IL‐1 signalling determines the fate of skin grafts expressing non‐self protein in keratinocytes

Please cite this paper as: IL‐1 signalling determines the fate of skin grafts expressing non‐self protein in keratinocytes. Experimental Dermatology 2010; 19 : 723–729. Abstract: Although IL‐1 is a known inflammatory cytokine during pathogen infection, the role of IL‐1 in skin graft rejection, particularly where foreign antigen is expressed exclusively in keratinocytes, is less understood. Here, we use a syngeneic skin graft system, where antigens are expressed in epithelial cells via either a keratin 14 or keratin 5 promoter, to explore the role of IL‐1 in graft rejection and induction of epithelial antigen‐specific effector CD8⁺ T‐cell function. Keratin 5 ovalbumin (K5mOVA) transgenic skin grafts destined for rejection demonstrated increased expression of IL‐1β and its receptors compared to K14 HPV16 E7 transgenic grafts that do not reject spontaneously. Rejection of OVA grafts lacking the IL‐1 receptor (IL‐1R1) was delayed and associated with decreased numbers of antigen‐specific CD8 T cells. In contrast, K14E7 grafts survived on immunocompetent, syngeneic recipients with decreased graft levels of IL‐1β and IL‐1R1 and 2. However, in the absence of the IL‐1 receptor antagonist, IL‐1Ra, skin grafts were spontaneously rejected and an E7‐specific CD8 T‐cell response was primed. Thus, expression of the HPV16E7 oncoprotein in epithelial cells prevents IL‐1β‐associated skin graft rejection and induction of antigen‐specific CD8 T‐cell responses. Enhancing IL‐1β signalling, via blocking of the IL‐1 receptor antagonist, may represent an alternative strategy for treatment of HPV16E7‐associated cancers.     

CD4+ T cells producing interleukin (IL)‐17, IL‐22 and interferon‐γ are major effector T cells in nickel allergy

Background It has been suggested that interleukin (IL)‐17 and IL‐22 play important roles in the elicitation of human allergic contact dermatitis; however, the frequencies of T cell subtypes producing IL‐17 and IL‐22 in human allergic contact dermatitis are unknown. Objectives To determine the frequencies of CD4⁺, CD8⁺ and γδ T cells producing IL‐17, IL‐22 and interferon (IFN)‐γ in the blood and skin from nickel‐allergic patients. Patients/materials/methods Blood samples were collected from 14 patients and 17 controls, and analysed by flow cytometry. Biopsies were taken from 5 patients and 6 controls, and analysed by immunohistochemistry and flow cytometry of skin lymphocytes. Results We found an increased frequency of γδ T cells in the blood, but no differences in the distribution of cytokine‐producing CLA⁺ T cell subtypes in nickel‐allergic patients as compared with controls. In nickel‐allergic patients, there was massive cellular infiltration dominated by CD4⁺ T cells producing IL‐17, IL‐22 and IFN‐γ in nickel‐challenged skin but not in vehicle‐challenged skin. Conclusion CD4⁺ T cells producing IL‐17, IL‐22 and IFN‐γ are important effector cells in the eczematous reactions of nickel‐induced allergic contact dermatitis in humans.     

Long‐term maintenance of skin immune system in a NOD‐Scid IL2rγnull mouse model transplanted with human skin

We developed a NOD‐Scid IL2rγ^null mouse model transplanted with human skin that brings fundamental insight on in vivo cellular mechanisms of intradermal immunization and antigen presentation by dermal dendritic and epidermal Langerhans cells for skin T‐cell immunity. Indeed, T‐cell immunity is a crucial checkpoint for the induction of in vivo rapid control of skin infection. With the long‐term preservation of a complete human skin immune system, this model offers the unique opportunity not only to better understand mechanisms of skin immune response but also to test new compounds and devices for cutaneous routes of vaccination, as well as new therapeutics approach for skin diseases, allergies or infections.     

Primary human keratinocytes efficiently induce IL‐1‐dependent IL‐17 in CCR6+ T cells

Abstract: Keratinocytes and activated T cells interact in skin inflammation by virtue of chemokines and cytokines. T cell‐derived IL‐17 has been described to play an important role in the course of psoriatic inflammation. In this study, we addressed how keratinocytes influence the secretion of IL‐17 in autologous T cell subsets. We found that a co‐culture of autologous keratinocytes and T cell‐receptor‐stimulated T cells markedly enhanced the production of IL‐17. Besides the importance of direct cell contact, this effect was mainly mediated by IL‐1 and could be blocked by the IL‐1 antagonist anakinra. An additional increase in IL‐17 production by IL‐23 was only seen in the presence of IL‐1, which thus plays a permissive role for the action of IL‐23. Importantly, co‐culture of keratinocytes with CCR6+ CD4+ T cells that are enriched for Th17 cells resulted in significantly higher IL‐17 production compared to co‐culture with CD4+ T cells.     

Role of epidermal γδ T‐cell‐derived interleukin 13 in the skin‐whitening effect of Ginsenoside F1

Ginsenoside F1 (GF1) is a metabolite of ginsenoside Rg1. Although GF1 has several benefits for skin physiology, the effect of GF1 on skin pigmentation has not been reported. We found that a cream containing 0.1% GF1 showed a significant whitening effect on artificially tanned human skin after 8 weeks of application. However, GF1 did not inhibit mRNA expression of tyrosinase or dopachrome tautomerase (DCT) in normal human epidermal melanocytes (NHEMs) or cocultured NHEMs/normal human epidermal keratinocytes. Interestingly, GF1 enhanced production of interleukin 13 (IL‐13) from human epidermal γδ T cells. IL‐13 significantly reduced the mRNA expression and protein amount of both tyrosinase and DCT and reduced melanin synthesis activities in NHEMs, resulting in visible brightening of NHEM pellet. These results suggest that enhancement of IL‐13 production by GF1 from epidermal γδ T cells might play a role in the skin‐whitening effect of GF1 via the suppression of tyrosinase and DCT.     

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.     

Granzyme A potentiates chemokine production in IL‐17‐stimulated keratinocytes

Plaque psoriasis presents with focal skin inflammation, partially maintained by IL‐17‐mediated interactions between infiltrating epidermal T cells and activated keratinocytes. Here we show that the majority of lesional epidermal CD8 T cells express granzyme A, alone or in combination with IL‐17. To assess proinflammatory properties of granzyme A in psoriasis, primary human keratinocytes were stimulated with granzyme A in the presence or absence of IL‐17. Out of 33 analysed keratinocyte‐derived inflammatory mediators, granzyme A potentiated IL‐17‐induced secretion of CXCL 1, CXCL 12 and CCL 4. Intriguingly, all three chemokines are implicated in psoriasis pathogenesis and are involved in recruitment of T cells, neutrophils and pDCs into inflamed tissues. Our results indicate that granzyme A produced by lesional CD8 T cells specifically increase the chemokine production from inflamed keratinocytes, thereby amplifying a chemotactic inflammatory loop that sustains psoriasis lesions.     

Effects of a ceramide‐containing water‐in‐oil ointment on skin barrier function and allergen penetration in an IL‐31 treated 3D model of the disrupted skin barrier

Atopic dermatitis (AD) is a chronically relapsing, pruritic inflammation of the skin with dryness and disturbed skin barrier function. Recently, we established that IL‐31 treatment of human 3D skin models resulted in a disrupted skin barrier phenotype resembling AD. In this model, we found that IL‐31 interferes with the differentiation of keratinocytes and inhibits the expression of terminal differentiation markers. In the present study, we investigated the effects of a ceramide‐containing water‐in‐oil skin care ointment on the physical skin barrier structure and function in disrupted skin barrier models, generated either by using primary normal human epidermal keratinocytes (NHEK) or HaCaT cells. We observed that the physical skin barrier of the models recovered after daily topical treatment with the ceramide‐containing ointment. Topical application of the ointment prevented downregulation of filaggrin and disorganization of other differentiation markers, such as keratin 10 and β4‐integrin, as demonstrated by immunohistological analysis. The expression of Ki67 was also upregulated in response to the ointment. Furthermore, functional studies revealed that local application of the ointment diminished the increased uptake of fluorescently labelled recombinant allergens of timothy grass (phl p1) in our model. In conclusion, our data revealed that topical application of a ceramide‐containing skin care ointment reduced IL‐31 induced impairments of the physical skin barrier and skin barrier function in an in vitro model of the disrupted skin barrier. This standardized model can be utilized in the future to monitor ex vivo effects of various topical therapies on skin morphology, physiology, and gene expression.     

Circulating CLA+ T lymphocytes as peripheral cell biomarkers in T‐cell‐mediated skin diseases

T lymphocytes expressing the CLA antigen constitute a subset of effector memory lymphocytes that are functionally involved in T‐cell‐mediated cutaneous diseases. Skin‐seeking lymphocytes recirculate between inflamed skin and blood during cutaneous inflammation. Many studies in different T‐cell‐mediated inflammatory cutaneous diseases have clearly related their pathologic mechanisms to CLA+ T cells. Based on common features of these cells in different cutaneous disorders mediated by T cells, we propose that circulating CLA+T cells could constitute very useful peripheral cellular biomarkers for T‐cell‐mediated skin diseases.     

Interleukin‐1 from epithelial cells fosters T cell‐dependent skin inflammation

Background Chronic inflammatory skin diseases such as atopic dermatitis and psoriasis are characterized by the infiltration of lymphocytes into the epidermal compartment. Several studies point to an active role of skin epithelial cells in the pathophysiology of such diseases. Objectives In this study we addressed the regulatory function of primary human keratinocytes in the interaction with autologous T cells and monocytes. Methods We used a human coculture model with keratinocytes grown from epidermal stem cells of the outer root sheath of human hair follicles and autologous T cells. Results In our coculture system we observed a high production of interferon (IFN)‐γ, but not Th2 cytokines, in the presence of superantigen or antigen‐pulsed autologous monocytes. Critical parameters for this effect were: (i) T‐cell receptor activation, (ii) an intercellular adhesion molecule‐1/lymphocyte function‐associated antigen (LFA)‐1‐dependent interaction between keratinocytes and T cells, and (iii) secretion of interleukin (IL)‐1β. Remarkably, in the presence of activated T cells, epithelial cells seemed to be a more significant source of IL‐1β than monocytes. Application of the LFA‐1 blocker efalizumab or IL‐1 receptor antagonist anakinra enabled us to suppress completely the production of IFN‐γ by T cells in the coculture. Conclusions IL‐1 secretion and the physical contact between keratinocytes and activated, infiltrating T cells may be central for the development of chronic inflammatory skin conditions.     

The crucial role of IL‐22 and its receptor in thymus and activation regulated chemokine production and T‐cell migration by house dust mite extract

House dust mite (HDM) is known as one of the factors that causes atopic dermatitis (AD). Interleukin (IL)‐22 and thymus and activation regulated chemokine (TARC) are related to skin inflammatory disease and highly expressed in AD lesions. However, the effects of HDM on IL‐22 production in T cells and on TARC production and IL‐22Rα receptor expression in keratinocytes are unknown. To identify the role of HDM in keratinocytes and T cells, we investigated IL‐22Rα expression and TARC production in the human keratinocyte cell line HaCaT and IL‐22 production in T cells treated with HDM extract as well as their roles in HDM‐induced skin inflammation. HDM extract not only increased IL‐22Rα expression and TARC production in HaCaT but also enhanced IL‐22, tumor necrosis factor (TNF)‐α and interferon (IFN)‐γ production in T cells. The HDM extract‐induced IL‐22 from T cells significantly increased the production of IL‐1α, IL‐6 and TARC in HaCaT cells. In addition, we found that TARC produced in HDM extract‐treated HaCaT induced T‐cell recruitment. These results suggest that there is a direct involvement of HDM extract‐induced IL‐22 in TARC production and T‐cell migration. Taken together, TARC production in HaCaT through the interaction between IL‐22 and IL‐22Rα facilitates T‐cell migration. These data show one of the reasons for inflammation in the skin lesions of AD patients.     

Signalling protein complexes isolated from primary human skin‐resident T cells can be analysed by Multiplex IP‐FCM

Studying signal transduction in skin‐resident T cells (sr‐T cells) can be limited by the small size of clinical biopsies. Here, we isolated sr‐T cells from clinical samples and analysed signalling protein complexes by multiplex immunoprecipitation detected by flow cytometry (mIP‐FCM). In samples from two independent donors, antigenic stimulation induced signalling proteins to join shared complexes that were observed in seven pairwise combinations among five proteins. This demonstrates that sr‐T cells isolated from small clinical samples provide sufficient material for mIP‐FCM‐based analysis of signalling‐induced protein complexes. We propose that this strategy may be useful for gaining improved mechanistic insight of sr‐T cell signal transduction associated with dermatological disease.     

Epstein–Barr virus‐associated T‐cell lymphoproliferative disorder affecting skin and lung in an elderly patient

A 70‐year‐old man presented with papular skin lesions and was diagnosed with Epstein–Barr virus (EBV)‐associated T‐cell lymphoproliferative disorder (T‐LPD). The patient showed infiltration of a large number of EBV‐encoded RNA‐positive T cells in the skin and lung, presence of EBV load in the peripheral blood, and expansion of clonal EBV‐infected γδ T cells and CD8⁺ T cells in the blood and skin, as assessed by EBV‐terminal repeat Southern blot, T‐cell receptor polymerase chain reaction and flow cytometric analyses. In the Japanese or East Asian fatal cases of EBV‐associated T/natural killer (NK)‐LPD, there are two peaks in age at death, approximately 20 years and 60 years. The former age group is associated with chronic active EBV infection (CAEBV), and the latter group typically suffers from extranodal NK/T‐cell lymphoma. Our case is characterized not only by the unique skin and lung manifestations but also the late onset age of the disease, indicating that the skin manifestation of CAEBV can be seen even in elderly patients.     

Elevated levels of the antimicrobial peptide LL‐37 in hidradenitis suppurativa are associated with a Th1/Th17 immune response

Hidradenitis suppurativa (HS) is an inflammatory skin disease with poorly understood immunopathogenic mechanisms. LL‐37 is an antimicrobial peptide, which is transcribed from the CAMP (cathelicidin antimicrobial peptide) gene. Previous reports showed upregulated levels of CAMP and LL‐37 in HS lesions, and therefore, the aim of this study was to compare levels of LL‐37 in HS to other inflammatory skin diseases and to establish immunomodulatory functions of LL‐37 in HS. We confirm an upregulation of the LL‐37 peptide in lesional HS skin with comparable levels as in psoriasis patients and are able to positively correlate the presence of LL‐37 in HS with the presence of T cells, macrophages, neutrophils, IFN‐γ, IL‐17, IL‐23, TNF‐α, IL‐32 and IL‐1β. Mechanistically, LL‐37 boosts the proliferation of unspecifically activated CD4⁺ T cells via an increased calcium signalling independent of antigen‐presenting cells. Targeting LL‐37 may therefore represent a new therapeutic option for the treatment of this recalcitrant disease, but it has to be kept in mind that LL‐37 also has an antimicrobial function.     

IL‐36γ has proinflammatory effects on human endothelial cells

Interleukin‐36 cytokines are predominantly expressed by epithelial cells. Significant upregulation of epidermal IL‐36 is now a recognised characteristic of psoriatic skin inflammation. IL‐36 is known to induce inflammatory responses in dendritic cells, fibroblasts and epithelial cells. Although vascular alterations are a hallmark of psoriatic lesions and dermal endothelial cells are well known to play a critical role in skin inflammation, the effects of IL‐36 on endothelial cells are unexplored. We here show that endothelial cells including dermal microvascular cells express a functionally active IL‐36 receptor. Adhesion molecules VCAM‐1 and ICAM‐1 are upregulated by IL‐36γ stimulation, and this is reversed by the presence of the endogenous IL‐36 receptor antagonist. IL‐36γ‐stimulated endothelial cells secrete the proinflammatory chemokines IL‐8, CCL2 and CCL20. Chemotaxis assays showed increased migration of T‐cells following IL‐36γ stimulation of endothelial cells. These results suggest a role for IL‐36γ in the dermal vascular compartment, and it is likely to enhance psoriatic skin inflammation by activating endothelial cells and promoting leucocyte recruitment.     

Decreased interleukin‐21 expression in skin and blood in advanced mycosis fungoides

Interleukin (IL)‐21 is regarded as a potent antitumor agent, which increases the cytotoxicity of both natural killer (NK) and CD8⁺ T cells. In this study, we investigated the role of IL‐21 in mycosis fungoides (MF). IL‐21 mRNA expression levels in patch and plaque MF were significantly higher than those in normal skin. IL‐21 mRNA expression levels in tumor MF were significantly decreased compared with those in patch and plaque MF. Interestingly, mRNA expression levels of IL‐21 in MF lesional skin significantly correlated with those of T‐helper type‐1 cytokines/chemokines such as CXCL10, CXCL11 and γ‐interferon. Immunohistochemistry showed that IL‐21 was expressed by keratinocytes in patch and plaque MF. Furthermore, serum IL‐21 levels in patients with tumor MF were significantly lower than those of healthy controls and plaque MF. Thus, IL‐21 expression was significantly downregulated in skin and blood of patients with tumor MF, which may contribute to progression of MF. Our study suggests that recombinant IL‐21 would be a promising therapy for MF.     

Gamma‐delta T‐cell lymphoma arising in a long‐standing cutaneous plaque

The precise classification and characterization of primary cutaneous gamma‐delta T‐cell lymphoma (PCGD‐TCL) has been hindered by clinical and morphologic features that overlap with other lymphomas, especially subcutaneous panniculitis‐like T cell lymphoma (SPTCL). The recent World Health Organization/European Organization for Research and Treatment of Cancer (WHO/EORTC) classification distinguishes the more aggressive PCGD‐TCL from the usually indolent SPTCL, however. We report a 30‐year‐old woman with an indurated violaceous plaque on the left cheek that had been present for several years. Biopsies showed a dense lymphocytic infiltrate involving the subcutis and dermis that consisted mostly of small and medium‐sized lymphocytes, some with irregular nuclear contours and dense chromatin. These cells were positive for TIA‐1, TCR‐gamma and CD8, but negative for beta‐F1 and granzyme‐B. Staging with positron emission tomography–computed tomography (PET/CT), CBC and bone marrow with flow cytometry identified lymphadenopathy as well as blood and marrow involvement by an abnormal TCRgd‐positive T‐cell proliferation (Ann Arbor Stage IV). The patient's history of a long‐standing lesion in this case is unusual, in that gamma‐delta T‐cell lymphomas are typically rapidly progressive neoplasms. As such, it raises the possibility of ‘transformation’ of a long‐standing inflammatory process into an overt lymphoma.     

Therapeutic potential of topically administered γ‐AlOOH on 2,4‐dinitrochlorobenzene‐induced atopic dermatitis‐like lesions in Balb/c mice

Boehmite (γ‐AlOOH) has a wide range of applications in a variety of industrial and biological fields. However, little is known about its potential roles in skin diseases. The current study investigated its effect on atopic dermatitis (AD). Following characterization, cytotoxicity, pro‐inflammatory response and oxidative stress associated with boehmite were assessed, using TNF‐α‐induced keratinocytes and mast cells. In addition, therapeutic effects of boehmite, topically administered to Balb/c mice induced by 2,4‐dinitrochlorobenzene (DNCB), were evaluated. Expression of cytokines (TLSP, IL‐25 and IL‐33) and the generation of ROS from keratinocytes induced by TNF‐α were significantly inhibited by boehmite without affecting cell viability. MAPKs (ERK, JNK and p38) required for cytokine expression were suppressed by boehmite treatment. Up‐regulation of cytokines (TSLP, IL‐4, IL‐5, IL‐13, RANTES) in human mast cells treated with phorbol 12‐myristate 13‐acetate and calcium ionophore was also suppressed by boehmite. Boehmite improved the AD severity score, epidermal hyperplasia and transepidermal water loss in DNCB‐induced AD‐like lesions. Moreover, Th2‐mediated cytokine expression, mast cell hyperplasia and destruction of the skin barrier were improved by boehmite treatment. Overall, we demonstrated that boehmite may potentially protect against AD.     

Papuloerythroderma‐like cutaneous involvement of a CD62L− subclone of T‐cell prolymphocytic leukemia

We report the case of an 88‐year‐old Japanese man with erythrodermic involvement of T‐cell prolymphocytic leukemia (T‐PLL). He had a history of pharyngeal diffuse large B‐cell lymphoma successfully treated with polychemotherapy including cyclophosphamide and epirubicin, 6 years before the current illness. He presented with numerous reddish, coalescing, flat‐topped papules on the trunk and extremities, sparing the skin folds of the abdomen, the features of which mimicked those of papuloerythroderma. Immunohistochemistry showed perivascular and epidermotropic infiltration of CD3⁺ CD4⁺ T cells in the cutaneous lesion. However, flow cytometric analysis revealed that the skin infiltrating T cells were negative for surface CD4, and that CD3⁺ CD4^? CD8^? cells made up 92% of the T‐cell fraction of peripheral blood. The circulating atypical T cells had a round or oval nucleus and prominent nucleoli, and the deletion of chromosomes 6q, 13 and 17. These cytological profiles were consistent with those of T‐PLL and distinct from those of Sézary cells. The same T‐cell clone was detected in the cutaneous lesion and peripheral blood, but the expression of CD62L was absent in the skin infiltrates and present in the circulating cells. No specific mutation was detected in STAT3 or STAT5B. Although low‐dose oral etoposide had a beneficial effect on the skin rash, a fatal crisis of marked leukocytosis (169 × 10³/μL) occurred 19 months after the illness onset. CD62L‐leukemic cells of T‐PLL may infiltrate the skin to form papuloerythroderma‐like cutaneous lesions.