Intrinsic alterations of pro-inflammatory mediators in unstimulated and TLR-2 stimulated keratinocytes from atopic dermatitis patients

Abstract: In many patients with atopic dermatitis (AD), the disease is complicated by their enhanced susceptibility to bacterial skin infections, especially with Staphylococcus aureus (S. aureus). Resistance to bacterial skin infections, e.g. S. aureus, is based on the function of intact innate immune mechanisms in the epidermis, mainly provided by keratinocytes. Toll‐like receptor (TLR)‐2 recognizes components of S. aureus and is known to be expressed on keratinocytes. The aim of this study was to investigate intrinsic TLR‐2 expression and cytokine secretion upon TLR‐2 stimulation with peptidoglycan (PGN), lipoteichoic acid (LTA) and N‐palmitoyl‐S‐[2,3‐bis(palmitoyl)‐(2RS)‐propyl]‐(R)cysteinyl‐alanyl‐glycine (Pam3Cys) in keratinocytes from patients with AD compared to healthy controls. Human primary keratinocytes (HPKs) were cultivated from hair follicles of patients with AD and non‐atopic healthy controls and stimulated with Pam3Cys, LTA and PGN. TLR‐2, TLR‐1 and TLR‐6 expression were investigated at the mRNA level. IL‐6, IL‐8, chemokine C‐C motif ligand (CCL)‐20 and MMP‐9 production were studied at the protein level. TLR‐2, TLR‐1 and TLR‐6 were expressed on both HPKs from patients with AD as well as healthy controls without significant differences between these groups. HPKs from patients with AD had an intrinsically reduced capacity to produce IL‐6, IL‐8, CCL‐20 and MMP‐9 and responded less to TLR‐2 stimulation compared to HPKs from healthy controls. Our findings show evidence for intrinsic alterations in HPKs from patients with AD compared to healthy controls and diminished responses upon TLR‐2 stimulation that might contribute to the enhanced susceptibility to skin infections with S. aureus.     

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.     

Heparinoid suppresses Der p‐induced IL‐1β production by inhibiting ERK and p38 MAPK pathways in keratinocytes

Epidermal keratinocytes initiate skin inflammation by activating immune cells. The skin barrier is disrupted in atopic dermatitis (AD) and epidermal keratinocytes can be exposed to environmental stimuli, such as house dust mite (HDM) allergens. We showed previously that HDM allergens activate the NLRP3 inflammasome of keratinocytes, thereby releasing pro‐inflammatory cytokines. Heparinoid is an effective moisturizer for atopic dry skin. However, a recent report showed that heparinoid treatment can improve inflammation of lichen planus. Therefore, we hypothesized that it acts on epidermal keratinocytes not only as a moisturizer, but also as a suppressant of the triggers of skin inflammation. We found that HDM allergen‐induced interleukin (IL)‐1β release from keratinocytes was inhibited significantly by heparinoid pretreatment without affecting cell viability. However, heparinoid did not affect caspase‐1 release, suggesting that heparinoid did not affect HDM allergen‐induced inflammasome activation. Heparinoid treatment not only decreased intracellular levels of pro‐IL‐1β, but also suppressed IL‐1β messenger RNA (mRNA) expression in keratinocytes. Among the intracellular signalling pathways, the activation of extracellular signal‐regulated kinase and p38 pathways, which are required for IL‐1β expression in keratinocytes, was inhibited by heparinoid treatment. The inhibitory effect of heparinoid on IL‐1β mRNA expression was also confirmed with living skin equivalents. Our results demonstrated that heparinoid suppresses the initiation of keratinocyte‐mediated skin inflammation.     

Dual oxidase 2 is essential for house dust mite‐induced pro‐inflammatory cytokine production in human keratinocytes

House dust mites (HDMs) are known to trigger chronic inflammation through Toll‐like receptors (TLRs) and their signalling cascades. In this study, we found that TLR2 ligation by HDMs induced the activation of dual oxidase 2 (Duox2) and nuclear factor‐κB (NF‐κB), leading to the production of pro‐inflammatory cytokines in human keratinocytes. Stimulation of human keratinocytes with HDMs resulted in increases in interleukin‐8 (IL‐8) and chemokine (C–C motif) ligand 20 (CCL20) levels. However, pro‐inflammatory cytokine production was abolished in keratinocytes transfected with TLR2 siRNA, indicating that HDM‐induced cytokine production was mediated via TLR2 signalling. We also examined the function of Duox1/2 isozymes, which are primarily expressed in keratinocytes, in HDM‐mediated pro‐inflammatory cytokine production. Human keratinocytes transfected with control siRNA or Duox1 siRNA showed no inhibition of IL‐8 or CCL20 production in response to HDMs, whereas the silencing of Duox2 expression resulted in a failure to induce cytokine production. Moreover, the phosphorylation and nuclear localization of RelA/p65, a component of NF‐κB, were induced by HDMs in human keratinocytes. Transfection of human keratinocytes with TLR2 siRNA or Duox2 siRNA resulted in the complete abolishment of RelA/p65 nuclear localization in response to HDMs. Taken together, these results indicate that the HDM‐dependent TLR2‐Duox2 signalling axis indeed promotes NF‐κB activation, which induces IL‐8 and CCL20 production and mediates epidermal keratinocyte inflammation.     

Aggravation of atopic dermatitis‐like symptoms by consecutive low concentration of formaldehyde exposure in NC/Nga mice

Formaldehyde (FA) has been known to be associated with development of asthma (AS) and atopic dermatitis (AD). In this study, we investigated whether FA inhalation would affect the provocation or exacerbation of AD‐like symptoms. Atopic‐prone NC/Nga mice were exposed to low (0.2 ppm) and high (1.0 ppm) concentration of FA by inhalation. Combined exposure to low concentration of FA inhalation and topical house dust mite (HDM) stimulation significantly upregulated HDM‐induced total plasma IgE and IgG2a production, Th1‐, Th2‐, Th17‐related cytokine as well as COX‐2 mRNA expressions in the skin. Interestingly, independent FA inhalation, especially at low concentration (0.2 ppm), increased the skin mRNA expressions of IL‐13, IL‐17E/IL‐25 and COX‐2, even though it failed to induce AD‐like skin inflammation. In conclusion, we suggest that increased skin mRNA expressions of IL‐13, IL‐25/IL‐17E and COX‐2 by independent low concentration of FA exposure might be a key factor to exacerbate HDM‐mediated AD‐like skin inflammation.     

DAMP molecules S100A9 and S100A8 activated by IL‐17A and house‐dust mites are increased in atopic dermatitis

S100A9 and S100A8 are called damage‐associated molecular pattern (DAMP) molecules because of their pro‐inflammatory properties. Few studies have evaluated S100A9 and S100A8 function as DAMP molecules in atopic dermatitis (AD). We investigated how house‐dust mites affect S100A9 and S100A8 expression in Th2 cytokine‐ and Th17 cytokine‐treated keratinocytes, and how secretion of these molecules affects keratinocyte‐derived cytokines. Finally, we evaluated expression of these DAMP molecules in AD patients. S100A9 expression and S100A8 expression were strongly induced in IL‐17A‐ and Dermatophagoides (D.) farinae‐treated keratinocytes, respectively. Furthermore, co‐treatment with D. farinae and IL‐17A strongly increased expression of S100A9 and S100A8 compared with D. farinae‐Th2 cytokine co‐treatment. The IL‐33 mRNA level increased in a dose‐dependent manner in S100A9‐treated keratinocytes, but TSLP expression did not change. S100A8/A9 levels were also higher in the lesional skin and serum of AD patients, and correlated with disease severity. Taken together, S100A9 and S100A8 may be involved in inducing DAMP‐mediated inflammation in AD triggered by IL‐17A and house‐dust mites.     

Study of pro‐ and anti‐inflammatory cytokine profile in the patients with parthenium dermatitis

Background: Parthenium dermatitis is a common airborne allergic contact dermatitis induced by exposures to the weed Parthenium hysterophorus. The disease manifests as itchy erythematous papules, papulovesicular and plaque lesions on exposed areas of the body. Objectives: The aim of this study was to show the alterations in pro/anti‐inflammatory cytokines in parthenium dermatitis. Methods: The study included 50 patients with parthenium dermatitis confirmed by patch testing using aqueous extracts of P. hysterophorus and 50 age‐matched healthy controls. The levels of pro‐inflammatory [tumour necrosis factor‐α (TNF‐α), interleukin (IL)‐6, IL‐8, and IL‐17] and anti‐inflammatory (IL‐4 and IL‐10) cytokines were estimated by commercially available high sensitivity enzyme‐linked immunosorbent assay (ELISA) kits. Results: All the dermatitis patients showed significantly (P < 0.001) elevated levels of TNF‐α, IL‐6, IL‐8, and IL‐17 levels as compared to healthy controls. In contrast, the anti‐inflammatory cytokine IL‐4 showed an insignificant decrease (P < 0.217) and a decrease in level of IL‐10 was statistically significant (0.001) compared with controls. Conclusions: The present study suggests the involvement of pro‐inflammatory cytokines in the pathogenesis of parthenium dermatitis. A decrease in levels of anti‐inflammatory cytokines was demonstrated, which could not downregulate pro‐inflammatory cytokines in parthenium dermatitis.     

Two arginine residues in the COOH‐terminal of human β‐defensin‐3 constitute an essential motif for antimicrobial activity and IL‐6 production

Human β‐defensin‐3 (HBD‐3) possesses antimicrobial activities and the potential to induce proinflammatory cytokines. HBD‐3 contains a unique motif of two arginine residues (Arg or R) in the COOH‐terminal region. To understand the bioactive properties of the Arg residues of HBD‐3, we examined antimicrobial activities against Staphylococcus aureus and Pseudomonas aeruginosa using synthetic HBD‐2, HBD‐3 and two variant peptides of HBD‐3: the Arg‐truncated variant designated desR HBD‐3 and NRR HBD‐3, in which both Arg residues were shifted to the N‐terminal region. IL‐6 production from keratinocytes was studied using the peptides. HBD‐3 possessed approximately five‐fold more potent antimicrobial activities, evaluated as the minimum inhibitory concentration (MIC), against S. aureus compared with desR and NRR HBD‐3, while no significant activity was observed in HBD‐2. The antimicrobial activity of HBD‐3 against S. aureus was well preserved even at high sodium chloride concentrations, but was attenuated in desR and NRR HBD‐3. All the peptides exhibited similar antimicrobial activities against P. aeruginosa, but HBD‐2 and desR HBD‐3 showed diminished antimicrobial activities against P. aeruginosa at high salt concentrations. IL‐6 production was significantly induced in keratinocytes with HBD‐3, but not remarkably with stimulation by other peptide. These Arg residues are essential for the antimicrobial and biological properties of HBD‐3.     

Interleukin-18 is elevated in the horny layer in patients with atopic dermatitis and is associated with Staphylococcus aureus colonization

Background An increase in interleukin (IL)‐18 production from epidermal cells has been reported in an atopic dermatitis (AD) mouse model, and subsequent topical application of Staphylococcus aureus results in severe dermatitis. Objectives To reveal the relationship between S. aureus colonization of skin lesions and keratinocyte IL‐18 production, particularly in AD with relatively low serum IgE levels. We also aimed to establish a simple and noninvasive method of assaying IL‐18 produced by epidermal keratinocytes to evaluate local skin inflammation and therapeutic effects in patients with AD. Methods IL‐18 in the horny layer of the skin was collected via a tape‐stripping method and measured in 95 patients with AD and 40 healthy controls by enzyme‐linked immunosorbent assay (ELISA). Clinical severity, blood data and S. aureus skin colonization were evaluated before and after treatment. Results IL‐18 levels in the horny layer were significantly higher in the skin lesions of patients with AD than in healthy controls and correlated with SCORAD, levels of serum IL‐18, IgE, lactate dehydrogenase, thymus and activation‐regulated chemokine, blood eosinophils and transepidermal water loss. In the AD group with serum IgE < 1500 IU mL^?1, significantly higher IL‐18 levels were observed in the horny layer of patients colonized with S. aureus compared with those who were not. Conclusions Epidermal IL‐18 production was associated with the severity of AD. Staphylococcus aureus colonization seems to contribute to this IL‐18 production, especially in the AD group with relatively low IgE production. Tape stripping provides an easy and noninvasive method to assess epidermal IL‐18 production by ELISA.     

Increased expression of the aryl hydrocarbon receptor in patients with chronic inflammatory skin diseases

Polychlorinated biphenyls (PCBs) and 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) are major environmental pollutants, and their effects on the human body critically depend on the aryl hydrocarbon receptor (AhR). The aim of this study was to evaluate the significance of the AhR and its ligands in chronic inflammatory skin diseases such as atopic dermatitis (AD) and psoriasis. Expression of AhR‐related mRNA was increased in lesional skin from patients with AD and psoriasis compared to those of normal skin from healthy controls. The AhR and aryl hydrocarbon receptor nuclear translocator were colocalized in the nuclei of keratinocytes at the lower epidermis of psoriatic lesions, which suggested activation of the AhR pathway. After treatment of normal human epidermal keratinocytes with TCDD or PCBs, IL‐6 and IL‐8 production were increased. The results of this study suggest that AhR is highly expressed in the acute lesional skin of patients with AD and psoriasis, and the AhR pathway is activated especially in psoriasis.     

Baseline sebum IL‐1α is higher than expected in afro‐textured hair: a risk factor for hair loss?*

Objective To investigate changes in sebum cytokines in response to hair cosmetics. Design and setting A prospective study at a University hospital. Methods We used a novel method for scalp surface sebum collection (Sebutape^®) on three visits, sequentially a week apart, to investigate changes in six cytokines in 36 healthy women before and after shampoo and compared various chemical treatments (ammonium thioglycolate, “lye” sodium hydroxide and “no‐lye” guanidine hydroxide relaxers) performed by a professional hairdresser. Results Significant levels detected were IL‐1 alpha (IL‐1α) and IL‐1 receptor antagonist (IL‐1ra), which were higher in untreated scalp vs. forehead: P < 0.001. Baseline levels of scalp sebum IL‐1α were 18 times higher than IL‐1ra. The levels of IL‐1α decreased uniformly after shampoo (visit 1) and various chemical treatments (both crown and vertex all P < 0.001 – visit 2) but increased on follow‐up at visit 3. Decreases in IL‐1ra mimicked IL‐1α at the vertex [after shampoo (P = 0.018) and visit 3 (P = 0.014)], but not on the crown, a finding which may suggest site‐specific scalp predisposition to inflammation. The ratio of IL‐1ra/IL‐1α increased in all groups after all chemical treatments and on follow‐up (all P < 0.001) but was surprisingly not significantly different from natural hair that underwent shampoo. Limitations A wider cytokine panel may reveal response differences in treatment groups. Conclusions Baseline inflammatory scalp cytokines are higher than expected and reduce with shampooing. Scrutiny of the influence of hair moisturizer formulations and shampoo intervals and studies investigating pro‐fibrotic cytokines are required. This may elucidate the predilection of afro‐textured hair to scarring alopecia.     

CCL7 contributes to the TNF‐alpha‐dependent inflammation of lesional psoriatic skin

Chemokines are small chemotactic proteins that have a crucial role in leukocyte recruitment into tissue. Targeting these mediators has been suggested as a potential therapeutic option in inflammatory skin diseases such as psoriasis. Using quantitative RT‐PCR, we found CCL7, a chemokine ligand known to interact with multiple C‐C chemokine receptors, to be markedly increased in lesional psoriasis as opposed to atopic dermatitis, lichen planus, non‐lesional psoriatic and normal control skin. Surprisingly, this increase in CCL7 mRNA expression exceeded that of all other chemokines investigated, and keratinocytes and dermal blood endothelial cells were identified as its likely cellular sources. In an imiquimod‐induced psoriasis‐like mouse model, CCL7 had a profound impact on myeloid cell inflammation as well as on the upregulation of key pro‐psoriatic cytokines such as CCL20, IL‐12p40 and IL‐17C, while its blockade led to an increase in the antipsoriatic cytokine IL‐4. In humans receiving the TNF‐α‐blocker infliximab, CCL7 was downregulated in lesional psoriatic skin already within 16 hours after a single intravenous infusion. These data suggest that CCL7 acts as a driver of TNF‐α‐dependent Th1/Th17‐mediated inflammation in lesional psoriatic skin.     

Cannabinoid 1 receptors in keratinocytes attenuate fluorescein isothiocyanate‐induced mouse atopic‐like dermatitis

Atopic dermatitis is a chronic inflammatory disease characterized by an impaired epidermal barrier function combined with a chronic Th2‐type inflammatory response and an intense pruritus. Here, we used an experimental mouse model for Th2‐type contact hypersensitivity (CHS) to fluorescein isothiocyanate (FITC) to investigate the potential role of cannabinoid 1 receptors (CB1) in the pathophysiology of mouse atopic‐like dermatitis. Mice lacking CB1 receptors globally (Cnr1^?/?) or specifically in keratinocytes (KC‐Cnr1^?/?) as well as wild‐type (WT) control mice were sensitized and challenged with FITC. We examined ear swelling responses, transepidermal water loss, Th2‐type skin inflammatory responses and serum IgE levels. Both Cnr1^?/? and KC‐Cnr1^?/? showed enhanced CHS responses to FITC and a delayed epidermal barrier repair when compared with WT mice. mRNA levels for IL‐4, thymic stromal lymphopoietin (TSLP) and CCL8, as well as eosinophil activity, were significantly increased in inflamed ear tissue of FITC‐challenged Cnr1^?/? and KC‐Cnr1^?/? mice. Importantly, CB1 receptor‐deficient keratinocytes secreted increased levels of TSLP, a proinflammatory mediator that drives Th2‐type skin inflammation in atopic dermatitis, under basal and Th2‐type inflammatory conditions. Taken together, our results demonstrate that CB1 receptors in keratinocytes help to maintain epidermal barrier homoeostasis and attenuate Th2‐type allergic inflammatory responses. Based on our work, we propose that enhanced epidermal allergen penetrance cooperates with increased production of TSLP and CCL8 by epidermal keratinocytes for the induction of type 2 CD4+ T helper cells. Our results place keratinocytes at the cross‐roads of outside‐in and inside‐out pathophysiologic mechanisms of atopic dermatitis.     

Naïve CD4+ T cells from patients with atopic dermatitis show an aberrant maturation towards IL-4-producing skin-homing CLA+ cells

Abstract: Overproduction of interleukin‐4 (IL‐4) has been reported in lesional and in peripheral T cells from patients with atopic dermatitis (AD). It is not clear whether the development of IL‐4‐producing T helper type 2 (Th2) cells from naïve precursors is an intrinsic phenomenon of T cells or whether other, extrinsic factors play a significant role. To analyze these alternatives, we investigated the IL‐4 production of effector T cells generated in vitro from highly purified CD4+ CD45RA+ naïve T cells in the absence of signals derived from antigen‐presenting cells. Effector T cells generated from naïve precursors from both AD and healthy donors produced comparable amounts of IL‐4 after restimulation. Priming in the presence of exogenous IL‐4 enhanced the production of IL‐4 while neutralizing endogenously produced IL‐4 abolished IL‐4 production similarly in atopic and healthy T cells. A subset of effector T cells acquired the expression of the cutaneous lymphocyte antigen (CLA). The frequency of CLA+ T cells was not different between atopic and healthy donors. CLA+ T cells, differentiated from naïve atopic, but not healthy T cells, showed a preferential Th2 cytokine profile as assessed by intracellular cytokine staining. Also effector T cells derived from atopic patients without dermatitis tended to show this imbalance, although it was not significantly different to healthy controls. This Th2 cytokine profile did not develop when naïve T cells were cultured in the presence of IL‐12. In conclusion, high IL‐4 production in developing T cells from AD patients was associated with CLA expression, the net IL‐4 production of all effector CD4+ T cells, however, was similar to IL‐4 production by T cells from healthy donors.     

Stimulated human melanocytes express and release interleukin‐8, which is inhibited by luteolin: relevance to early vitiligo

Vitiligo is a disorder of depigmentation, for which the pathogenesis is as yet unclear. Interleukin (IL)‐8 (CXCL8) is a key inflammatory chemokine. We investigated the regulation of IL‐8 production in human melanocytes, and the IL‐8 serum levels and skin gene expression in patients with vitiligo and in controls. Cultured melanocytes were stimulated for 24 h with tumour necrosis factor (TNF) 100 ng/mL and IL‐1β 10 ng/mL, with or without pretreatment with luteolin 50 μmol/L for 30 min, and IL‐8 release was measured by ELISA. Serum cytokines were measured by a microbead array. Skin biopsies were taken from healthy subjects (n = 14) as well as from marginal lesional and nonlesional skin from patients with vitiligo (n = 15). IL‐8 gene expression was evaluated by quantitative real time PCR. Both TNF and IL‐1β stimulated significant IL‐8 release (P < 0.01) from melanocytes, whereas pretreatment with luteolin significantly inhibited this effect (P < 0.01). IL‐8 gene expression was significantly increased in vitiligo compared with control skin (P < 0.05). IL‐8 may be involved in vitiligo inflammation. Inhibition by luteolin of IL‐8 release could be useful for vitiligo therapy.     

Increased expression of the histamine H4 receptor following differentiation and mediation of the H4 receptor on interleukin‐8 mRNA expression in HaCaT keratinocytes

Recent in vivo studies have demonstrated involvement of the histamine H4 receptor in pruritus and skin inflammation. We previously reported that an H4 receptor antagonist attenuated scratching behaviour and improved skin lesions in an experimental model of atopic dermatitis. We also reported the expression of the H4 receptor in human epidermal tissues. In this study, we investigated the expression of H4 receptor mRNA and the function of the receptor in a culture system that mimics in vivo inflammation on the HaCaT human keratinocyte cell line. Increased expression of the H4 receptor was observed in HaCaT cells following differentiation. Treatment of HaCaT cells with histamine and TNFα enhanced the mRNA expression of interleukin (IL)‐8. These increases in expression were significantly inhibited by the H4 receptor antagonist JNJ7777120. Our results indicate that IL‐8 mRNA expression might be enhanced by histamine and TNFα via H4 receptor stimulation in keratinocytes.     

Expression of toll-like receptor 2, NOD2 and dectin-1 and stimulatory effects of their ligands and histamine in normal human keratinocytes

Background Epidermal keratinocytes are involved in the skin innate immunity and express toll‐like receptors (TLRs) and other innate immune proteins. The epidermis is continuously exposed to pathogenic Gram‐positive bacteria or fungi. However, few studies have examined the function and expression of innate immune proteins in keratinocytes. Histamine, which is well known for itch and allergy, is closely associated with innate immunity, but its influence on epidermal innate immunity is still unclear. Objectives To clarify the expression of innate immune proteins in keratinocytes stimulated by ligand pathogen‐associated molecules, and the function of histamine in this process. Methods We investigated the effects of lipopeptide (MALP‐2, 1–100 ng mL^?1; ligand for TLR2), peptidoglycan (PGN, 0·02–2 μg mL^?1; ligand for NOD2) and β‐glucan (1–100 μg mL^?1; ligand for dectin‐1) in the presence or absence of histamine on mRNA expression of TLR2, NOD2 and dectin‐1 as well as human β‐defensin 2 by quantitative real‐time polymerase chain reaction in cultured normal human epidermal keratinocytes. TLR2 expression was also examined at the cell surface and intracellularly, as determined by flow cytometry and confocal microscopy. The quantities of interleukin (IL)‐1α and IL‐8 produced by keratinocytes were measured using enzyme‐linked immunosorbent assay. Results At the mRNA level, TLR2 was enhanced by PGN but not by its ligand MALP‐2 or by β‐glucan; NOD2 was easily induced by all three ligands; and dectin‐1 was enhanced by its ligand β‐glucan. These enhanced expressions were further augmented by histamine at 1 μg mL^?1. While the surface expression of TLR2 was barely detectable by flow cytometry even after stimulation, the intracellular expression of TLR2 was apparently elevated by PGN and further promoted by histamine. A confocal microscopic analysis also revealed the enhanced expression of TLR2 in the cytoplasm. The expression of TLR2, NOD2 and dectin‐1 was functional, as these pathogen‐associated molecules induced the production of IL‐1α, IL‐8 and defensin, and again, histamine greatly enhanced this production. Conclusions Our study demonstrated that the expression of functional innate immune receptors is augmented by the pathogen‐associated molecules in a ligand‐feed forward or nonrelated manner in keratinocytes, and histamine promotes their expression and the resultant production of cytokines and defensins.     

Serum cytokines and growth factor levels in Japanese patients with psoriasis

Background. Although the precise pathomechanism of psoriasis is still unknown, various cytokines and growth factors derived from T cells, dendritic cells or keratinocytes, are critically involved in this disease. There have been several studies determining the serum levels of cytokines in patients with psoriasis, but with conflicting results. The levels of various cytokines and growth factors were measured in the sera of patients with psoriasis and compared with those of healthy controls. The correlation with disease severity was also determined. Methods. Sera were collected from 122 patients with psoriasis and 78 healthy controls for ELISA analysis to evaluate the levels of cytokines and growth factors. The severity of psoriasis was determined by the Psoriasis Area and Severity Index (PASI). Results. Serum levels of tumour necrosis factor (TNF)‐α, interferon (IFN)‐γ, interleukin (IL)‐2, IL‐6, IL‐7, IL‐8, IL‐12, IL‐17, IL‐18 and vascular endothelial growth factor (VEGF) were significantly increased in patients with psoriasis compared with those of healthy controls. The serum levels of IL‐2, soluble intercellular adhesion molecule‐1, epidermal growth factor, hepatocyte growth factor and amphiregulin were not significantly different from those of healthy controls. Increased serum levels of TNF‐α, IFN‐γ, IL‐12, IL‐17, IL‐18 and VEGF correlated with PASI. Furthermore, these cytokine levels were decreased after psoriasis treatment. In contrast, serum levels of IL‐10 were decreased in psoriasis and negatively correlated with PASI. Discussion. Serum levels of TNF‐α, IFN‐γ, IL2, IL‐6, IL‐7, IL‐8, IL‐12, IL‐17, IL‐18 and VEGF were positively correlated and that of IL‐10 was negatively correlated with PASI in Japanese patients with psoriasis. These parameters might be useful for determining the disease activity of psoriasis.