Skin-derived antileukoproteinase (SKALP), an elastase inhibitor from human keratinocytes. Purification and biochemical properties.

Recently we reported a preliminary characterization of anti-elastase activity which is found in cultured keratinocytes and in epidermis from psoriasis patients, but not in normal human epidermis. Here we present evidence that this inhibitory activity is derived from a cationic protein with a molecular mass of 18 kDa. In psoriatic scales the inhibitor is mainly present as a biologically active 11 kDa fragment. Inhibition of human leukocyte elastase is strong (Ki = 2 x 10(11) M) and fast (kon = 10(7) M-1.s-1). Using chromatofocusing, affinity chromatography and gel-permeation FPLC, the 11 kDa fragment was purified from psoriatic scales. This preparation was reduced and carboxymethylated, blotted onto poly(vinylidene difluoride) membrane and subjected to N-terminal gas-phase sequencing. Within a stretch of 16 amino acids a 40% homology was found with the active site of antileukoproteinase (ALP) a known serine proteinase inhibitor present in mucous secretions. We therefore propose the acronym SKALP (skin-derived antileukoproteinase) as a name for this elastase inhibitor.

Immunohistochemical localization of SKALP/elafin in psoriatic epidermis.

Recently we have reported the purification and biochemical characterization of a new, inducible elastase inhibitor [skin-derived antileukoproteinase (SKALP)], which could be extracted in high amounts from psoriatic skin but not from normal human skin. Here we demonstrate the immunohistochemical localization of SKALP in psoriatic epidermis. SKALP was found exclusively in the upper layers of the suprabasal compartment and stratum corneum of lesional psoriatic epidermis. Basal keratinocytes were always negative. No immunoreactive SKALP was found in normal epidermis and non-lesional psoriatic epidermis, in accordance with findings in functional assays. Western blots of skin extracts from psoriatic and normal skin confirmed the immunohistochemical findings and revealed two major bands with apparent molecular weights of 10.5 and 11.5 kDa. We would hypothesize that SKALP could act as a modulator of epidermal inflammation by interfering with polymorphonuclear leukocyte trafficking, and that it could protect structural proteins against elastase-mediated damage.

Levels of skin-derived antileukoproteinase (SKALP)/elafin in serum correlate with disease activity during treatment of severe psoriasis with cyclosporin A.

The epidermal serine proteinase inhibitor SKALP (also known as elafin), directed against human leukocyte elastase and proteinase 3, is strongly induced in suprabasal keratinocytes during inflammation. The presence of SKALP/elafin in urine has been demonstrated for several inflammatory skin disorders, such as psoriasis, erythroderma, and erysipelas. In this study we investigated whether SKALP/elafin levels in serum and urine of psoriatic patients can be used as a marker for disease activity during treatment. Patients with severe chronic disabling psoriasis were treated for 16 weeks with cyclosporin A, which resulted in a marked clinical improvement as measured with the PASI score. SKALP/elafin levels both in serum and urine were determined with an enzyme-linked immunosorbent assay (ELISA). Measurements were performed at the start of the cyclosporin A treatment, and after regular intervals up to 16 weeks. The results indicate that 1) SKALP/elafin determination in serum rather than in urine is the preferred method, because the decrease in serum SKALP levels during therapy is more pronounced and correlated better with the clinical course of the patients; 2) SKALP/elafin levels in serum decreased during cyclosporin A treatment (p < 0.05); and 3) SKALP/elafin levels in serum correlate with the PASI score (p < 0.01). We conclude that SKALP/elafin measurement in serum of patients with severe psoriasis provides a tool for monitoring disease activity.

Tenascin-C expression in human epidermal keratinocytes is regulated by inflammatory cytokines and a stress response pathway.

Recently we showed that human epidermal keratinocytes express the extracellular matrix protein tenascin-C (TN-C) during wound healing, but not in normal adult skin. To gain further insight into the regulation of epidermal TN-C expression, we tested the effect of various stimuli on TN-C expression by cultured keratinocytes. Our results indicate that IL-4 is a very strong inducer of TN-C protein and mRNA expression in normal keratinocytes. Furthermore, TNFalpha and IFNgamma moderately increased TN-C expression. No other cytokines and growth factors that we tested, including various factors that stimulate TN-C expression in mesenchymal cells, significantly affected TN-C secretion by cultured keratinocytes. The regulation of TN-C expression in keratinocytes is distinct from that of fibronectin, since IL-4 and IFNgamma did not affect fibronectin expression in our experiments, and TNFalpha only slightly increased fibronectin levels. To investigate the role of cellular stress response pathways that can be activated by TNFalpha in the regulation of TN-C expression, we tested the effect of different inhibitors and an activator of these intracellular signalling cascades. The results show that the p38 MAP-kinase pathway is not involved in TNFalpha-induced TN-C expression in cultured keratinocytes. Activation of the JNK/SAPK-1 pathway by the addition of sphingomyelinase resulted in a dose-dependent increase of TN-C expression. TN-C expression by squamous carcinoma cell lines was differentially affected by the cytokines that stimulated TN-C expression in normal keratinocytes: TNFalpha again increased TN-C secretion, but IL-4 and IFNgamma had little effect. We conclude that there are distinct regulation mechanisms for TN-C expression in normal keratinocytes, tumor-derived keratinocytes and mesenchymal cells. The observation that TN-C is abundant in inflamed skin is a strong indication that inflammatory cytokines such as IL-4, TNFalpha and IFNgamma could also be involved in the regulation of epidermal TN-C expression in vivo.

Cell kinetic characterization of the epidermal growth factor dependent BALB/MK line using flow cytometric analysis of DNA content and iododeoxyuridine incorporation.

Epidermal hyperproliferation (psoriasis, wound repair) is the result of quiescent (G0) keratinocytes being recruited into the cell cycle. A detailed characterization of the cell cycle kinetic parameters of the mouse keratinocyte line (Balb/MK) has been carried out with the aid of bivariate iododeoxyuridine (IdUrd) and DNA analysis using flow cytometry, in order to establish whether it might provide a useful model for the study of the biochemical events controlling recruitment into the cell cycle. Balb/MK keratinocytes were cultured using low Ca2+ Dulbecco's modified Eagle's medium/F 12 in the presence of 10% dialysed fetal bovine serum and 4 ng/ml epidermal growth factor (EGF). IdUrd labelling followed by flow cytometric analysis of trypsinized cells showed that about 95% of the population were actively cycling, with a cell cycle time of around 14h and no significant contact inhibition. Omission of serum and EGF followed by IdUrd pulse-labelling indicated that the cells progressively withdrew from the cycle and, after 16h, less than 10% incorporated IdUrd. Subsequent restimulation with serum resulted in a synchronized cohort of cells being recruited. Entry into the S phase of the cell cycle (IdUrd incorporation) started at 8 h and was maximal between 12 h and 16h after stimulation. Restimulation with EGF alone reached a growth fraction of 87% after 24 h continuous labelling compared with 97% using serum together with EGF. Epidermal growth factor already showed a significant stimulation at 10 pg/ml compared with the controls. There is a broad plateau centred on 5 ng/ml, followed by a slight decline above this level. We conclude that the use of a cell line with defined cell cycle kinetic parameters which can be switched between the quiescent and cycling states in a fully defined medium will provide an ideal model for biochemical studies of the relevant signal transduction pathways in keratinocytes.

Expression of tenascin-C splice variants by human skin cells.

Tenascin-C is an extracellular matrix glycoprotein that is expressed in a spatially and temporally restricted pattern. Various functionally different tenascin-C isoforms can be expressed as a result of alternative splicing of the pre-mRNA. Previously we identified human epidermal keratinocytes as a source of tenascin-C in healing wounds. In this study, we investigated whether different tenascin-C transcripts are expressed by epidermal keratinocytes and dermal fibroblasts. In addition, we compared expression of tenascin-C splice variants at the mRNA and protein levels in tissue samples of normal and diseased skin. Northern blot analysis revealed two major tenascin-C mRNA transcripts of approximately 7500 and 5800 nucleotides in cultured epidermal keratinocytes and fibroblasts, and in biopsies. Although both dermal fibroblasts and epidermal keratinocytes predominantly expressed the larger tenascin-C mRNA, epidermal keratinocytes expressed smaller transcripts at higher levels than dermal fibroblasts. In keratinocytes the levels of the two mRNAs were differentially affected by inflammatory cytokines that increased tenascin-C expression in these cells. The addition of IFN gamma slightly increased the proportion of large transcripts. In contrast, TNF alpha favoured expression of smaller tenascin-C transcripts, and IL-4 equally affected the expression of large and small tenascin-C mRNAs. To enable detection of tenascin-C transcripts that are expressed at very low levels, we amplified by polymerase chain reaction the fibronectin type III repeats whose expression is regulated by alternative splicing. In cDNA of cultured keratinocytes and fibroblasts, and in skin biopsies, several tenascin-C transcripts could be detected that corresponded to tenascin-C variants including different numbers of fibronectin type III repeats. Distribution of tenascin-C isoforms at the protein level was studied immunohistochemically in healthy skin, wounds, psoriatic lesions and epidermal tumours and hyperplasia. No differences were observed in reactivity between an antibody that binds all tenascin-C isoforms and antibodies that bind fibronectin type III repeats that can be spliced out from smaller tenascin-C isoforms. We conclude that the tenascin-C isoforms that are translated from transcripts that we identified at the mRNA level seem to be distributed similarly in the conditions investigated.

Flow cytometric and microscopic characterization of the uptake and distribution of phosphorothioate oligonucleotides in human keratinocytes.

Gene-specific inhibition by antisense oligonucleotides has been successful in a large number of systems. In an attempt to use this strategy for the modulation of skin disease-specific gene expression, we studied oligonucleotide uptake in cultured human keratinocytes. This study revealed a heterogeneous uptake of fluorescently labeled phosphorothioate oligonucleotides. Flow cytometric and microscopic analysis showed two fluorescent cell populations with differences in intensity: a 'bright' population of highly fluorescent small cells and a 'dim' population of less fluorescent but larger cells. The heterogeneity in uptake between these two populations was not a result of differences in cell cycle phases of the keratinocytes, as shown by flow cytometric sorting and measurements of relative DNA content. In both populations the oligonucleotides were transported intracellularly and were mainly located in the cytoplasm. A typically speckled localization pattern was demonstrated by confocal laser scanning microscopy. We used propidium iodide (PI) to assess viability, and showed that in nonviable (PI-permeable) keratinocytes the oligonucleotides accumulated in the nucleus. The use of a lipidfection reagent also changed the intracellular distribution of oligonucleotides from a punctate cytoplasmic pattern to an intense nuclear localization. The process of uptake by the viable keratinocytes was dependent on oligonucleotide concentration, incubation time and temperature. This study underlines the importance of kinetic studies on oligonucleotide uptake in human keratinocytes which must be considered when specific oligonucleotides are used against skin disease-specific genes.

The growth and differentiation of human keratinocytes in vitro: a combined immunohistochemical and flow cytometric study.

In this study we performed a cell kinetic characterization of the growth and differentiation of human keratinocytes. Using a combination of immunohistochemical and flow cytometric techniques it was possible to obtain a detailed description of these processes. The proliferative activity of the cell cultures was analysed with flow cytometric techniques, measuring relative DNA content, iododeoxyuridine incorporation and the expression of the antigen recognized by Ki-67. In addition to a standard monolayer culture technique, cells were maintained in suspension. Under these conditions these cells were not capable of dividing, started to lose their nuclei, and the expression of differentiation-related proteins such as involucrin and filaggrin was induced, suggesting that the cells changed towards a differentiated phenotype. Binding of the antibody Ks8.12, recognizing keratins 13 and 16, occurred under all culture conditions, independent of cell density, and also in suspension, suggesting that it is a marker for abnormal differentiation rather than for hyperproliferation.

Skin-derived antileukoproteinase (SKALP) is decreased in pustular forms of psoriasis. A clue to the pathogenesis of pustule formation?

Skin-derived antileukoproteinase (SKALP, also known as elafin) is an inducible epidermal serine proteinase inhibitor, that we have recently characterized at the protein and DNA levels. SKALP is a strong and specific inhibitor of PMN elastase, and is putatively involved in the regulation of cutaneous inflammatory processes. In order to investigate the role of SKALP in the control of elastase in psoriatic epidermis, we compared SKALP expression in normal skin, and in skin from patients with chronic plaque psoriasis and pustular forms of psoriasis. Epidermal scales and biopsies were collected and SKALP expression was studied at the mRNA level and at the protein level both functionally and immunochemically. In epidermal scales, we found that the levels of both free and total SKALP activity in pustular psoriasis were far lower than in plaque psoriasis. A significant number of pustular psoriasis patients showed latent SKALP activity, which represents the amount of SKALP putatively complexed to elastase. In addition, we found free elastase activity in 25% of the pustular psoriasis patients, indicating a total saturation of epidermal SKALP activity. In epidermal biopsies from pustular psoriasis patients, SKALP activity was significantly decreased compared with those from plaque psoriasis patients. Northern blot analysis did not reveal differences in epidermal mRNA levels between chronic plaque psoriasis and pustular psoriasis. We hypothesize that a reduced amount of epidermal SKALP contributes to an imbalance between elastase and its inhibitor, thereby promoting the formation of epidermal pustules. We suggest that these findings could provide a rationale for the treatment of pustular psoriasis with inhibitors of PMN-derived proteinases, as a new therapeutic modality.

MON-150, a versatile monoclonal antibody against involucrin: characterization and applications.

A monoclonal antibody, designated MON-150, was found serendipitously to react strongly with the granular layer of normal human epidermis and with the upper spinous layers of psoriatic epidermis. From analysis by flow cytometry of cultured human keratinocytes, it appeared that the percentage of MON-150-positive cells strongly increased when the cells reached confluence and the growth fraction declined. To identify the antigen recognized by MON-150, a lysate of human keratinocytes was subjected to affinity chromatography using a MON-150 Sepharose column. This yielded a single protein of approximately 350 kDa as measured on Superose 6 FPLC gel permeation chromatography using non-denaturing conditions. In Western blot analysis under denaturing and reducing conditions, a 140-kDa protein was detected. The subcellular localization and the molecular weight of the antigen recognized by MON-150 suggested that the antigen involved might be involucrin. This was confirmed using a commercial polyclonal antiserum against involucrin. We conclude that MON-150 is a new, versatile antibody against human involucrin.

The effect of the PDE-4 inhibitor (cipamfylline) in two human models of irritant contact dermatitis.

BACKGROUND: New therapeutic approaches have to be considered in the treatment of irritant contact dermatitis (ICD). Recently, phosphodiesterase 4 (PDE-4) inhibitors have been introduced as nonsteroidal, antiinflammatory agents. These agents inhibit the secretion of the cytokines thought to be involved in the pathogenesis of ICD. We investigated the effect of a new selective PDE-4 inhibitor (cipamfylline) in human models using single and repeated exposures to an irritant in a blind, randomized pilot study with healthy volunteers. We compared the effect of cipamfylline ointment with a strong corticosteroid (betamethasone-17-valerate) and with a placebo ointment. METHODS: Ten volunteers were patch tested at four investigation sites with sodium dodecyl sulphate (1%) for 24 h. In a model that simulates chronic damage, 11 volunteers were patch tested with sodium dodecyl sulphate (0.2%) for 4 h daily for four consecutive days. The investigation sites were treated once a day with the above-mentioned agents. One site was left untreated. We used erythema scoring, measurements of transepidermal water loss (TEWL) and several immunohistochemical markers for epidermal proliferation and differentiation. RESULTS: Repeated application revealed that betamethasone-17-valerate caused a statistically significant reduction in erythema and TEWL compared to cipamfylline and placebo. We also observed a significant suppression of proliferating cells and cytokeratin 16 expression at sites treated with betamethasone compared to the other sites. In the model for acute ICD, no significant differences were seen between the investigated sites. CONCLUSIONS: Our results show that betamethasone-17-valerate may modulate the response in ICD. In this human model of ICD we could not confirm the efficacy of cipamfylline. Clinical studies are needed before the effect of PDE-4 inhibitors in ICD can be refuted with certainty.

Reliability of the proximal skin paddle of the osteocutaneous free fibula flap: a prospective clinical study.

The vascularization of the skin paddle of 20 osteocutaneous fibula free flaps in 20 patients was studied. All skin paddles were designed over the proximal and middle third of the fibula. A parallel vascularization of the skin was found in 10 cases. In these cases, an axial (septo)musculocutaneous perforator was found to originate high in the peroneal artery or even in the popliteal artery. This branch runs parallel to the peroneal artery without any further connections with it. In 5 of these 10 cases, no other skin perforators were located within the boundaries of the skin paddle. Harvesting such a flap in the traditional way by blind inclusion of a muscle cuff results in ligation of the supplying vessel of the skin paddle and subsequent loss of the skin. In this series, this would have been the case in 5 of the 20 patients (25 percent). This might explain the bad reputation of the skin paddle of this flap. The high prevalence of the described vascular configuration in a proximally designed skin paddle justifies à vue dissection of all musculocutaneous perforators up to their origin, unless one or more septocutaneous perforators are found within the boundaries of the flap.

Factors influencing nickel dermatitis. II.

The nickel concentrations in urine and other data of a very hypersensitive female patient have been followed during two periods exceeding 30 days each. Only a limited degree of correlation was found between the course of the nickel concentration in urine and the clinical activity of the dermatitis. In order to better evaluate the measure of the correlation and the influence of some other factors upon the activity of the dermatitis, a pathway analysis scheme has been constructed. Consideration of this scheme reveals the need for more extensive data regarding the nickel ion climate in the body.

Dithiocarbamate therapy for nickel dermatitis.

Increased internal exposure to nickel can cause an exacerbation of nickel contact dermatitis. Nickel ions are chelated by diethyldithiocarbamate (DDC) and thereby inactivated. An oral dose of about 1 g DDC/day was given to a patient. The nickel excretion in the urine increased about tenfold; the nickel elimination in scalp hair did not increase. The slightly negative nickel balance did not exhaust the nickel content of the organs appreciably with a dose of 1.2 g DDC/day for 2 months. At the end of this experiment patch tests with nickel sulphate were still positive though less local therapy was needed, and the cross correlation between the activity of the eczema and the nickel concentration in the urine had lost its former periodicity. It is therefore not yet possible to conclude whether or not DDC may be really of help in the very nickel hypersensitive patient by reducing the exposure to nickel originating in food and other environmental sources.

Factors influencing nickel dermatitis. I.

The nickel concentrations in urine and blood plasma of a very hypersensitive female patient have been followed during two periods of 34 and 42 days each. A limited degree of correlation was found between the course of the nickel concentration in plasma, the nickel concentration in urine and the clinical activity of the dermatitis. Evidently other factors also influence the activity of the dermatitis; among these menstruation and stress might be expected to play a role.

Flow cytometry as a tool for the study of cell kinetics in epidermis.

Flow cytometric measurements of the DNA content were performed on a large number of skin biopsies by an automated technique. Expressed as a percentage of all viable cells in the epidermis, the figures for cells in S-phase averaged 1.8% and for G2M 0.9%. No significant differences due to sex were found. Concomitantly with age the ratio S/G2M (representing the duration of S to the duration of G2M) increased. Also seasonal effects were clear, showing higher values for S and G2M in June compared to November and December. Lastly we found small differences dependent on body-site, the ratio S/G2M being greater in legs than in arms. The present status is discussed together with future lines of development.

Skin-derived antileucoproteases (SKALPs): characterization of two new elastase inhibitors from psoriatic epidermis.

Elastase inhibiting activity (EIA) was demonstrated in the epidermis from lesions and in psoriatic scale, whereas normal epidermis did not contain significant EIA. Two new elastase inhibitors were partially purified and characterized using psoriatic scale as a source. The two species (approximate molecular weights 10 and 20 kDa) were shown to be stable, and high-affinity inhibitors of human leucocyte elastase (Ki less than 10(-10) M). No activity against human cathepsin G could be demonstrated. Cultured human keratinocytes were shown to contain EIA activity similar to that found in psoriatic scale. EIA could also be demonstrated in human epidermis following the induction of an experimental inflammatory response by sellotape-stripping. We propose the acronym SKALP (skin-derived antileucoprotease) as a name for these new proteinase inhibitors.

Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy-SNARF-1 and flow cytometry.

In this study we describe a method to measure intracellular pH in cultured human keratinocytes using flow cytometry. Keratinocytes pose a technical problem because the population is heterogeneous with respect to size and metabolic activity (nonspecific esterase activity), resulting in variability in dye uptake. In order to compensate for this, dyes were selected that change colour with pH. The ratio of fluorescence intensities at two wavelengths was recorded and used as a measure of intracellular pH by reference to the pH in the presence of the proton ionophore nigericin. However, methods published till now do not routinely combine the ratiometric technique and excitation with an argon ion laser set at 488 nm. Therefore we have tested the recently developed pH-sensitive dye carboxyseminaphthorhodafluor-1 (SNARF-1) as a possible candidate for flow cytometric pH measurements and compared it with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and 2,3-dicyanohydroquinone (DCH) with respect to emission spectra, resolution, range, and stability of cellular fluorescence. SNARF-1 had a practical and stable excitation wavelength of 488 nm rather than UV, it offered the possibility of ratiometric measurements on the basis of a real emission shift, and had superior resolution for the pH range 7-8. With SNARF-1 we found that keratinocytes cultured under low serum conditions (0.2%) contain a higher proportion of cells with relatively low intracellular pH compared to high serum cultures (6%). Furthermore, pH changes were followed by changes in relative DNA content. These findings suggest that intracellular pH can be an early functional proliferation marker for human keratinocytes.