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.     

Inhibition of scratching behaviour caused by contact dermatitis in histidine decarboxylase gene knockout mice

A neuronal system dedicated to itch consists of primary afferent and spinothalamic projection neurons. Histamine is thought to be one of the main mediators for the transmission of itch sensation. However, there are little available information on the role of histamine in scratching behaviour and sensory transmission of atopic dermatitis and chronic eczema. In the present study, the role of histamine in scratching behaviour and neural conduction of sensation in the chronic eczema model was investigated by using l-histidine decarboxylase (HDC) gene knockout mice lacking histamine. The chronic contact dermatitis was induced with daily application of diphenylcyclopropenone (DCP) on a hind paw of HDC (+/+) and HDC (-/-) mice for 2 months. The observation of scratching behaviour and the hot-plate test were performed in both mice. Histological studies were performed in the skin and spinal cord tissues. Histological examination revealed that both HDC (+/+) and HDC (-/-) mice displayed the similar extent of inflammatory cell infiltration, hyperplastic epidermis and newly spreading of neuronal processes in the skin tissue. Scratching behaviour was exclusively induced in HDC (+/+) mice, whereas it was barely observed in HDC (-/-) mice. The expression of c-Fos was specifically upregulated in HDC (+/+) mice in lamina I of the spinal dorsal horn following repeated DCP application. Scratching behaviour in chronic contact dermatitis in mice was thought mainly mediated with histamine. The afferent pathway of sensation in chronic contact dermatitis model may connect with the central nervous system through lamina I of the spinal dorsal horn.     

Platelets are important for the development of immune tolerance: Possible involvement of TGF‐β in the mechanism

Platelets have diverse roles in immune processes in addition to their key functions in haemostasis and thrombosis. Some studies imply that platelets may be possibly related to the immune tolerance induction. However, the role of platelets in the development of immune tolerance is not fully understood. The purpose of this study was to investigate the role of platelets in the development of regulatory mechanisms responsible for cutaneous inflammation using a mouse model of low zone tolerance (LZT). Mice were treated with 2,4,6‐trinitro‐1‐chlorobenzene (TNCB) 8 times every other day for tolerance induction with administration of anti‐platelet antibody or control antibody during the tolerance induction phase every 3 days. After the treatment for the tolerance induction, mice were sensitized and then challenged with TNCB. The contact hypersensitivity (CHS) was significantly decreased at 24 hours after challenge in the mice with LZT than in those without LZT. Platelet depletion via administration of anti‐platelet antibody reversed the inhibition of CHS and reduced the frequency of Foxp3⁺ Tregs in the inflamed skin and draining lymph nodes in mice with LZT. In addition, repeated low‐dose skin exposure resulted in elevated plasma levels of transforming growth factor (TGF)‐β1. Interestingly, platelet depletion reduced plasma TGF‐β1 levels of mice with LZT. Furthermore, the CHS response was reduced by administration of recombinant TGF‐β1 during platelet depletion in mice with LZT. Administration of anti‐TGF‐β antibody reversed the inhibition of the CHS responses. These results suggest that platelets are involved in the induction of immune tolerance via the release of TGF‐β1.     

Induced histamine regulates Ni elution from an implanted Ni wire in mice by downregulating neutrophil migration

Histamine regulates various inflammatory reactions. We have reported that the expression of histidine decarboxylase (HDC) was induced by subcutaneous implantation of nickel (Ni) wire. However, the source and functions of histamine in Ni elution and Ni wire‐induced inflammation have not been completely studied. We aimed to elucidate the effects of de novo synthesized histamine on leucocyte infiltration and Ni elution. Implantation of Ni wire induced an increase in the Ni ion content of the surrounding tissues and serum and in the mRNA levels of HDC, a histamine‐producing enzyme, macrophage inflammatory protein‐2 (MIP‐2), a chemoattractant for neutrophils, and monocyte chemoattractant protein‐1 (MCP‐1), a chemoattractant for monocytes. The Ni wire induced HDC expression even in mast cell‐deficient WBB6F1‐W/W^V mice. In HDC knockout (HDC KO) mice, the Ni wire‐induced increase in MIP‐2 mRNA expression was significantly higher than that in wild‐type mice but not MCP‐1. MIP‐2 expression was enhanced in histamine H2 receptor knockout (H2R KO) mice but not in WBB6F1‐W/W^V mice. Histamine inhibited NiCl₂‐induced MIP‐2 mRNA expression in mouse bone marrow‐derived macrophages (BMDMs) obtained from wild‐type mice; this inhibition was not observed in BMDMs from H2R KO mice. Ni elution increased in HDC KO mice, in which leucocyte infiltration also increased, and was suppressed in mice treated with neutrophil‐specific antibody. These results suggest that the Ni wire induced HDC expression in non‐mast cells and that, in the chronic phase of inflammation, endogenous histamine reduced Ni elution, probably through regulation of MIP‐2 expression and neutrophil migration.     

The accelerating effect of histamine on the cutaneous wound-healing process through the action of basic fibroblast growth factor

This study revealed that the absence of histamine in histidine decarboxylase gene-knockout (HDC(-/-)) mice resulted in delayed cutaneous wound healing and that exogenously administered histamine compensated this process. With the overproduction of histamine in HDC gene-transgenic mice, the healing was accelerated compared to the HDC(+/+) mice. These results indicate that histamine positively accelerated the cutaneous wound healing. Macrophage recruitment and angiogenesis at the wound edge were specifically impaired in HDC(-/-) mice, and histamine-treated wounds in HDC(-/-) mice demonstrated increased macrophage recruitment and angiogenesis. The amount of basic fibroblast growth factor (bFGF) in protein level at the wound edge was higher in HDC(+/+) mice, especially on the 3rd and 5th day of wound healing compared to those in HDC(-/-) mice. Topically administered SU5402, a specific antagonist to fibroblast growth factor receptor-1 tyrosine kinase, to the wound surface suppressed the wound healing in HDC(+/+) mice but not in HDC(-/-) mice. Moreover, SU5402 reduced macrophage recruitment and angiogenesis in HDC(+/+) mice. From these observations, it was concluded that the accelerated wound-healing activity of histamine was mediated by the activity of bFGF, which leads to angiogenesis, and macrophage recruitment in the wound-healing process.     

CD109, a novel TGF‐β antagonist,decreases fibrotic responses in a hypoxic wound model

Excessive extracellular matrix deposition that occurs in many fibrotic skin disorders such as hypertrophic scarring and scleroderma is often associated with hypoxia. CD109 is a novel TGF‐β co‐receptor and TGF‐β antagonist shown to inhibit TGF‐β‐induced extracellular matrix protein production in vitro. We examined whether CD109 is able to regulate extracellular matrix deposition under low oxygen tension in vivo using transgenic mice overexpressing CD109 in the epidermis. By creating dorsal bipedicle skin flaps with centrally located excisional wounds in these mice and their wild‐type littermates, we generated a novel murine hypoxic wound model. Mice were sacrificed on 7 or 14 days post‐wounding, and tissues were harvested for histological and biochemical analysis. Hypoxic wounds in both transgenic and wild‐type mice showed increased levels of HIF‐1α and delayed wound closure, validating this model in mice. Hypoxic wounds in CD109 transgenic mice demonstrated decreased collagen type 1 and fibronectin expression, and reduced dermal thickness on day 7 post‐wounding as compared to those in wild‐type mice and to non‐hypoxic control wounds. These results suggest that CD109 decreases extracellular matrix production and fibrotic responses during hypoxic wound healing. Manipulating CD109 levels may have potential therapeutic value for the treatment of fibrotic skin disorders associated with poor oxygen delivery.     

Loureirin B inhibits fibroblast proliferation and extracellular matrix deposition in hypertrophic scar via TGF‐β/Smad pathway

The ethanolic extract of Resina Draconis (RDEE) has been reported beneficial to normal wound healing yielding more regularly arranged collagen fibres. Loureirin B, a major component in RDEE, has been supposed to be effective on the prevention and treatment of pathological scars. To investigate the therapeutic effects of loureirin B on hypertrophic scar (HS), fibroblasts from human HS and normal skin (NS) were isolated. Results showed that loureirin B dose‐dependently downregulated both mRNA and protein levels of type I collagen (ColI), type III collagen (ColIII) and α ‐ smooth muscle actin (α ‐ SMA) in HS fibroblasts. Loureirin B also suppressed fibroblast proliferative activity and redistributed cell cycle, but did not affect cell apoptosis. In vivo rabbit ear scar model, loureirin B significantly improved the arrangement and deposition of collagen fibres, decreased protein levels of ColI, ColIII and α ‐ SMA and suppressed myofibroblast differentiation and scar proliferative activity. In NS fibroblasts, loureirin B effectively inhibited TGF‐β1‐induced upregulation of ColI, ColIII and α‐SMA levels, myofibroblast differentiation and the activation of Smad2 and Smad3. Loureirin B also affected mRNA levels of major MMPs and TIMPs in TGF‐β1‐stimulated fibroblasts. Taken together, this study demonstrates that loureirin B could downregulate the expression of fibrosis‐related molecules by regulating MMPs and TIMPs levels, inhibit scar fibroblast proliferation and suppress TGF‐β1‐induced fibrosis, during which TGF‐β1/Smad2/3 pathway is likely involved. These findings suggest that loureirin B is a potential therapeutic compound for HS treatment.     

Histamine helps development of eczematous lesions in experimental contact dermatitis in mice

Histamine is released from mast cells in the skin, causing urticaria and itching. However, little is known about the roles of histamine in development of eczematous lesions in contact dermatitis. Effects of histamine on development of eczematous lesions in contact dermatitis were assessed using histamine-deficient mice in which contact dermatitis was developed by repeated application of diphenylcyclopropenone. Development of eczematous lesions in contact dermatitis was suppressed in histamine-deficient mice compared to wild-type mice. H₁ agonist ((6-12-(4-imidazol)ethylamino)-N-(4-trifluoro- methylphenyl)hepatanecarboxamide) promoted development of eczematous lesions in histamine-deficient mice. H₁ receptor antagonist (loratadine) suppressed development of eczematous lesions in wild-type mice, whereas H₂ agonist (dimaprit) and receptor antagonist (cimetidine) were ineffective. These results suggest that histamine facilitates the development of eczematous lesions in a murine model of contact dermatitis via H₁ receptors.     

Integrin αE(CD103) is induced but plays no pathogenic role in psoriasiform skin lesions of TGFβ1 transgenic mice

T‐cells expressing α_E(CD103), an integrin induced by TGFβ on T‐cells in vitro, accumulate within epithelia in inflammatory disorders, including psoriasis. However, it is unclear, if and how α_E(CD103) contributes to skin inflammation. Using two complementary approaches, we have investigated α_E(CD103) in psoriasis‐like skin inflammation of mice with transgenic epidermal expression of human TGFβ1: α_E(CD103) was inhibited by function‐blocking antibodies in vivo, and double‐mutants with additional α_E(CD103)‐depletion were generated in two different genetic backgrounds. Epidermal hTGFβ1 expression was associated with prominent expression of α_E(CD103) on infiltrating cells. However, neither treatment with α_E(CD103)‐blocking antibodies nor deficiency of α_E(CD103) in double‐mutant mice altered the psoriasis‐like phenotype. In addition, histopathological and flow cytometric analyses revealed similar pathological skin alterations and lymphocyte subgroups in the different mouse strains. Thus, while α_E(CD103) expression is indeed associated with hTGFβ1 in vivo, it has little, if any, influence on the course of the psoriasis‐like phenotype in K5.hTGFβ1 transgenic mice.     

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.     

Mast cell histamine‐mediated transient inflammation following exposure to nickel promotes nickel allergy in mice

We previously reported that allergic responses to nickel (Ni) were minimal in mice deficient in the histamine‐forming enzyme histidine decarboxylase (HDC‐KO), suggesting an involvement of histamine in allergic responses to Ni. However, it remains unclear how histamine is involved in the process of Ni allergy. Here, we examined the role of histamine in Ni allergy using a murine model previously established by us. Mice were sensitized to Ni by intraperitoneal injection of a NiCl₂‐lipopolysaccharide (LPS) mixture. Ten days later, allergic inflammation was elicited by challenging ear‐pinnas intradermally with NiCl₂. Then, ear‐swelling was measured. Pyrilamine (histamine H1‐receptor antagonist) or cromoglicate (mast cell stabilizer) was intravenously injected 1 h before the sensitization or the challenge. In cell‐transfer experiments, spleen cells from Ni‐sensitized donor mice were intravenously transferred into non‐sensitized recipient mice. In both sensitized and non‐sensitized mice, 1 mm or more NiCl₂ (injected into ear‐pinnas) induced transient non‐allergic inflammation (Ni‐TI) with accompanying mast cell degranulation. LPS did not affect the magnitude of this Ni‐TI. Pyrilamine and cromoglicate reduced either the Ni‐TI or the ensuing allergic inflammation when administered before Ni‐TI (at either the sensitization or elicitation step), but not if administered when the Ni‐TI had subsided. Experiments on HDC‐KO and H1‐receptor‐KO mice, and also cell‐transfer experiments using these mice, demonstrated histamine's involvement in both the sensitization and elicitation steps. These results suggest that mast cell histamine‐mediated Ni‐TI promotes subsequent allergic inflammatory responses to Ni, raising the possibility that control of Ni‐TI by drugs may be effective at preventing or reducing Ni allergy.     

Role of IL‐10 and TGF‐β in melanoma

IL‐10 and TGF‐β are immunosuppressive cytokines expressed in tumors including melanoma and, therefore, deemed major cause for failing antitumor immune responses. Re‐evaluating their role, we compared their expression by quantitative RT‐PCR in melanoma and skin of healthy individuals, tested their induction in dendritic cells and T cells co‐cultured with tumor cells, and their effects on the immune cells. Both cytokines as well as their receptors were expressed in melanoma at significantly lower levels than in healthy skin. Consequently, the expressions of IL‐10‐responsive SOCS‐3 and TGF‐β‐responsive Smad‐7 were low in tumors but high in healthy skin. T cells co‐cultured with tumor cells developed an anergic state without increased IL‐10 or TGF‐β expression. In vitro tumor‐induced immature dendritic cells produced high IL‐10 levels and less efficiently induced T‐cell proliferation. Nonetheless, they could be induced to mature, and blocking IL‐10 did not alter the capacity of the resulting mature dendritic cells to stimulate T cells. Mature dendritic cells co‐cultured with tumor cells produced increased IL‐10 but decreased TGF‐β and more efficiently induced T‐cell proliferation. The lack of correlation of IL‐10 and TGF‐β with immune deficits in situ and in vitro suggests re‐evaluating their roles in cancer.     

NADPH oxidase‐2 is a key regulator of human dermal fibroblasts: a potential therapeutic strategy for the treatment of skin fibrosis

The proliferation of human skin dermal fibroblasts (HDFs) is a critical step in skin fibrosis, and transforming growth factor‐beta1 (TGF‐β1) exerts pro‐oxidant and fibrogenic effects on HDFs. In addition, the oxidative stress system has been implicated in the pathogenesis of skin disease. However, the role of NADPH oxidase as a mediator of TGF‐β1‐induced effects in HDFs remains unknown. Thus, our aim was to investigate the role of NADPH in human skin dermal fibroblasts. Primary fibroblasts were cultured and pretreated with various stimulants. Real‐time Q‐PCR and Western blotting analyses were used for mRNA and protein detection. In addition, siRNA technology was applied for gene knock‐down analysis. Hydrogen peroxide production and 2′,7′‐dichlorofluorescein diacetate (DCFDA) measurement assay were performed. Here, our findings demonstrated that HDFs express key components of non‐phagocytic NADPH oxidase mRNA. TGF‐β1 induced NOX2 and reactive oxygen species formation via NADPH oxidase activity. In contrast, NOX3 was barely detectable, and other NOXs did not display significant changes. In addition, TGF‐β1 phosphorylated MAPKs and increased activator protein‐1 (AP‐1) in a redox‐sensitive manner, and NOX2 suppression inhibited baseline and TGF‐β1‐mediated stimulation of Smad2 phosphorylation. Moreover, TGF‐β1 stimulated cell proliferation, migration, collagen I and fibronectin expression, and bFGF and PAI‐1 secretion: these effects were attenuated by diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and NOX2 siRNA. Importantly, NOX2 siRNA suppresses collagen production in primary keloid dermal fibroblasts. These findings provide the proof of concept for NADPH oxidase as a potential target for the treatment of skin fibrosis.     

Regulatory T‐cell cytokines in patients with nonsegmental vitiligo

In the etiopathogenesis of vitiligo, the role of suppressor cytokines, such as transforming growth factor‐β (TGF‐β) and interleukin‐10 (IL‐10), associated with regulatory T‐cells (Treg) is not completely known. In this study, the role of Treg‐cell functions in the skin of patients with nonsegmental vitiligo was investigated. Lesional and nonlesional skin samples from 30 adult volunteers ranging in age from 18 to 36 years with nonsegmental vitiligo were compared with normal skin area excision specimens of 30 benign melanocytic nevus cases as controls. All samples were evaluated staining for forkhead box P3 (Foxp3), TGF‐β, and IL‐10 using the standardized streptavidin–biotin immunoperoxidase immunohistochemistry method. Foxp3 expression was lower in lesional vitiligo skin specimens compared to controls; it was also lower in lesional vitiligo specimens than nonlesional vitiligo specimens. IL‐10 levels were lower in lesional vitiligo specimens compared to the controls, whereas IL‐10 expression was significantly lower in lesional specimens compared with nonlesional specimens. TGF‐β expression was higher in both lesional and nonlesional skin specimens of patients with vitiligo compared to controls. TGF‐β expression was lower in lesional skin specimens than nonlesional skin specimens. In addition, there was no significant correlation between Foxp3 expression with TGF‐β and IL‐10 expressions in lesional skin specimens in the vitiligo group. In this study, results supporting the contribution of Treg cells and IL‐10 deficiency to the autoimmune process were obtained. Therefore, future studies are necessary to demonstrate the definitive role of Treg‐cell functions in the etiopathogenesis of vitiligo.     

Upregulation of histidine decarboxylase mRNA expression in scleroderma skin

Abstract The involvement of histamine in the pathogenesis of systemic sclerosis (SSc) has been suggested. Possible involvement of histamine and histidine decarboxylase (HDC), the synthesizing enzyme for histamine, in the formation of the skin abnormalities in SSc was studied. Skin histamine concentrations in SSc were significantly lower than in normal controls (P < 0.02). In situ hybridization with an HDC probe revealed that the expression of the HDC gene in SSc was greater than in normal controls. The number of cells and the mean grain number per cell expressing HDC mRNA were both significantly greater in SSc than in normal controls (both P < 0.01). These results show a reduction in histamine concentration and an elevated HDC gene expression in SSc skin, indicating an increase in both histamine release and HDC gene expression. The upregulation of histamine turnover appears to be involved in the formation of the skin abnormalities of SSc. Received: 4 July 2000 / Revised: 27 October 2000 / Accepted: 20 December 2000     

Effect of pedunculagin investigated by non‐invasive evaluation on atopic‐like dermatitis in NC/Nga mice

Background/purpose: Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disorder that is becoming increasingly prevalent. Experimental animal models have been an indispensable tool for studying its pathological mechanisms and for in vivo testing of novel therapeutic approaches. AD‐like lesions can be induced experimentally in NC/Nga mice. Pedunculagin, an ellagitannin purified from the Manchurian alder, Alnus hirsuta var. microphylla, Betulaceae, is a novel immunomodulator. To evaluate the effect of pedunculagin for AD‐like lesions in NC/Nga mice, using clinical and non‐invasive methods. Methods: AD‐like lesions were induced in NC/Nga mice using 2,4,6‐trinitrochlorobenzene (TNCB). A cream containing 0.1% or 0.5% pedunculagin was applied to the positive treatment group, and the base cream without pedunculagin was applied to the negative treatment group. The control group did not receive any kind of topical agents. We evaluated the therapeutic efficacy of pedunculagin for AD by statistical evaluation of the clinical severity score using non‐invasive biomedical engineering tools before treatment, and 1 day, 3 days, 1 week, 2 weeks and 4 weeks afterwards. Results: An AD‐like skin rash was successfully induced using TNCB in NC/Nga mice. The group receiving higher concentrations of pedunculagin showed faster and greater improvement. Conclusion: Our results suggest that remedies made from natural materials like pedunculagin are now showing promise for medical applications, and many new studies are expected to explore this potential.     

Bleomycin‐induced fibrosis in MC1 signalling‐deficient C57BL/6J‐Mc1re/e mice further supports a modulating role for melanocortins in collagen synthesis of the skin

The melanocortin‐1 receptor (MC₁) binds α‐melanocyte‐stimulating hormone (α‐MSH) with high affinity and has a physiological role in cutaneous melanin pigmentation. Previously, we reported that human dermal fibroblasts also express functional MC₁. α‐MSH suppressed transforming growth factor‐β₁‐ and bleomycin (BLM)‐induced collagen synthesis in vitro and in vivo. Using MC₁ signalling‐deficient C57BL/6J‐Mc1r^e/e mice, we tested as to whether MC₁ has a regulatory role on dermal collagen synthesis in the BLM model of scleroderma. Notably, mice with a C57BL/6J genetic background were previously shown to be BLM‐non‐susceptible. Interestingly, treatment of C57BL/6J‐Mc1r^e/e but not of C57BL/6J‐wild‐type mice with BLM increased cutaneous collagen type I content at RNA and protein level along with development of skin fibrosis. Cutaneous levels of connective tissue growth factor and monocyte chemotactic protein‐1 were also increased in BLM‐treated C57BL/6J‐Mc1r^e/e mice. Primary dermal fibroblasts from C57BL/6J‐wt mice further expressed MC₁, suggesting that these cells are directly targeted by melanocortins to affect collagen production of the skin. Our findings support the concept that MC₁ has an endogenous regulatory function in collagen synthesis and controls the extent of fibrotic stress responses of the skin.     

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.