The identification of CCL18 as biomarker of disease activity in localized scleroderma.

BACKGROUND: Localized Scleroderma (LoS) encompasses a group of idiopathic skin conditions characterized by (sub)cutaneous inflammation and subsequent development of fibrosis. Currently, lack of accurate tools enabling disease activity assessment leads to suboptimal treatment approaches. OBJECTIVE: To investigate serum concentrations of cytokines and chemokines implicated in inflammation and angiogenesis in LoS and explore their potential to be utilized as biomarker of disease activity. Additionally, to investigate the implication of potential biomarkers in disease pathogenesis. METHODS: A 39-plex Luminex immuno-assay was performed in serum samples of 74 LoS and 22 Healthy Controls. The relation between a validated clinical measure of disease activity (mLoSSI) and serum analytes was investigated. Additionally, gene and protein expression were investigated in circulating cells and skin biopsies. RESULTS: From the total of 39, 10 analytes (CCL18, CXCL9, CXCL10, CXCL13, TNFRII, Galectin-9, TIE-1, sVCAM, IL-18, CCL19) were elevated in LoS serum. Cluster analysis of serum samples revealed CCL18 as most important analyte to discriminate between active and inactive disease. At individual patient level, CCL18 serum levels correlated strongest with mLoSSI-scores (rs = 0.4604, P < 0.0001) and in longitudinal measures CCL18 concentrations normalised with declining disease activity upon treatment initiation. Additionally, CCL18 was elevated in LoS serum, and not in (juvenile) dermatomyositis or spinal muscular atrophy. Importantly, CCL18 gene and protein expression was increased at the inflammatory border of cutaneous LoS lesions, with normal expression in unaffected skin and circulating immune cells. CONCLUSION: CCL18 is specific for disease activity in LoS thereby providing relevance as a biomarker for this debilitating disease.

Late cornified envelope (LCE) proteins: distinct expression patterns of LCE2 and LCE3 members suggest nonredundant roles in human epidermis and other epithelia.

BACKGROUND: Deletion of the late cornified envelope (LCE) proteins LCE3B and LCE3C is a strong and widely replicated psoriasis risk factor. It is amenable to biological analysis because it precludes the expression of two epidermis-specific proteins, rather than being a single-nucleotide polymorphism of uncertain significance. The biology of the 18-member LCE family of highly homologous proteins has remained largely unexplored so far. OBJECTIVES: To analyse LCE3 expression at the protein level in human epithelia, as a starting point for functional analyses of these proteins in health and disease. METHODS: We generated the first pan-LCE3 monoclonal antibody and provide a detailed analysis of its specificity towards individual LCE members. LCE2 and LCE3 expression in human tissues and in reconstructed human skin models was studied using immunohistochemical analyses and quantitative polymerase chain reaction. RESULTS: Our study reveals that LCE2 and LCE3 proteins are differentially expressed in human epidermis, and colocalize only in the upper stratum granulosum layer. Using an in vitro reconstructed human skin model that mimics epidermal morphogenesis, we found that LCE3 proteins are expressed at an early time point during epidermal differentiation in the suprabasal layers, while LCE2 proteins are found only in the uppermost granular layer and stratum corneum. CONCLUSIONS: Based on the localization of LCE2 and LCE3 in human epidermis we conclude that members of the LCE protein family are likely to have distinct functions in epidermal biology. This finding may contribute to understanding why LCE3B/C deletion increases psoriasis risk.

Mast-cell interleukin-1ß, neutrophil interleukin-17 and epidermal antimicrobial proteins in the neutrophilic urticarial dermatosis in Schnitzler's syndrome.

BACKGROUND: Schnitzler's syndrome (SchS) is an autoinflammatory disease characterized by a chronic urticarial rash, a monoclonal component and signs of systemic inflammation. Interleukin (IL)-1ß is pivotal in the pathophysiology. OBJECTIVES: Here we investigated the cellular source of proinflammatory mediators in the skin of patients with SchS. METHODS: Skin biopsies of lesional and nonlesional skin from eight patients with SchS and healthy controls, and patients with cryopyrin-associated periodic syndrome (CAPS), delayed-pressure urticaria (DPU) and cold-contact urticaria (CCU) were studied. We studied in vivoIL-1ß, IL-17 and antimicrobial protein (AMP) expression in resident skin cells and infiltrating cells. In addition we investigated the in vitro effect of IL-1ß, IL-17 and polyinosinic-polycytidylic acid (poly:IC) stimulation on cultured epidermal keratinocytes. RESULTS: Remarkably, we found IL-1ß-positive dermal mast cells in both lesional and nonlesional skin of patients with SchS, but not in healthy control skin and CCU, and fewer in CAPS. IL-17-positive neutrophils were observed only in lesional SchS and DPU skin. In lesional SchS epidermis, mRNA and protein expression levels of AMPs were strongly increased compared with nonlesional skin and that of healthy controls. When exposed to IL-1ß, poly:IC or IL-17, patient and control primary human keratinocytes produced AMPs in similar amounts. CONCLUSIONS: Dermal mast cells of patients with SchS produce IL-1ß. This presumably leads to activation of keratinocytes and neutrophil influx, and further amplification of inflammation by IL-17 (from neutrophils and mast cells) and epidermal AMP production leading to chronic histamine-independent neutrophilic urticarial dermatosis.

Expression profile of cornified envelope structural proteins and keratinocyte differentiation-regulating proteins during skin barrier repair.

BACKGROUND: Recent studies have emphasized the importance of heritable and acquired skin barrier abnormalities in common inflammatory diseases such as psoriasis and atopic dermatitis (AD). To date, no comprehensive studies on the effect of experimental barrier disruption on cornified envelope protein expression have been performed. OBJECTIVES: To analyse the effect of experimental skin barrier disruption on the expression of cornified envelope structural proteins and keratinocyte differentiation-regulating proteins. METHODS: We examined mRNA (day 1, 3 and 7) and protein (day 1, 2, 4 and 9) expression levels of structural proteins and regulatory molecules after sodium dodecyl sulphate (SDS) application on normal skin, and tape stripping of uninvolved epidermis of patients with psoriasis and AD and healthy controls. RESULTS: Upon tape stripping, several structural molecules were significantly downregulated (at the mRNA level as well as the protein level), including LCE5A, LCE2B, FLG, FLG2 and LOR, whereas others were upregulated: IVL, SPRR1, SPRR2, HRNR and most notably LCE3A. The epidermal crosslinking enzymes TGM1, TGM3 and TGM5 were all upregulated, whereas proteases involved in the desquamation process (CTSV, KLK5 and KLK7) were downregulated or unaffected. Most results were similar in SDS-instigated irritant contact dermatitis. There was no significant difference in response between normal epidermis and nonlesional skin of patients with psoriasis and AD. CONCLUSIONS: Skin barrier disruption induces a temporary barrier repair response composed of increased expression of several cornification-related proteins, and decreased expression of some structural and desquamation-related proteins.

Molecular diagnostics of psoriasis, atopic dermatitis, allergic contact dermatitis and irritant contact dermatitis.

BACKGROUND: Microarray studies on the epidermal transcriptome in psoriasis and atopic dermatitis (AD) have revealed genes with disease-specific expression in keratinocytes of lesional epidermis. These genes are possible candidates for disease-specific pathogenetic changes, but could also provide a tool for molecular diagnostics of inflammatory skin conditions in general. OBJECTIVES: To analyse if gene expression signatures as found in purified epidermal cells from AD are also present in other eczematous conditions such as allergic and irritant contact dermatitis. METHODS: We used real-time quantitative polymerase chain reaction, immunohistochemistry and bioinformatics to investigate gene expression in different forms of eczema. Normal epidermis and psoriatic epidermis were analysed for comparison. RESULTS: Carbonic anhydrase II was highly induced in epidermis from all forms of eczema but not in psoriasis. Remarkably, the presumed neuron-specific Nel-like protein 2 showed a strong induction only in AD epidermis. Interleukin-1F9, elafin, beta-defensin-2 and vanin-3 were strongly induced in psoriasis, but not in any type of eczema. High levels of the chemokines CCL17 and CXCL10 were predominantly found in epidermis of allergic contact dermatitis. The chemokine CXCL8 was highly expressed in psoriasis, AD and allergic contact dermatitis, but not in irritant contact dermatitis. Cluster analysis or multinomial logistic regression indicated that expression levels of a set of seven genes are a strong predictor of the type of inflammatory response. CONCLUSIONS: These observations contribute to molecular diagnostic criteria for inflammatory skin conditions.

The cystatin M/E-controlled pathway of skin barrier formation: expression of its key components in psoriasis and atopic dermatitis.

BACKGROUND: The antiprotease activity of cystatin M/E regulates skin barrier formation, as it inhibits the activity of cathepsin V, cathepsin L and legumain, thereby controlling the processing of transglutaminase 3. Misregulation of this pathway by unrestrained protease activity, as seen in cystatin M/E-deficient mice, leads to abnormal stratum corneum and hair follicle formation, and severe disturbance of skin barrier function. OBJECTIVES: Our major aim was to make a quantitative analysis of the expression of all players of this pathway in the epidermis of patients with inflammatory skin diseases. A second aim was to determine if reconstructed human skin could be used as an in vitro model system to investigate this pathway. METHODS: Autopsy material from normal human tissues, biopsies from normal skin of healthy volunteers, and lesional skin from patients with atopic dermatitis and psoriasis were used to study the expression of the above-mentioned molecules at the mRNA level by quantitative real-time polymerase chain reaction. Localization of the protein was performed by immunofluorescence microscopy, and expression was quantitated by image analysis. RESULTS: In skin, cystatin M/E is expressed at relatively higher levels than its target proteases, when compared with other tissues, which emphasizes its prominent role in cutaneous biology. We found decreased expression of cystatin M/E and cathepsin V in lesional atopic dermatitis and psoriasis epidermis at the mRNA level as well as the protein level. Cathepsin L and transglutaminase 3 were increased at the transcriptional level; however, this was not reflected by higher protein levels. Interestingly, the expression of all these molecules in reconstructed skin was qualitatively and quantitatively similar to the in vivo situation. CONCLUSIONS: Disturbance of the cystatin M/E-cathepsin pathway could contribute to the dysregulated skin barrier function observed in inflammatory dermatoses. Human reconstructed skin appears to be a valuable model to study this novel biochemical pathway in vitro.

Oral retinoic acid metabolism blocking agent Rambazole for plaque psoriasis: an immunohistochemical study.

BACKGROUND: The novel systemic all-trans retinoic acid metabolism blocking agent (RAMBA) R115866 (Rambazole(TM); Barrier Therapeutics, Geel, Belgium; further referred to as rambazole) increases intracellular levels of endogenous all-trans retinoic acid (RA). Well-known effects of RA are normalization of aberrant epithelial growth and differentiation. Hence, rambazole might be beneficial in the treatment of plaque psoriasis. OBJECTIVES: The dynamics of epidermal proliferation, keratinization, lesional T-cell subsets and cells expressing natural killer (NK)-receptors in plaque psoriasis were assessed during treatment with rambazole, as part of a phase IIa open-label clinical trial. METHODS: Six patients were treated with rambazole, 1 mg, once daily, for 8 weeks. At weeks 0, 2 and 8, psoriatic plaque severity scores (SUM) and biopsies from a target lesion were assessed. Epidermal proliferation (Ki67), keratinization markers (K10, K13, K19), T-cell subsets (CD3, CD4+, CD8+, CD45RO+, CD45RA+, CD2+, CD25+, GITR+) and cells expressing NK-receptors (CD94, CD161) were immunohistochemically stained and quantified with image analysis. RESULTS: At week 2 the mean SUM-score was virtually equal to baseline, which was accompanied immunohistochemically by equal epidermal hyperproliferation, a nonsignificant decrease in K10 positive epidermis and, overall, a nonsignificant increase in immunocyte subsets. At week 8, in contrast, plaque severity was reduced by 34% from baseline (P < 0.05). Improvements were also detected for epidermal proliferation (-63%; P < 0.01) and K10 expression (+29%; P < 0.01), compared with baseline. No induction of retinoid-specific keratinization (K13, K19) was observed. A nonsignificant reduction of all pathogenic T-cell subsets and cells expressing NK-receptors was observed at week 8 of treatment (P > 0.05). CONCLUSIONS: Clinical efficacy of rambazole is primarily the result of restoring proliferation (Ki67) and differentiation (K10) of epidermal keratinocytes. Secondly, relevant T-cell subsets and cells expressing NK-receptors showed nonsignificant reductions after 8 weeks of treatment with rambazole.

Identification of lesional CD4+ CD25+ Foxp3+ regulatory T cells in Psoriasis.

BACKGROUND: Depletion of CD4+ CD25+ Foxp3+ naturally occurring regulatory T cells (T(reg)) induces autoimmune phenomena. These cells have not yet been fully characterized in the skin of psoriatic patients. OBJECTIVES: To prove that the Zenon immunofluorescent labeling technique is suitable for the demonstration of co-localization of T-cell markers and in particular to show the distribution of T(reg) in psoriatic skin. METHODS: In biopsies derived from normal and psoriatic skin, CD4+ CD25+, CD4+ CD45RO+, CD8+ CD25+, CD8+ CD45RO+ and CD4+ CD25+ Foxp3+ cells in the dermis and in the epidermis were immunophenotyped, using a quantitative immunofluorescent labeling technique (Zenon), analyzed and compared using image analysis. RESULTS: The immunofluorescent labeling technique was shown to be an easy and reliable tool to demonstrate co-localization of T-cell markers. In psoriasis, all pathogenic T-cell subsets (CD4+ CD25+, CD4+ CD45RO+, CD8+ CD25+ and CD8+ CD45RO+ cells) were significantly increased in the dermis and in the epidermis, as compared to normal skin (all p < 0.05). Using this labeling technique we were able to reveal CD4+ CD25+ Foxp3+ T(reg) in psoriatic dermis, but not in the dermis of normal skin (p < 0.0001). CONCLUSIONS: The Zenon immunofluorescence technique in combination with image analysis is suitable for the demonstration of co-localization of T-cell markers in tissue. Increased numbers of pathogenic T cells (CD4+ CD25+, CD4+ CD45RO+, CD8+ CD25+ and CD8+ CD45RO+) were shown in the dermis and epidermis, whereas CD4+ CD25+ Foxp3+ T(reg) were identified in psoriatic skin with a predilection for the upper dermis.

Effect of calcipotriol on epidermal cell populations in alefacept-treated psoriatic lesions.

BACKGROUND AND OBJECTIVES: The effect of the established antipsoriatic treatment with topical calcipotriol (with a maximum of 100 g per week) in addition to systemic treatment with alefacept, a new biological agent for psoriasis, on epidermal cell populations in the psoriatic lesion was investigated using a combination of the Zenon labelling technique and microscopic image analysis. Epidermal cell populations were measured quantitatively with this sensitive method. PATIENTS/METHODS: Frozen sections of non-treated psoriatic epidermis and psoriatic epidermis treated with either alefacept intramuscular or alefacept intramuscular in combination with topical calcipotriol for 12 weeks were compared immunohistochemically. Antibodies against keratin 6, 10 and 15 were labelled with the Zenon technique, whereas antibodies against the Ki-67 antigen and beta-1 integrin were covalently Fluorescein Isothiocyanate (FITC)-labelled. Using image analysis, these markers were measured in the epidermis in a standardized manner. RESULTS AND CONCLUSIONS: Treatment of psoriasis with alefacept resulted in a good clinical response in several patients and in a normalization of epidermal expression of the immunohistochemical parameters for differentiation and proliferation. The addition of topical calcipotriol resulted in a faster clinical improvement with a similar overall clinical response and a similar response of epidermal cell populations as compared to treatment with alefacept monotherapy after 12 weeks of treatment. This study also suggests that the appearance of keratin 15 has a predictive value for the duration of remission. It can be concluded that the addition of a low-dose calcipotriol treatment does not contribute to the clinical efficacy of alefacept, both at the clinical level and with respect to markers for epidermal proliferation and differentiation.

Cystatin M / E expression in inflammatory and neoplastic skin disorders.

BACKGROUND: Cystatins are natural and specific inhibitors of endogenous mammalian lysosomal cysteine proteinases and exogenous microbial cysteine proteinases. Cystatins were shown to provide regulatory and protective functions against uncontrolled proteolysis in several disease processes. Recently we reported that cystatin M/E, which is a novel member of the cystatin gene family, has an unusually restricted expression pattern that is limited to skin. Although cystatin M/E possesses two distinct biochemical properties (it is a proteinase inhibitor and a substrate for transglutaminase) its physiological function is unknown. Disturbance of the balance between proteinases and their inhibitors can lead to irreversible damage as in chronic inflammatory reactions and tumour invasion. OBJECTIVES: To examine the expression pattern of cystatin M/E in inflammatory conditions and neoplastic skin disorders in order to obtain possible clues on its function. Furthermore, we wished to determine whether cystatin M/E expression could discriminate between various types of neoplasia. METHODS: Biopsy material of normal skin, atopic dermatitis and psoriatic lesional skin, healing excisional wounds in healthy volunteers, and several types of epidermal neoplasia (keratoacanthoma, actinic keratosis, basal cell carcinoma and squamous cell carcinoma) were used in this study. For comparison we studied the expression of cystatin M/E in squamous neoplasias from non-cutaneous origin. Affinity-purified polyclonal antibodies against cystatin M/E were used for immunohistochemical detection. RESULTS: Cystatin M/E is constitutively expressed in the stratum granulosum of normal skin, sebaceous glands, eccrine sweat glands and the infundibular epithelium of hair follicles. Expression in atopic dermatitis and psoriasis was found to extend to several layers of the stratum spinosum. In wound healing, cystatin M/E was not found in the edge of migrating keratinocytes, but it was strongly expressed in the suprabasal layers of the neo-epidermis. In epidermal neoplasias cystatin M/E expression was only found in differentiated cells and keratinized cell nests. CONCLUSIONS: Inflammation causes cystatin M/E to be expressed in the spinous cell layers where it colocalizes with transglutaminase for which it serves as a substrate. Speculatively, increased expression of cystatin M/E is compatible with a role in controlling increased levels of cysteine proteinases during inflammation and infection. Cystatin M/E expression in neoplastic epidermis is confined to well-differentiated cells and as such does not discriminate between benign and (pre)malignant epidermal neoplasias.

An immunohistochemical assessment of the response of the psoriatic lesion to single and repeated applications of high-dose dithranol cream.

Dithranol, although a time-honoured treatment and from the beginning of the previous century still going strong, remains an empirical treatment. There is growing evidence that the biochemical basis for the mechanism of action of dithranol at the molecular level is related to the redox activity leading to the production of active oxygen species, which include singlet oxygen, superoxide anion radical and hydroxyl radical. Some authors suggest that epidermal proliferation and/or keratinisation may be the target for dithranol, while others refer to aspects of cutaneous inflammation as crucial in the antipsoriatic effect of dithranol. The present study aims to analyse the effect of single and repeated applications of dithranol on aspects of epidermal proliferation, keratinisation and inflammation in the psoriatic plaque. The most marked effect of dithranol proved to be that on epidermal proliferation (the number of Ki-67-positive nuclei) with an early reduction already 1 day following the single application. This reduction lasted for 16 days. However, such an application induced only a modest clinical improvement. Repeated challenges, resulting in a decrease in the number of Ki-67-positive nuclei of 66%, led to a substantial clinical improvement after 12 days. Repeated challenges resulted in a significant reduction of the number of polymorphonuclear leucocytes. However, this reduction was less pronounced as compared to the effect on epidermal proliferation. It is concluded that epidermal proliferation is a sensitive marker to demonstrate an early effect of dithranol. The dynamics of the cell-biological responses suggest that intermittent applications might be a promising new approach. As dithranol does not reduce the number of T lymphocytes, it is attractive to speculate that the combination of dithranol with immunosuppressive treatments might be a very effective combination.

The response of uninvolved skin of patients with psoriasis to single and repeated applications of dithranol cream: an immunohistochemical assessment.

Dithranol is one of the most effective topical treatments for patients with psoriasis. The well-known irritation is a serious limitation. In an earlier study we investigated the inflammatory response to single and repeated applications with dithranol 2% cream in skin from healthy volunteers. In the present study, we assessed the clinical and cell-biological response of single and repeated challenges with dithranol 2% cream in uninvolved skin of patients with psoriasis. A striking difference between the two studies is the late phase in the single-challenge group after 8 days, showing a longer-lasting response in the uninvolved skin compared to normal skin with respect to proliferation and inflammation markers. A controlled and synchronised irritation by dithranol might induce anti-inflammatory processes and as such constitute an antipsoriatic principle. It is attractive to speculate that in psoriasis the induction of anti-inflammatory responses is defective. Following repeated applications of dithranol, a more uniform course of proliferation, differentiation and inflammation markers was observed in the uninvolved psoriatic skin as compared to the skin of healthy volunteers. Again a defect in the induction of anti-inflammatory responses might account for this event. In view of these differences between normal skin and psoriatic uninvolved skin, it may be advisable to use the uninvolved skin of patients with psoriasis in further studies on the interference between dithranol irritancy and various anti-inflammatory agents.