Up-regulation of macrophage inflammatory protein-3 alpha/CCL20 and CC chemokine receptor 6 in psoriasis

Autoimmunity plays a key role in the immunopathogenesis of psoriasis; however, little is known about the recruitment of pathogenic cells to skin lesions. We report here that the CC chemokine, macrophage inflammatory protein-3 alpha, recently renamed CCL20, and its receptor CCR6 are markedly up-regulated in psoriasis. CCL20-expressing keratinocytes colocalize with skin-infiltrating T cells in lesional psoriatic skin. PBMCs derived from psoriatic patients show significantly increased CCR6 mRNA levels. Moreover, skin-homing CLA+ memory T cells express high levels of surface CCR6. Furthermore, the expression of CCR6 mRNA is 100- to 1000-fold higher on sorted CLA+ memory T cells than other chemokine receptors, including CXCR1, CXCR2, CXCR3, CCR2, CCR3, and CCR5. In vitro, CCL20 attracted skin-homing CLA+ T cells of both normal and psoriatic donors; however, psoriatic lymphocytes responded to lower concentrations of chemokine and showed higher chemotactic responses. Using ELISA as well as real-time quantitative PCR, we show that cultured primary keratinocytes, dermal fibroblasts, and dermal microvascular endothelial and dendritic cells are major sources of CCL20, and that the expression of this chemokine can be induced by proinflammatory mediators such as TNF-alpha/IL-1 beta, CD40 ligand, IFN-gamma, or IL-17. Taken together, these findings strongly suggest that CCL20/CCR6 may play a role in the recruitment of T cells to lesional psoriatic skin.     

In vitro and in situ expression of IL-23 by keratinocytes in healthy skin and psoriasis lesions: enhanced expression in psoriatic skin

Keratinocytes contribute to cutaneous immune responses through the expression of cytokines. We investigated whether human keratinocytes can express IL-23, a newly defined IFN-gamma-inducing cytokine composed of a unique p19 subunit and a p40 subunit shared with IL-12. Cultured keratinocytes from normal and lesional psoriatic skin were found to express constitutively mRNA for both subunits of IL-23. Low but significant levels of the heterodimeric IL-23 protein could be detected in cell lysates and supernatants from stimulated keratinocytes by immunoblotting and ELISA. Functional analysis showed that these low levels of keratinocyte-derived IL-23 were sufficient to enhance the IFN-gamma production by memory T cells. Immunostaining of skin sections confirmed expression of both subunits of IL-23 by keratinocytes in situ and also revealed expression of this cytokine in the dermal compartment. IL-23 expression was significantly higher in psoriatic lesional skin, compared with normal and psoriatic nonlesional skin. The immunostained preparations of cultured cells and IL-23 levels in culture supernatants did not show any difference between normal and psoriatic keratinocytes indicating no intrinsic aberration of IL-23 expression in keratinocytes from psoriatic skin. Double staining of cytospin preparations demonstrated that IL-23 p19 is also expressed by epidermal Langerhans cells, dermal dendritic cells, and macrophages. Psoriasis is a chronic inflammatory skin disease mediated by IFN-gamma-expressing type 1 memory T cells. As IL-23 is important to activate memory T cells to produce IFN-gamma, its augmented expression of IL-23 by keratinocytes and cutaneous APC may contribute to the perpetuation of the inflammation process in this disease.     

Human beta-defensin-2.

Human beta-defensin-2 (HBD-2) is a cysteine-rich cationic low molecular weight antimicrobial peptide recently discovered in psoriatic lesional skin. It is produced by a number of epithelial cells and exhibits potent antimicrobial activity against Gram-negative bacteria and Candida, but not Gram-positive Staphylococcus aureus. HBD-2 represents the first human defensin that is produced following stimulation of epithelial cells by contact with microorganisms such as Pseudomonas aeruginosa or cytokines such as TNF-alpha and IL-1 beta. The HBD-2 gene and protein are locally expressed in keratinocytes associated with inflammatory skin lesions such as psoriasis as well as in the infected lung epithelia of patients with cystic fibrosis. It is intriguing to speculate that HBD-2 is a dynamic component of the local epithelial defense system of the skin and respiratory tract having a role to protect surfaces from infection, and providing a possible reason why skin and lung infections with Gram-negative bacteria are rather rare.     

CD57 Expression and Cytokine Production by T Cells in Lesional and Unaffected Skin from Patients with Psoriasis

Background

The immunopathogenic mechanisms leading to psoriasis remain unresolved. CD57 is a marker of replicative inability and immunosenescence on CD8+ T cells and the proportion of CD57 expressing CD8+ T cells is increased in a number of inflammatory conditions.

Methodology

We examined the expression of CD57 on T cells in the skin of patients affected with psoriasis, comparing lesional and unaffected skin. We also assessed functionality of the T cells by evaluating the secretion of several inflammatory cytokines (IL-17A, IFN-gamma, IL-2, IL-33, TNF-alpha, IL-21, IL-22, and IL-27), from cell-sorted purified CD4+ and CD8+ T cells isolated from lesional and unaffected skin biopsies of psoriasis patients.

Principal Findings

We observed that the frequency of CD57+CD4+ and CD57+CD8+ T cells was significantly higher in unaffected skin of psoriasis patients compared to lesional skin. Sorted CD4+ T cells from psoriatic lesional skin produced higher levels of IL-17A, IL-22, and IFN-gamma compared to unaffected skin, while sorted CD8+ T cells from lesional skin produced higher levels of IL-17, IL-22, IFN-gamma, TNF-alpha, and IL-2 compared to unaffected skin.

Conclusions/Significance

These findings suggest that T cells in unaffected skin from psoriasis patients exhibit a phenotype compatible with replicative inability. As they have a lower replicative capacity, CD57+ T cells are less frequent in lesional tissue due to the high cellular turnover.     

Cytokine milieu of atopic dermatitis,as compared to psoriasis,skin prevents induction of innate immune response genes

Atopic dermatitis (AD) and psoriasis are the two most common chronic skin diseases. However patients with AD, but not psoriasis, suffer from frequent skin infections. To understand the molecular basis for this phenomenon, skin biopsies from AD and psoriasis patients were analyzed using GeneChip microarrays. The expression of innate immune response genes, human beta defensin (HBD)-2, IL-8, and inducible NO synthetase (iNOS) was found to be decreased in AD, as compared with psoriasis, skin (HBD-2, p = 0.00021; IL-8, p = 0.044; iNOS, p = 0.016). Decreased expression of the novel antimicrobial peptide, HBD-3, was demonstrated at the mRNA level by real-time PCR (p = 0.0002) and at the protein level by immunohistochemistry (p = 0.0005). By real-time PCR, our data confirmed that AD, as compared with psoriasis, is associated with elevated skin production of Th2 cytokines and low levels of proinflammatory cytokines such as TNF-alpha, IFN-gamma, and IL-1beta. Because HBD-2, IL-8, and iNOS are known to be inhibited by Th2 cytokines, we examined the effects of IL-4 and IL-13 on HBD-3 expression in keratinocyte culture in vitro. We found that IL-13 and IL-4 inhibited TNF-alpha- and IFN-gamma-induced HBD-3 production. These studies indicate that decreased expression of a constellation of antimicrobial genes occurs as the result of local up-regulation of Th2 cytokines and the lack of elevated amounts of TNF-alpha and IFN-gamma under inflammatory conditions in AD skin. These observations could explain the increased susceptibility of AD skin to microorganisms, and suggest a new fundamental rule that may explain the mechanism for frequent infection in other Th2 cytokine-mediated diseases.     

Genetically predetermined limitation in HaCaT cells that affects their ability to serve as an experimental model of psoriasis

Proinflammatory cytokines TNF, IFNG, and IL17 play an important role in eruption of psoriasis. The activation of epidermal keratinocytes with the named cytokines alters their terminal differentiation program and causes their hyperproliferation in the diseased skin. HaCaT cells, which are immortalized human keratinocytes, are often used as a cellular model of psoriasis. The aim of this study was to evaluate changes in gene expression and the proliferation rates in cultured HaCaT cells treated with TNF, IFNG, and IL17. We found that HaCaT cells decrease their proliferation rate in response to either IL17 or a combination TNF and IFNG. The analysis of microarray data discovered a group of 12 genes, which were downregulated in HaCaT after treatments with the named cytokines and upregulated in psoriatic lesional skin. Eight genes were important for DNA replication and they also contributed to two larger networks that regulated cell progression through the cell cycle. We conclude that HaCaT cells have a sufficient limitation as a cellular model of psoriasis due to their treatment with proinflammatory cytokines, namely TNF, IFNG, and IL17 does not increase their proliferation rate. Thus, the studies of psoriasis based on HaCaT cells as an experimental model shall take in account this important phenomenon.     

Inhibition of keratinocyte apoptosis by IL-15: a new parameter in the pathogenesis of psoriasis?

Keratinocytes (KC) are important source of and targets for several cytokines. Although KC express IL-15 mRNA, the functional effects of IL-15 on these epithelial cells remain to be dissected. Investigating primary human foreskin KC and HaCaT cells, we show here by semiquantitative RT-PCR and flow cytometric analysis that both translate IL-15 and IL-15R mRNA and express IL-15 and IL-15Ralpha protein on the cell surface, suggesting that human KC can employ IL-15 for juxtacrine signaling. While IL-15 exerted no significant effect on KC proliferation and IL-6 or IL-8 secretion, IL-15 inhibited both anti-Fas and methylcellulose-induced KC apoptosis in vitro. This is in line with the recognized potent anti-apoptotic effects of IL-15. IL-2, whose receptor shares two components with the IL-15R, failed to inhibit KC apoptosis. Together with the role of IL-15 in sustaining chronic immune reactions, this invited the question of whether a reduction of KC apoptosis by IL-15 may be involved in the pathogenesis of psoriasis, a chronic hyperproliferative inflammatory skin disease characterized by abnormally low KC apoptosis in the epidermis. Remarkably, compared with nonlesional psoriatic skin and skin of healthy volunteers, lesional psoriatic epidermis showed high IL-15 protein expression in the epidermis and enhanced binding activity for IL-15. Therefore, antagonizing the inhibitory effects of IL-15 on KC apoptosis deserves exploration as a novel therapeutic strategy in psoriasis management.     

Crosstalk between keratinocytes and T lymphocytes via Fas/Fas ligand interaction: modulation by cytokines.

Apoptosis mediated by Fas/FasL interaction plays an important role during many inflammatory skin disorders. To estimate whether the expression of FasL, the ligand for Fas, might be regulated by cytokines we stimulated primary human keratinocytes with several pro- and anti-inflammatory cytokines. Keratinocytes cultured to subconfluence expressed FasL constitutively. Cells stimulated with the proinflammatory cytokines IL-1beta, TNF-alpha, IFN-gamma, and IL-15, respectively, increased significantly their intracellular as well as cell surface-bound FasL expression in a time- and dose-dependent manner. This cytokine-induced FasL expression was dependent on new protein synthesis. Despite enhanced expression of cell surface-bound FasL, no release of soluble FasL was measured in the cell supernatants determined by ELISA. Stimulation of the cells with IL-6, IL-10, IL-12, TGF-beta1, and GM-CSF did not modulate the constitutive FasL expression, but IFN-gamma-mediated FasL up-regulation was significantly diminished by IL-10 and TGF-beta1, respectively. Up-regulation of FasL on IFN-gamma-stimulated keratinocytes led to increased apoptosis within monolayers cultured for 48 h. Moreover, coculture experiments performed with Fas+ Jurkat T cells revealed that enhanced FasL expression on IFN-gamma-stimulated keratinocytes induced apoptosis in cocultured T cells, demonstrating that up-regulated FasL was functionally active. In summary, our data suggest the important regulatory role of cytokine-controlled Fas/FasL interaction in the cross-talk between keratinocytes and skin-infiltrating T cells for maintenance of homeostasis in inflammatory skin processes.     

Regulation of Involucrin in Psoriatic Epidermal Keratinocytes: The Roles of ERK1/2 and GSK-3β

Psoriasis, a common inflammatory skin disease, is characterized by epidermal hyperplasia, abnormal differentiation, angiogenesis, immune activation, and inflammation. Involucrin is an early terminal differentiation marker of epidermal keratinocytes. In this study, we determined the immunolocalization of involucrin in psoriatic lesions and normal skin of individuals without psoriasis by means of immunofluorescence (IF) assay. Furthermore, the regulation of involucrin by interleukin (IL)-13, IL-17A, endothelin (ET)-1, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ was investigated by Western blot. Extracellular regulate protein kinases 1/2 (ERK1/2) and glycogen syntheses kinase-3β (GSK-3β) inhibitors were also included to define the roles of these signals in the production of involucrin in both psoriatic and normal keratinocytes. In psoriatic lesional skin, involucrin was detected in the stratum spinosum, but not in the basal or the cornified layer. In normal skin, involucrin was restricted to the granular layer and the upper stratum spinosum. IL-13, IL-17A, ET-1, TNF-α, and IFN-γ up-regulate expression of involucrin in both psoriatic and normal keratinocytes. However, this effect was abolished by ERK1/2 and GSK-3β inhibitors. In conclusion, involucrin is up-regulated in psoriatic keratinocytes. IL-13, IL-17A, ET-1, TNF-α, and IFN-γ could increase involucrin protein levels in psoriatic and normal keratinocytes. The ERK1/2 and GSK-3β signaling pathways may play positive roles in regulating epidermal differentiation as observed in psoriasis.     

Arginase-1 overexpression induces cationic amino acid transporter-1 in psoriasis

Regulated uptake of extracellular l-arginine by cationic amino acid transporters (CATs) is required for inducible nitric oxide synthase and arginase activity. Both enzymes were recently recognized as important in the pathophysiology of psoriasis because of their contribution to epidermal hyperproliferation. We here characterize the expression pattern of CATs in psoriatic skin compared to healthy skin. CAT-1 mRNA expression was strongly upregulated in lesional and nonlesional areas of psoriatic skin compared to healthy skin, whereas expression of CAT-2A and the inducible isoform CAT-2B was unaltered in psoriatic skin. Furthermore, we tested the hypothesis that arginase-1 overexpression regulates CAT expression via intracellular l-arginine concentration. In in vitro experiments with arginase-1 overexpressing HaCaT cells, CAT-1 mRNA expression was increased. Likewise, this occurs in l-arginine-starved HaCaT cells. Both CAT-2 isoforms were not affected. Arginase-1 overexpression limits the synthesis of NO at physiological, but not supraphysiological, l-arginine levels. Plasma l-arginine concentration was diminished in psoriasis patients and the arginase product l-ornithine was significantly increased compared to healthy controls. In summary, arginase-1 overexpression leads to upregulated CAT-1 expression in psoriatic skin, which is due to lowered intracellular l-arginine levels and limits NO synthesis at physiological l-arginine concentrations.     

Cytokines in skin lesions of psoriasis

Cytokine levels were compared in aqueous extracts of stratum corneum from psoriatic lesions and normal heel. Samples from heel contained high levels of interleukin-1 alpha (IL-1 alpha) and beta measured in immunoassays, although only the IL-1 alpha was biologically active. No other cytokines could be detected in heel samples. Interleukin-1 (IL-1) levels were dramatically reduced in lesional samples. A neutrophil chemoattractant was found in all lesional extracts, and was demonstrated to be mainly interleukin-8 (IL-8) using a specific neutralizing antiserum. Tumor necrosis factor alpha (TNF-alpha) and beta (TNF-beta), and interferon alpha (IFN-alpha) and gamma (IFN-gamma) were detected in lesional extracts using immunoassays, however, no equivalent biological activities could be detected. Interleukins 2 (IL-2), 4 (IL-4), and 6 (IL-6), granulocyte and granulocyte/macrophage colony stimulating factor (GM-CSF), could not be detected in any samples. IL-8 is therefore the only biologically active cytokine shown in this study to be elevated in psoriatic lesional extracts, and may therefore play a role in the pathogenesis of the disease.