)₂D₃ increased their response to TNCB by 40% compared with 24,25(OH)₂D₃-treated mice as control (P<0.01). There were no findings suggesting that the pretreatment of the challenge site with 1,25(OH)₂D₃ induced an irritant dermatitis that was superimposed on a subsequent CHS reaction. The 1,25(OH)₂D₃ modulation of CHS response to TNCB in mice suggests that the hormone may play a role in the regulation of the immune response in vivo.     

Modulating T cell functions does not alleviate chronic inflammatory skin lesions in K5.TGFβ1 transgenic mice

Please cite this paper as: Modulating T cell functions does not alleviate chronic inflammatory skin lesions in K5.TGFβ1 transgenic mice. Experimental Dermatology 2009; 19: 406–415. Abstract: To use mice with chronic hyperproliferative skin inflammation as psoriasis models, their thorough phenotypic and functional characterization is indispensable. Mice with keratin 5 promoter‐controlled overexpression of latent human Transforming Growth Factor (TGF)β1 within the basal epidermis (K5.TGFβ1 mice) show a psoriasiform phenotype, but the underlying pathogenic mechanisms are not entirely clear. To elucidate the contribution of T lymphocytes to the pathogenesis in K5.TGFβ1 mice, we used three complementary approaches: first, peripheral T cells were eradicated via systemic treatment with CD3‐ or CD4‐depleting antibodies. However, this elimination did not alleviate the chronic inflammatory disorder. Second, bone marrow transplantation from transgenic mice into wildtype recipients and vice versa resulted in the expected reconstitution of both adaptive and innate immune system but had little effect on the cutaneous phenotype both in wildtype and transgenic chimeras. Third, based on the hypothesis that the disease course could be modulated by regulatory T cells (Tregs), we expanded Tregs in vivo using a superagonistic anti‐CD28 antibody. While this treatment achieved a threefold increase in Foxp3‐expressing Tregs, there was little, if any, effect on the chronic skin inflammation. We conclude from our findings that T cells play little, if any, role in the skin lesions of K5.TGFβ1 mice.