Distribution of IL-18 and IL-18 receptor in human skin: various forms of IL-18 are produced in keratinocytes

Abstract Human interleukin-18 (IL-18) enhances IL-12-mediated IFN-γ production by lymphocytes and Fas/ perforin-mediated cytolysis by NK cells. IL-18 is synthesized as a 24 kDa proform, and the proform is processed in the cytoplasm into an 18 kDa mature form. Active and precursor forms of IL-18 have been detected in immunocompetent cells, and active IL-18 exerts its functions through its receptor. We sought to determine which human skin cells are responsible for production of IL-18 and which express its receptor. Monoclonal antibodies against human IL-18 and polyclonal antibody against IL-18 receptor were provided for this analysis. Formalin-embedded and frozen sections of human epidermis were analyzed by immunoperoxidase and immunofluorescence staining. IL-18 was detected in all living cell layers of the epidermis, hair follicles, arrectores pilorum, eccrine ducts and endothelial cells. IL-18 was localized in the cytoplasm of cells in living epidermal cell layers. In contrast, IL-18 receptor was mainly detected in keratinocytes and expressed in the cell periphery in living cell layers. Since keratinocytes were the main source of IL-18 and its receptor, cultured human keratinocytes were further analyzed by immunoblotting. IL-18 receptor was identified as an 80 kDa single band. The mature 18 kDa and precursor 24 kDa forms of IL-18 were detected by our monoclonal antibody (mAb) 21 and mAb 132, respectively, while only the 18 kDa form was detected by a commercial mAb, 125-2H. Cultured keratinocytes showed positive granular staining for IL-18 in the cytoplasm and positive staining for IL-18 receptor mainly in the cell periphery. Our findings indicate that mature IL-18, precursor IL-18 and IL-18 receptor are simultaneously expressed with different localizations by human epidermal keratinocytes. Keratinocytes might be activated by their own IL-18 in an autocrine or paracrine fashion. Received: 14 August 2000 / Revised: 10 December 2000 / Accepted: 24 April 2001     

白介素8及其受体CXCR2在银屑病角质形成细胞中的表达

目的 观察白介素8（IL－8）及基受体CXCR2在银屑病角质形成细胞中的表达及在银屑病的临床及病理表现中的作用。方法 培养银屑病患者皮损处角质形成细胞，通过微孔小室实验检测其上清液的趋化功能，通过酶联免疫吸附法（ELISA）检测上清液中IL－8的表达，通过流式细胞仪分析银屑病患者皮损处角质形成细胞的趋化因子受体CXCR2的表达。结果 银屑病患者皮损处角质形成细胞分泌上清液对中性粒细胞的趋化能力明显强于正常对照组，其分泌的IL－8水平也高于正常人，皮损处角质形成细胞CXCR2的表达也明显强于正常对照组。结论 银屑病患者皮损局部表面出的角质形成细胞高度增殖与角质形成细胞高分泌、高表达具有促增殖作用的IL－8及其受体CXCR2有关，同时皮损局部大量的炎性细胞的浸润部分可能是由于角质形成细胞高分泌具有趋化能力的IL－8，它们可能在银屑病的发生、发展中起着重要作用。     

In situ expression of messenger RNA of interleukin-1 and interleukin-6 in psoriasis: interleukin-6 involved in formation of psoriatic lesions

Summary Psoriasis is a disease of abnormal proliferation and differentiation of epidermal cells. Several cytokines released by keratinocytes are implicated as factors responsible for this pathological condition of the epidermis. In order to elucidate the role of these cytokines in psoriasis, messenger RNA (mRNA) expression of interleukin-1 (IL-1) and IL-6 in psoriatic epidermis was investigated using biotin-labelled complementary DNA (cDNA) of the cytokines. Messenger RNA of IL-1 was weakly detected in some normal healthy epidermis specimens and more strongly in all the perilesional uninvolved psoriatic epidermis specimens. It was also expressed in the transitional zone between uninvolved and fully developed psoriatic skin, but was not expressed in lesional skin. In contrast, IL-6 mRNA was rarely expressed in normal healthy epidermis, but was expressed in perilesional uninvolved psoriatic epidermis, in the transitional zone and in the fully developed lesional epidermis, with the maximum intensity in the transitional zone. Expression of mRNA of IL-6 receptor showed a similar tendency to that of IL-6. It was expressed in psoriatic epidermis, most strongly in the transitional zone, but not in normal healthy epidermis. IL-6 was demonstrated immunohistochemically in psoriatic epidermis, but IL-6 receptor was demonstrated only in the transitional zone. Thus IL-6 and its receptor expression correlated well with the formation of psoriatic lesions where IL-1 may initiate their expression. IL-6 may play an important role in the pathogenesis of psoriasis.     

The effect of IFN-γ on healthy and psoriatic keratinocytes in a skin equivalent model is influenced by the source of the keratinocytes and by their interactions with fibroblasts

We investigated the effect of interferon-gamma (IFN-γ) on skin equivalents. Keratinocytes from involved and uninvolved skin from psoriatic subjects and from healthy subjects were grown on preproduced dermal equivalents (DE) containing fibroblasts from healthy skin or psoriatic lesions. Healthy keratinocytes were added when the dermal equivalents were either 22 days (DE(22)) or 37 days old (DE(37)) and psoriatic keratinocytes when the dermal equivalents were 28–52 days old (DE(28–52)). The skin equivalents were cultured for 11 days in a serum-free medium, and then with or without 500 U/ml IFN-γ for 6 days. The expression of markers associated with differentiation and proliferation were investigated by immunohistochemistry. Differentiation was assessed by computed scores for the expression of cytokeratin 16, involucrin, filaggrin and the receptor for epidermal growth factor. The differentiating effect of IFN-γ on healthy keratinocytes grown on DE(37) was significantly stronger than on psoriatic keratinocytes grown on DE(28–52). In healthy keratinocytes, the differentiating effect of IFN-γ was significantly stronger in skin equivalents containing DE(37) than in those containing DE(22). The proliferation rate, i.e. the percentage of Ki-67 ⁺ keratinocytes in the basal layer, was studied in healthy keratinocytes grown on DE(22). In these cultures IFN-γ increased the proliferation rate in the presence of psoriatic fibroblasts but not in the presence of healthy fibroblasts. HLA-DR expression was induced only in healthy keratinocytes grown on DE(22). We conclude that the influence of IFN-γ on epidermal differentiation and proliferation is influenced by the origins of both the keratinocytes and the fibroblasts. These findings suggest that interactions between keratinocytes and fibroblasts might be involved in the pathogenesis of psoriasis. Received: 12 March 1996     

Interleukin 1 alpha and beta in psoriatic skin: enzymoimmunoassay, immunoblot studies and effect of systemic retinoids

Interleukin 1 alpha (IL-1 alpha) and interleukin 1 beta (IL-1 beta) proteins were studied by enzymoimmunoassay (EIA) and Immunoblot analysis in the 10,000 g supernatant of normal and psoriatic (lesional and nonlesional) human skin specimens. By EIA IL-1 alpha was the principal form detected in all the specimens, which contrasts with the predominance of IL-1 beta in human blood monocytes. In psoriatic plaques relatively less IL-1 alpha and more IL-1 beta were detected. On Immunoblot analysis the mature form (17 kD) was not detected in normal skin, which showed only 52-kD immunoreactive forms. In contrast the 17-kD form was found in psoriatic skin. This indicates either a distinct processing of IL-1 molecules or a contribution of inflammatory cells infiltration to the IL-1 pool in psoriatic plaques. During systemic retinoids therapy the amount of both IL-1 species decreased in lesional and nonlesional psoriatic skin.     

寻常性银屑病皮损及非皮损中IL-23(p19/p40)和IL-12(p35/p40)mRNA的表达

目的检测寻常性银屑病患者皮损及非皮损处IL-23(p19/p40)和IL-12(p35/p40)mRNA表达,探讨其临床意义。方法采用逆转录-聚合酶链反应(RT-PCR)检测寻常性银屑病患者皮损、非皮损处及正常人皮肤中IL-23(p19/p40)和IL-12(p35/p40)mRNA的表达水平。结果寻常性银屑病患者皮损中IL-23p19及p40(IL-23/IL-12)mRNA的表达均高于非皮损组织和正常皮肤组织(P<0.05),且非皮损处高于正常对照组,差异有显著性(P<0.05);IL-12p35mRNA在银屑病皮损处、非皮损处和正常对照中表达水平差异无显著性(P>0.05)。结论在寻常性银屑病发病过程中,IL-23可能发挥较IL-12更重要的作用。     

银屑病皮损中IL—1受体拮抗物的蛋白和mRNA表达

采用抗人ＩＬ－１ｒａ单抗和地高辛标记的ＲＮＡ探针,在银屑病破损和正常人皮肤切片上进行ＡＰＡＡＰ染色和原位杂交,检测并比较ＩＬ－Ｉｒａ的蛋白表达和ｍＲＮＡ转录水平。结果发现,正常人皮肤中ＩＬ－１ｒａ蛋白染色阴性,ＩＬ－１ｒａｍＲＮＡｗ信号微弱;相比之下,银屑病皮损中ＩＬ－１ｒａ蛋白和ｍＲＮＡ的表达均异常增高,统计学比较相差显著。上述结果显示：ＩＬ－１ｒａ在银屑病皮损中增强表达,提示银屑病病理过程中Ｉ     

Suppression of IL-17A-induced CCL20 production by cytokine inducible SH2-containing protein 1 in epidermal keratinocytes

BackgroundLesions of atopic dermatitis have fewer Th17 cells than those of psoriasis, resulting in frequent skin infections. Expression of CCL20, a chemokine that is important for recruiting Th17 cells, is suppressed in the lesions of atopic dermatitis. We previously reported that IL-4 induces the expression of cytokine-inducible SH2-containing protein 1 (CIS1), a member of the CIS/SOCS family, in epidermal keratinocytes.ObjectiveTo investigate whether CIS1 influences CCL20 production in epidermal keratinocytes.MethodsExpression of CIS1 was examined in atopic dermatitis skin and in cultured keratinocytes. The effects of overexpression of CIS1 on CCL20 production by IL-17A, and on signaling pathways inhibited by CIS1, were assessed in vitro.ResultsExpression of CIS1 was enhanced in the basal layer of the lesional epidermis of skin with atopic dermatitis. When CIS1 was expressed in keratinocytes using adenoviral vectors, IL-17A-induced CCL20 expression, but not HBD2 or S100A7 expression, was significantly suppressed. TNF-α/IL-1-induced CCL20 production was not altered by CIS1. Overexpression of CIS1 attenuated IL-17A-induced ERK phosphorylation. ERK phosphorylation was mediated by the Act1 and Src family kinase pathways. CIS1 overexpression suppressed Src phosphorylation. Among the Src family kinases, the Yes kinase may have an important role because knockdown of Yes in epidermal keratinocytes resulted in suppression of ERK phosphorylation and CCL20 mRNA expression by IL-17A.ConclusionCIS1 induced by Th2 cytokines has the ability to change the response of epidermal keratinocytes to IL-17A by suppression of Src family kinases.     

IL-18在寻常型银屑病患者皮损中的表达及其意义

目的：探讨白细胞介素18（Interleukin 18,IL-18）在寻常型银屑病患者皮损中的表达及其意义。方法：采用荧光定量聚合酶链反应法测定20例寻常型银屑病患者皮损中和17例正常对照者皮肤中IL-18mRNA的定量表达,采用直线相关回归分析方法分析IL-18mRNA的表达与寻常型银屑病病情严重指数（PASI）的关系。结果：寻常型银屑病皮损内IL-18mRNA明显高于正常对照者皮肤中的表达水平（U=-4．7698,P〈0．001）。寻常型银屑病皮损内IL-18mRNA的表达与患者PASI呈显著的正相关（r=0．96258,P〈0．001）。结论：IL-18的表达上调可能与寻常型银屑病的发生、发展有关。     

Psoriatic skin reveals the in vivo presence of an epidermal IL-1 inhibitor

Summary Production of inhibitor(s) of IL-1 activity can be induced in keratinocytes by exposure to UVB. We describe in this study the characterization of an endogenous constitutively expressed IL-1 inhibitor which is present in extracts of human psoriatic epidermal keratome biopsies. Size-fractionated extracts of normal human epidermis did not reveal IL-1 inhibitory factor(s) activity in normal epidermis. Psoriatic epidermal extracts, however, contained virtually no IL-1 bioactivity and inhibited the activity of recombinant human IL-1. This IL-1 inhibitor has a molecular weight of approximately 30 kDa and a pI of 5.3, as revealed by fast protein liquid chromatography size fractionation and chromatofocusing of psoriatic epidermal extracts. IL-1 inhibitory activity was not blocked by neutralizing anti-TGF monoclonal antibody. It did not have any inhibitory effect upon normal cellular proliferation but could block the IL-1 induction of IL-2 production by LBRM.33 cells as late as 4 h after exposure of LBRM.33 cells to IL-1. Thus, in vivo human psoriatic epidermis expresses an IL-1 inhibitor that specifically inhibits IL-1 activity but which appears distinct from previously described UV-induced epidermal IL-1 inhibitory activity or TGF.     

Osteopontin: a new emerging role in psoriasis

Osteopontin (OPN) is a phosphorylated acidic glycoprotein produced by cells of the immune system, epithelial tissue, smooth muscle cells, osteoblasts, and tumor cells. OPN interacts with integrins and CD44 to enhance Th1 and inhibit Th2 cytokine expression. The involvement of this molecule in the onset of psoriasis has not previously been studied. Here, we demonstrate that OPN is expressed in peripheral blood mononuclear cells and in skin biopsies of psoriatic patients. The study was conducted on 30 patients affected with plaque psoriasis, and on 11 healthy donors. Two blood samples and two skin samples from patients affected with atopic dermatitis were used as control for Th2 typical inflammatory skin disease. The analysis of IL-1β, IFN-γ, ΤΝF-α, IL-8, and ICAM-1 showed the characteristic Th1 pattern in all the psoriatic blood and skin samples analyzed. This study offers an opportunity for understanding inflammation in psoriasis and supports the hypothesis that OPN could represent a potential target for therapeutic intervention in psoriatic patients.     

Mast cell density and IL-8 expression in nonlesional and lesional psoriatic skin

BACKGROUND: An important cellular aberration at sites of psoriatic inflammation is an increase in the number of dermal mast cells. Being multifactorial immune effector cells, it is believed that mast cells play an essential role in perpetuating the inflammatory process of psoriasis. However, factors responsible for the infiltration and accumulation of mast cells in psoriatic lesions are largely unknown. Recent studies have demonstrated that Interleukin-8 (IL-8) exerts strong chemotactic effects on mast cells in vitro. Overexpression of IL-8 has also been reported in psoriatic lesions. In this study, we have found a correlation between the expression of IL-8 and dermal mast cell density in lesional psoriatic skin as compared to nonlesional psoriatic skin. METHODS: Four-mm punch biopsies were taken from 14 psoriatic patients and eight healthy volunteers. Using immunohistochemical techniques, 8 microm sections of lesional psoriatic, nonlesional psoriatic, and normal control samples were evaluated for dermal mast cell density and the density of IL-8 expressing keratinocytes. RESULTS: It was found that dermal mast cell density in lesional psoriatic, nonlesional psoriatic, and normal skin was 105.4 +/- 71.2, 42.3 +/- 30.1, and 47.5 +/- 32.5 mast cells/mm(2), respectively. IL-8+ keratinocyte density in lesional psoriatic, non lesional psoriatic, and normal skin was 171.5 +/- 67.1, 25.4 +/- 14.9 and 20.6 +/- 8.7 IL-8+ Keratinocytes/mm(2), respectively. CONCLUSIONS: The results of this study suggest that increased levels of IL-8 in the keratinocytes of psoriatic plaques play a contributing role in the migration of mast cells to lesion sites.     

Lithium and psoriasis: cytokine modulation of cultured lymphocytes and psoriatic keratinocytes by lithium

The predominant cutaneous side effect of lithium is the exacerbation or aggravation of psoriasis, but the pathogenesis is still unclear. The hyperproliferation of keratinocytes and a dense lesional infiltrate of mononuclear cells are the hallmarks of psoriatic skin lesions. Interactions between keratinocytes and T cells are thought to be one reason for an increased secretion of proinflammatory cytokines and growth factors. To investigate whether lithium influences cytokines of the ‘psoriatic cytokine network’, we established a coculture model with keratinocytes from psoriatic patients and from healthy controls cultured with HUT 78 lymphocytes and measured the cytokine levels of Il-2, Il-6, Il-8, IFNγ and TGFα in the culture supernatants after treatment with lithium. Il-6 levels were slightly elevated in the supernatants obtained from psoriatic and control keratinocyte cultures after lithium treatment, but IFNγ and Il-2 levels were elevated only in the lithium-treated cocultures with psoriatic keratinocytes. In contrast, these two cytokines were not affected by lithium in HUT 78 monocultures or in cocultures with normal epidermal cells. We also found slightly elevated TGFα levels in lithium-treated psoriatic cocultures but not in control cultures. We therefore demonstrated that lithium influences the cell communication of psoriatic keratinocytes with HUT 78 lymphocytes by triggering the secretion of TGFα, Il-2 and, massively, IFNγ. It seems possible that lithium also influences similar parts of the psoriatic cytokine network in vivo. Received: 19 May 1995     

Interleukin 2, soluble interleukin 2 receptor,and interferon-γ in the suction blister fluids from psoriatic skin

Summary Psoriasis represents inflammatory skin disorders characterized by significant changes in cellular immunity, particularly exhibiting alterations in T lymphocyte-related functions. Early psoriatic lesions have been reported to show an infiltration of activated helper T cells. Elevated levels of interleukin 2 (IL-2), IL-2 receptor (IL-2R), and interferon- (INF-) are associated with an early activation of T cells. To examine local activation of T cells in psoriatic skin, the amounts of activated T cell products, IL-2, secretory form of IL-2R (sIL-2R) and INF- were measured in the fluids of suction blisters raised on psoriatic skin. sIL-2R levels were significantly elevated in the suction blister fluids raised on psoriatic involved skin compared with those on normal and psoriatic uninvolved skin. On the other hand, neither IL-2 or IFN- was detected in the suction blister fluids either from normal, psoriatic uninvolved, or involved skin. However, we could detect IFN- and IL-2 in the psoriatic scale extracts. Although we failed to detect IL-2 and IFN- in the suction blister fluids, the increased levels of sIL-2R in the suction blister fluids from the psoriatic lesional skin indicate local activation of T cells in psoriatic lesional skin.This work was supported by grants-in-aid for scientific research nos. 01570558 and 63480243 from the Ministry of Education, Science and Culture, Japan and a grant from the Lydia O'Leary Memorial Foundation     

T cells in psoriatic lesional skin that survive conventional therapy with NB-UVB radiation display reduced IFN-γ expression

The type 1 T cell-derived cytokine interferon (IFN-) is overexpressed in psoriatic lesional skin. Recently, we have shown that a single high erythemal dose of broad-band ultraviolet B (UVB) irradiation reduces type 1 and favors type 2, i.e. interleukin-4 (IL-4), cytokine expression in normal and psoriatic skin. In this study, we wanted to see whether conventional narrow-band UVB (NB-UVB) therapy (i.e. repeated exposure to nonerythemal doses) also affects type 1/type 2 cytokine expression of T cells present in chronic plaque type psoriatic lesions. Staining of cryostat sections showed decreased expression of both IFN- and IL-4 in situ after NB-UVB therapy. CD4⁺ dermal T cell lines, derived from psoriatic lesional skin, displayed significantly decreased intracellular IFN- expression during and after NB-UVB therapy as compared to pretreatment values. Intracellular IL-4 expression was increased in most patients after therapy. Analysis of the supernatants of these stimulated dermal T cells revealed that IFN- production decreased significantly following NB-UVB therapy, whereas IL-4 expression increased in the T cell supernatants from most patients, confirming the intracellular determinations. In addition, IL-10 and transforming growth factor- levels in the supernatants appeared to be increased in the majority of patients following UVB therapy. Apart from the well-known killing effect of UVB on T cells, our results show that the improvement in psoriatic skin following NB-UVB therapy is also due to a reduced capacity of the surviving dermal T cells to express the proinflammatory cytokine IFN-.     

Reappraisal of in situ immunophenotypic analysis of psoriasis skin: interaction of activated HLA-DR+ immunocompetent cells and endothelial cells is a major feature of psoriatic lesions

Psoriasis is an inflammatory skin disease of unknown aetiology. Many observations indicate that T cells play an important role in the pathogenesis of the disease. Upregulation of MHC class-II molecules on immunocompetent cells, endothelial cells and keratinocytes on lesional psoriatic skin has been regarded as a hallmark of the disease. However, there is some controversy in the literature regarding the cell types expressing class-II molecules and there is limited information about the presence of immune cells other than T cells and antigen presenting cells in the cellular infiltrates of psoriatic skin. We therefore reinvestigated the subject using immunocytochemical single and multiple staining techniques. In agreement with earlier reports, our studies showed that the cellular infiltrates in lesional skin consist largely of HLA-DR⁺/IL-2R⁺ T cells, HLA-DR⁺/CD1a⁺ Langerhans cells, and HLA-DR⁺/CD68⁺ macrophages. We found increased HLA-DR expression mostly on immunocompetent cells and endothelial cells, but no prominent HLA-DR expression on keratinocytes in lesional psoriatic skin. Upregulation of HLA-DR on endothelial cells and in mononuclear infiltrates was also evident in the non-lesional skin of psoriatic patients as compared with normal controls. B cells and natural killer cells were also found in the cellular infiltrates in lesional psoriatic skin. In spite of the presence of a large amount of activated T cells in the epidermis, we found that HLA-DR expression on keratinocytes was not a major feature of psoriatic skin.