Increased production of transforming growth factor-alpha in psoriatic epidermis

To elucidate the involvement of transforming growth factor-alpha (TGF-alpha) in the pathogenesis of psoriasis, we measured TGF-alpha levels in the extracts from six normal epidermis and four psoriatic involved epidermis samples by enzyme-linked sorbent assay using monoclonal antibody specific for TGF-alpha. The amount of TGF-alpha in the extracts of normal epidermis was 1.45 +/- 1.06 ng/g of wet tissue, while the amount in psoriatic involved epidermis was 6.71 +/- 0.75 ng/g of wet tissue. The TGF-alpha level in psoriatic involved epidermis was thus 4.62 fold higher than that of normal epidermis (P less than 0.001). TGF-alpha binds to epidermal growth factor receptors and functions as an autocrine growth factor for epidermal keratinocytes. Therefore, the increased levels of TGF-alpha may be involved in the induction or the maintenance of hyperproliferation of psoriatic epidermal keratinocytes.     

Phorbol ester decreases cytosolic retinoic acid binding protein (CRABP) capacity in normal but not psoriatic epidermis

The retinoic acid binding protein (CRABP) characterized in human epidermal cytosol exhibits a three-fold increased binding capacity in lesional psoriatic epidermis as compared to normal or uninvolved skin. Treatment of epidermal homogenate from normal subjects by phorbol ester + ATP decreases the specific binding capacity of CRABP without affecting its dissociation constant (Kd=10 nM). The same effect was not observed in involved and uninvolved psoriatic epidermis. These results could be related to the previously reported decreased protein kinase C activity in psoriatic skin. They also suggest that posttranslational events could be responsible for pathological and pharmacological variations in CRABP binding capacity.     

Alterations in the desquamation-related proteolytic cleavage of corneodesmosin and other corneodesmosomal proteins in psoriatic lesional epidermis

Background  Desquamation occurs after proteolysis of corneodesmosomal proteins, including corneodesmosin (CDSN), by proteases of the kallikrein family, particularly KLK7. Impaired desquamation is one of the features of psoriasis, and psoriasis-associated single nucleotide polymorphisms of the CDSN gene may potentially modify the proteolysis of the encoded protein.
Objectives  To test whether the proteolysis of CDSN and other corneodesmosomal components is altered in psoriatic epidermis.
Methods  Total protein extracts obtained by tape-stripping of nonlesional and lesional skin from 11 patients were compared by immunoblotting experiments.
Results  An almost intact form of CDSN that has never been observed previously in the normal upper stratum corneum was detected in the lesional skin extracts, showing an altered proteolytic processing of the protein. This form was also observed in the nonlesional skin extracts, but in lower amounts. For most patients, increased amounts of desmoglein 1, plakoglobin and of high molecular weight fragments of desmocollin 1 were detected in the lesional skin. For most of them, similar amounts of KLK7 were immunodetected in both nonlesional and lesional skin extracts. No particular differences were observed related to the psoriasis type, the HLA-Cw6 status of the patients or any particular CDSN polymorphisms.
Conclusions  We detected a near full-length form of CDSN that has not been previously observed in normal stratum corneum. The results suggest a reduced degradation of all corneodesmosomal proteins in psoriatic lesions which probably reflects the persistence of corneodesmosomes.     

Influence of calcium on the proteolytic degradation of the calmodulin-like skin protein (calmodulin-like protein 5) in psoriatic epidermis

The calmodulin-like skin protein (CLSP) or so-called calmodulin-like protein 5, a recently discovered skin-specific calcium-binding protein, is closely related to keratinocyte differentiation. The 16-kDa protein is proteolytically degraded in the upper layers of the stratum corneum (SC) of healthy skin. With the use of specific new monoclonal antibodies to CLSP, we were able to demonstrate that the abnormal elevated levels of CLSP, characteristic of psoriatic epidermis, were probably not due to an overexpression of the protein, but most likely the result of its non-degradation. Further in vitro experiments using recombinant CLSP and in situ data clearly showed that calcium protected and chelator accelerated CLSP degradation. These data indicate that CLSP degradation in the SC of psoriatic skin might be hindered by the abnormally elevated calcium concentration. No degradation of CLSP in psoriatic epidermis keeping its ability to bind protein as transglutaminase 3 may have a physiological role in skin diseases such as psoriasis.     

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.     

Abnormal distribution of epidermal protein antigens in psoriatic epidermis.

The immunohistochemical distribution of the epidermal proteins filaggrin, involucrin, and cytokeratins is characteristic in normal epidermis. This distribution may change as a result of malignant transformation or abnormal differentiation. The present study was conducted to determine the patterns of reactivity of psoriatic epidermis to antibodies against various epidermal proteins and to clarify abnormal differentiation or maturation of the keratinocytes in psoriatic epidermis. Anti-human filaggrin, anti-human involucrin, and twelve kinds of anti-cytokeratin antibodies were used in this study. Cryostat or paraffin-embedded sections were stained with these antibodies by the avidin-biotin peroxidase technique. The epidermis of the noninvolved skin of patients with psoriasis vulgaris showed the distribution seen in normal skin. However, involved psoriatic skin revealed little or no reaction in the stratum corneum or in the granular layer with the anti-filaggrin antibody. Cells positively staining with anti-involucrin antibody paradoxically appeared in the lower cell layers of involved psoriatic epidermis. An anti-keratin antibody, AE1, stained suprabasal cells in involved psoriatic epidermis, although this antibody selectively stained epidermal basal cells in normal skin. The other anti-keratin antibodies, especially KL1, PKK1, and a polyclonal anti-keratin antibody, were less reactive with involved psoriatic skin than with normal skin. These observations suggest that the maturation pathway of keratinocytes in active psoriatic lesions differs qualitatively from that in normal epidermis.     

Perforin expression is upregulated in the epidermis of psoriatic lesions

BACKGROUND: There are currently very few data regarding the role of cell-mediated cytotoxicity in psoriasis. Both cytotoxic T lymphocytes and natural killer (NK) cells mediate cytotoxicity reactions, mainly by two distinct pathways, the perforin/granzyme and the Fas/Fas ligand pathway. OBJECTIVES: To study the expression and distribution of perforin, T- and NK-cell subsets in psoriatic lesional and nonlesional skin. METHODS: Skin biopsy specimens from both lesional and nonlesional skin of 11 patients with chronic plaque psoriasis and eight healthy controls were analysed by immunohistochemistry. RESULTS: We found a significant increase in CD4+ and CD8+ cells in psoriatic lesions compared with nonlesional and healthy skin. The expression of CD16+ NK cells was significantly lower in lesions compared with healthy skin. Perforin expression was significantly enhanced in the epidermis of psoriatic lesions. CONCLUSIONS: Perforin expression is upregulated in the epidermis of psoriatic lesions, suggesting a potential role for perforin in the creation of the psoriatic plaque.     

Studies of cell and organ culture of psoriatic and normal epidermis in vitro

Involved and noninvolved epidermis from 15 psoriatic patients, and epidermis from 12 normal individuals were grown under various conditions in vitro as cell and organ cultures. In the organ cultures of normal or noninvolved psoriatic skin, epidermal cells migrated around the dermis and differentiated. In psoriatic organ cultures, progressive necrotic changes were observed in the spinous cells including gradual detachment of the spinous layer from the dermis. In spite of these necrotic changes, newly-grown basal cells which appeared on the dermal tissue in the 9 to 12-day psoriatic explants clearly incorporated ³H-thymidine into nuclear DNA. By electron microscopy these cells were observed to have numerous ribosomes, a few mitochondria, tonofilament-like structures in the cytoplasm, and no hemi-desmosomes. Dissociated epidermal cells from the involved psoriatic skin failed to grow on plastic dishes. When these cells were plated on dishes coated with increasing amounts of collagen, the rate of cellular attachment was proportional to the thickness of the collagen coating, but they were unable to grow and form epidermal monolayers. In contrast, normal and noninvolved psoriatic epidermal cells attached to the dishes regardless of the presence of collagen and formed differentiated epidermal monolayers.     

Enzymatic dissociation of keratinocytes from human skin biopsies for in vitro cell propagation

Four techniques for dissociation of skin biopsies were compared to identify the method of choice for optimal expansion of isolated keratinocytes. Equivalent biopsies were obtained from 4 healthy human subjects and each divided into four parts. One part was minced and placed in a trypsinizing flask containing 0.05% trypsin and 0.01% ethylenediaminetetraacetic acid (EDTA). Released cells were harvested hourly. With the other parts, the epidermis was separated from the dermis after treatment with 0.5 mg/nml thermolysin, 2.5 mg/ml Dispase, or 0.17% trypsin and the epidermal portions were minced and incubated for 1 h in trypsin:EDTA. The cells were cocultivated with irradiated 3T3 fibroblasts to study the keratinocytes proliferative capacity. Freshly isolated cells were immunostained with anti-vimentin antibodies or grown in fibroblast-supportive conditions to detect the presence of human dermal fibroblasts. The mean number of cells dissociated per cm2 biopsy was higher after trypsin:EDTA digestion of a dermis-containing biopsy using a trypsinizing flask (4.0x 10(6) cells/cm2) compared to a biopsy where dermis-epidermis had been separated by thermolysin (2.8x 10(6) cells/cm2), Dispase (2.3x 10(6) cells/cm2) or trypsin (1.1 x 10(6) cells/cm2). Between 0.5% and 4% of the cells dissociated from a dermis-containing biopsy were human fibroblasts. This comprised more than twice the number of fibroblasts obtained by using epidermal/dermal split techniques. The proliferative capacity in primary and secondary culture was higher in cells isolated by trypsin:EDTA incubation in the trypsinizing flask or after epidermal-dermal separation using thermolysin, suggesting that Dispase or trypsin may have a more detrimental effect on the isolated keratinocytes. Our results show that dissociating the cells by trypsin:EDTA incubation in a trypsinizing flask or after epidermal-dermal separation using thermolysin, are preferable methods for isolating keratinocytes from human skin.     

Partial characterization of phospholipase C activity in normal, psoriatic uninvolved, and lesional epidermis

Arachidonic acid (AA), the precursor of prostaglandins and leukotrienes, can be directly liberated from membrane phospholipids by phospholipase A2 or indirectly by phospholipase C. One or both of these enzymes may be responsible for the increased content of AA found in psoriatic lesional epidermis. Keratome biopsies were obtained from normal and psoriatic individuals. After homogenization and sonication, a 10,000 g supernatant was used as the enzyme source. The activities of both phospholipase A2 and C were assayed in each sample using phosphatidylcholine and phosphatidylinositol, respectively, as substrates. Phospholipase A2 activity was found to be significantly higher than normal in both uninvolved and lesional psoriatic epidermis. In contrast, phospholipase C activity was significantly higher than normal in only the psoriatic plaque on the basis of wet weight (p less than 0.001), protein (p = 0.01), and DNA (p = 0.004) content. Phospholipase C activity in pmol diacylglycerol formed/min/microgram DNA was: normal 4.96 +/- 0.80, n = 13; uninvolved 7.29 +/- 1.06, n = 18; plaque 14.44 +/- 2.50, n = 18. Analysis (pH profile, calcium requirement, substrate specificity, and saturation kinetics) of pooled epidermal extracts showed no inherent differences in phospholipase C from normal and psoriatic epidermis, suggesting either a higher concentration or the presence of an activated form of the enzyme in psoriatic plaque. Since phospholipase C activity, in contrast to phospholipase A2 activity, is elevated only in lesional epidermis, it is possible that this enzyme contributes to AA accumulation observed in this tissue.     

Immunohistological detection of proliferating cells in normal and psoriatic epidermis using Ki-67 monoclonal antibody

Ki-67 monoclonal antibody (mAb) has been suggested to react only with proliferating cells. In order to study epidermal proliferating cells, immunohistological staining was performed with Ki-67 on 14 skin specimens from normal subjects and 30 from patients suffering from psoriasis. Staining of nuclei by Ki-67 was clearly observed in the epidermis. In normal epidermis, Ki-67+ cells were sparse in the basal layer (L1) and next upper layer (L2), while in psoriatic epidermis, they were abundant in L1, L2 and a few layers above L2. The percentage of positive cells in L1 of normal and psoriatic skin were 4.5 and 54%, respectively. Double staining (Ki-67 and S phase stain using bromodeoxyuridine) was also performed, and the results showed that: 1) the distribution patterns of Ki-67+ cells and those of S phase cells were similar in every section examined; 2) Ki-67 stained almost all S phase cells, and 3) Ki-67 also recognized considerable numbers of non-S phase cells. Therefore our data indicate that Ki-67 can detect a certain population of epidermal proliferating cells that includes S phase cells. Although it remains unclear whether all the proliferating cells in the epidermis can be detected by this mAb, we suggest that Ki-67 staining is an easy and useful technique for evaluating the proliferative activity of the epidermis.     

Histochemistry of the intercellular substance of the normal and psoriatic human epidermis

Summary The intercellular substance of skin samples obtained from normal subjects and from psoriatic patients has been studied with histochemical methods for carbohydrate containing substances and checked with enzymatic extractions. The surface coat which makes up most of the intercellular substance was stained with colloidal iron and with Alcian Blue solutions containing up to 0.20 M magnesium chloride; the stainings were heavily affected by the previous treatment of the sections with testicular hyaluronidase, but not with neuraminidase. The staining of the intercellular substance with Alcian Blue solutions containing up to 0.20 M magnesium chloride and the action of the hyaluronidase gives strength to the hypothesis that hyaluronic acid is contained in the substance. In the skin of psoriatic patients intercellular spaces wider than in normal skin and a reduced surface coat, particularly in the higher layers, has been observed.Paper presented at the V th International Congress of Histochemistry and Cytochemistry, Bucarest, 1976     

A multiparameter flow cytometric analysis of the effect of bexarotene on the epidermis of the psoriatic lesion

BACKGROUND: A new retinoid, bexarotene (Targretin), was recently investigated in a large multicentre trial for its efficacy and safety in psoriasis. Bexarotene is a novel retinoid X receptor (RXR)-selective ligand. OBJECTIVES: The aim was to study the effect of bexarotene in psoriasis by analysing markers for epidermal differentiation, proliferation and inflammation in epidermal single cell suspensions from lesions of patients with psoriasis treated with various doses of bexarotene. METHODS: Thirty-four patients with moderate to severe plaque psoriasis participated in this study and were assigned in sequence to four different dose regimens: 0.5, 1, 2 and 3 mg kg-1 once daily. Before and after 12 weeks of bexarotene treatment, punch biopsies were taken from lesional skin from which epidermal single cell suspensions were prepared using an optimized thermolysin protocol. A sum of scores was determined for each biopsy site, based on a four-point scale for erythema, induration and desquamation. An improved multiparameter flow cytometric assay was used that enabled simultaneous assessment of epidermal proliferation, various aspects of differentiation and epidermal inflammation. The following variables were measured simultaneously: relative DNA content, relative cell size, keratin (K) 10, K6 and vimentin expression. RESULTS: The psoriasis area and severity index (PASI) and sum of scores for the individual psoriatic lesion each showed a statistically significant decrease of 28% after 12 weeks of bexarotene treatment (P < 0.001). However, no significant dose-response effect was found. The total percentage of K10+ cells showed a significant increase of 43% (P < 0.01). The total population of K6 expressing cells did not show significant changes. Regarding the subpopulations of K6 single, K10 single and K6 and 10 co-expressing cells, a significant increase of 77% was seen in the K10+ K6- cells (P < 0.05), a significant decrease of 33% in K10- K6+ cells (P < 0.01), and no significant changes in the remaining population of K10+ K6+ cells. After 12 weeks of treatment with bexarotene no significant changes in epidermal proliferation and inflammation were shown. CONCLUSIONS: The present study indicates a direct effect of RXR activation by bexarotene on the transition of proliferation-associated keratinization into normal keratinization. Although no direct effect of bexarotene on DNA content in the total K10- cells was shown, further studies on subpopulations within the germinative layer such as stem cells and transit amplifying cells might be worthwhile.     

Protein kinase D distribution in normal human epidermis, basal cell carcinoma and psoriasis

BACKGROUND: Keratinocytes undergo a defined programme of proliferation and differentiation during normal stratification of the epidermis. Anomalies in the signalling pathways controlling this process probably contribute to the pathogenesis of hyperproliferative dermatological diseases, including psoriasis and basal cell carcinoma (BCC). We have previously proposed that protein kinase D (PKD) is a proproliferative signalling enzyme in keratinocytes and have speculated that abnormalities in its levels or regulation may contribute to hyperproliferative disorders of the skin. OBJECTIVES: To determine if hyperproliferative human skin disorders are characterized by abnormal protein expression or distribution of PKD, normal human epidermis was compared with BCC and uninvolved and involved psoriatic epidermis. METHODS: To examine protein expression, immunohistochemical analysis of human samples and Western blotting of neoplastic mouse keratinocytes was performed. Western analysis of neoplastic mouse cells using a phosphospecific PKD antibody allowed estimation of PKD activation status. RESULTS: Normal human epidermis demonstrated predominant PKD protein expression in the stratum basalis, the proliferative epidermal compartment, with decreased relative expression throughout the suprabasal strata. Uninvolved psoriatic skin showed a similar pattern, but in contrast, psoriatic lesions demonstrated a diffuse distribution of PKD staining throughout all strata. The majority of BCCs examined showed significant PKD protein levels and, in those biopsies in which the levels could be compared, elevated PKD levels relative to normal epidermis. PKD levels and activation status were also increased in a neoplastic mouse keratinocyte cell line. CONCLUSIONS: PKD was elevated or misdistributed in the hyperproliferative human skin disorders, BCC and psoriasis, as well as neoplastic mouse keratinocytes. We speculate that PKD exerts proproliferative and/or antidifferentiative effects in the epidermis, and that anomalous distribution and/or activation of PKD may be involved in precipitating or sustaining the disease process in BCC and psoriasis.     

In vivo quantification of microvessels in clinically uninvolved psoriatic skin and in normal skin

BACKGROUND: Microvascular abnormalities (capillary elongation, widening and tortuosity) are a characteristic feature of psoriasis and form one of the pathological diagnostic criteria. However, it is still not entirely clear when these microcirculatory changes appear in the skin of psoriatic subjects. Some studies suggest that capillary dilatation and elongation are present in the clinically uninvolved skin of psoriatic patients even at sites at which psoriatic plaques rarely occur. OBJECTIVES: To determine, using noninvasive techniques in vivo, the nature of any microvascular changes in the clinically uninvolved skin of psoriatic subjects and to quantify the dermal microvasculature in the clinically uninvolved skin of psoriatic subjects, in vivo. METHODS: Dermal microvessels in both the clinically uninvolved skin of psoriatic subjects and in the skin of normal volunteers (i.e. individuals without any clinical evidence of psoriasis or other inflammatory dermatoses) were directly visualized by native video-capillaroscopy, in vivo. Images were analysed using a combination of nonstereological and stereological measurements. The findings in each group were then compared to determine if there were any differences in microvascular parameters. RESULTS: Quantitative analysis of capillaroscopic images showed that there were no significant differences in microvessel density (P = 0.9), image area fraction (P = 0.6), microvessel length density (P = 0.7) and vessel image width (P = 1.0) in the clinically uninvolved skin of psoriatic subjects and the normal skin of healthy volunteers, when extensor forearm skin was examined. CONCLUSIONS: These findings indicate that prior to the development of clinical lesions there are no significant morphological differences between the dermal microvessels in the clinically uninvolved skin of psoriatic subjects and the dermal microvessels in the normal skin of healthy volunteers. However, during plaque formation, the superficial papillary microvessels in plaque skin undergo a striking, characteristic change, i.e. elongation, widening and tortuosity. These blood vessels must therefore, at least in part, play an important, necessary, but probably secondary role in the pathogenesis of clinical lesions in psoriasis.