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.

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 characterization of normal versus psoriatic epidermis using improved cell separation methodology.

Recently a new approach for epidermal cell characterization was developed: three-parameter flow cytometrical analysis of pure and complete epidermal cell suspensions prepared from punch biopsies followed by dermoepidermal separation by thermolysin. The aim of the present communication is the comparison between psoriatic lesional skin and normal skin using this new approach with respect to the percentage of suprabasal keratinocytes (keratin 10+ cells), mesenchymal cells, including the infiltrate cells (vimentin+ cells) and the percentage of basal cells in SG2 M phase, in order to validate this methodology in studies on psoriatic skin. Punch biopsies were taken from 7 healthy volunteers and in 7 psoriatic patients 4 biopsies were taken in each of them from comparable lesions. The present study reconfirmed that the percentage of basal keratinocytes in psoriasis was increased and the percentage of keratin 10+ cells was substantially decreased as compared to normal skin. The new methodology revealed data with a narrow range. In psoriatic lesional skin the intra individual variation was less compared to the inter individual variation.

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.

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.

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.

Multi parameter flow cytometric assessment of regenerative epidermis with special reference to the antiproliferative effect of occlusion.

Multi parameter flow cytometrical assays permit simultaneous assessment of proliferation, differentiation, and inflammation parameters. In this study, the validation of TO-PRO-3 iodide (TP3) compared to propidium iodide (PI) and DE-K10 compared to RKSE60 were evaluated in tape stripping induced hyperproliferation. No occlusion, Duoderm (intermediate occlusion) and Blenderm (maximal occlusion) were used as a model to evaluate the effect of occlusion on epidermal regeneration. Proliferation in the keratin 10-negative compartment measured with TP3 proved to be a good approximation of proliferation measured with PI. Other epidermal subpopulations (keratin 10-dim and -bright cells) did not make a relevant contribution to hyperproliferation. DE-K10 is probable more sensitive than RKSE60 to distinguish populations that differ in degree of differentiation. Occlusion of tape stripped skin resulted in decreased proliferation and increased differentiation. This effect was most pronounced with maximal occlusion. This study showed that occlusion is a therapy, which realises normalisation of hyperproliferative skin disorders.

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.

A novel dithranol formulation (Micanol): the effects of monotherapy and UVB combination therapy on epidermal differentiation, proliferation and cutaneous inflammation in psoriasis vulgaris.

Micanol, in which dithranol is micro-encapsulated in crystalline monoglycerides, is easy to wash off and staining and irritation are inconspicuous. These features make it appropriate to use in an out-patient setting. In this study the immunohistochemical effects of this new dithranol formulation were studied and compared with UVB and the combination of these therapies in skin biopsies of 8 patients with psoriasis. Markers for epidermal differentiation, proliferation and cutaneous inflammation were assessed. The present study suggests that Micanol predominantly had diminishing effects on inflammation markers, hardly affecting the epidermis. UVB had a broad spectrum of reductions. It is feasible that the combination resulted in various synergistic effects. Previous studies, however, revealed a relative persistence of the inflammatory infiltrate with more effects on epidermal processes following dithranol treatment. Based on the present study and on previous studies, it is hypothesised that Micanol delivers the active substance more directly in the dermal infiltrate, leaving the epidermis relatively unaffected. This might explain the low irritancy of Micanol treatment.

Cell kinetic characterization of cultured human keratinocytes from normal and psoriatic individuals.

Psoriasis is a chronic skin disease characterized by epidermal hyperproliferation, disturbed differentiation, and inflammation. It is still a matter of debate whether the pathogenesis of psoriasis is based on immunological mechanisms, on defective growth control mechanisms, or possibly on a combination of both. Several in vivo cell biological differences between psoriatic lesional epidermis and normal epidermis have been reported. However, it is not clear whether these changes are causal or consequential. In case that keratinocytes from psoriatic patients have genetically determined deficiencies or polymorphisms with respect to autocrine growth regulation and the response to inflammatory cytokines, we hypothesize that these differences should be maintained in culture. Here we have started a systematic comparison of first passage keratinocytes cultured from normal skin and uninvolved psoriatic skin to address the question whether there are intrinsic differences in basic cell cycle parameters. In an established, defined culture system using keratinocyte growth medium (KGM) we have determined: (i) cell cycle parameters of exponentially growing keratinocytes, (ii) induction of quiescence by transforming growth factor beta 1 (TGF-beta 1) and (iii) restimulation from the G0-phase of the cell cycle. Bivariate analysis of lodo-deoxyuridine incorporation and relative DNA content was performed by flow cytometry. Within the limitations of this model no gross differences were found between normal and psoriatic keratinocytes with respect to S-phase duration (Ts), total cell cycle duration (Tc), responsiveness to TGF-beta 1 and the kinetics for recruitment from G0. In psoriatic keratinocytes we found a lower amount of cell in S-phase and a shorter duration of G1, compared to normal keratinocytes. The methodology developed here provides us with a model for further studies on differences between normal and psoriatic keratinocytes in their response to immunological and inflammatory mediators.

Induction of normal and psoriatic phenotypes in submerged keratinocyte cultures.

Lesional psoriatic epidermis displays a number of phenotypic changes that are distinct from the differentiation program found in normal interfollicular epidermis. In psoriatic epidermis, keratinocytes are hyperproliferative and several differentiation-associated molecules are expressed that are absent in normal skin (e.g., cytokeratins (CK) 6, 16, and 17, and the epidermal proteinase inhibitor SKALP/ elafin). In addition, several molecules which are normally restricted to the stratum granulosum are strongly upregulated in the stratum spinosum (e.g., psoriasis-associated fatty acid binding protein (PA-FABP), psoriasin, involucrin, and transglutaminase). The aim of this study was to develop in vitro culture systems which (a) would allow to study the induction of normal and psoriatic differentiation pathways, and (b) would be amenable for screening of antipsoriatic drugs. Here we have investigated several models for induction of differentiation with respect to the expression of markers for the normal and psoriatic phenotype. Cell cycle parameters and expression levels of CK1, CK10, CK16, SKALP/elafin, transglutaminase, involucrin, psoriasin, and PA-FABP were assessed in these models using flow cytometry, immunocytochemistry, and Northern blot analysis. We observed that induction of differentiation with fetal calf serum resembled the psoriatic phenotype (sustained hyperproliferation; high levels of CK16, SKALP/elafin, transglutaminase, and involucrin; moderate psoriasin expression), whereas differentiation induced by growth factor depletion in a confluent culture resembled the normal differentiation phenotype (low proliferative rate; high expression levels of CK1 and CK10; moderate expression of involucrin and transglutaminase; low expression levels of SKALP/elafin and CK16; absence of psoriasin). We propose that these models can be used to study expression and pharmacological modulation of selected differentiation genes and the coordinated expression of sets of genes associated with epidermal differentiation programs.

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.

Cell kinetic characterization of growth arrest in cultured human keratinocytes.

In this study we have performed a cell kinetic characterization of growth and growth arrest of keratinocytes derived from normal human skin. Proliferative activity of the cell cultures was analysed with a flow cytometric technique, measuring relative DNA content and iododeoxyuridine (IdUrd) incorporation simultaneously. Normal human keratinocytes were grown in keratinocyte growth medium (KGM) and growth arrest was induced by using either keratinocyte basal medium (KBM) or KGM supplemented with TGF-beta 1. It was found that human keratinocytes grown in KGM plus TGF-beta 1 were growth-arrested within 52 hours. The rate of IdUrd incorporation into DNA decreased by more than 95% after 52 hours and paralleled the decrease of cells in S-phase. Within 52 hours after addition of TGF-beta 1, 79% of the growth-arrested cells were in the G0/G1-phase of the cell cycle, a situation that approaches that of the normal epidermis. Growth arrest of human keratinocytes in KBM showed a similar decrease in the rate of IdUrd incorporation. However, the decrease in IdUrd incorporation was not reflected in a decrease in cells in S-phase, suggesting that the cells were blocked in G0/G1, S or G2/M-phase rather than selectively in the physiological growth arrest state of G0/G1. Secondly, we investigated the kinetics of the cells when they were restimulated after growth arrest. We found that after termination of the growth arrest in KGM supplemented with TGF-beta 1 the cells require 6 to 8 hours to initiate DNA synthesis, with a continued decrease in the G0/G1 population, suggesting that the cells are recruited as a cohort. After growth arrest induced by KBM, cells also require 6 to 8 hours in KGM to initiate DNA synthesis, but the cells are not recruited as a cohort. We conclude that growth arrest induced by TGF-beta 1 is the preferred system in which to study induction of keratinocyte proliferation, since it induces a state of quiescence that approaches that of normal human epidermis.

SKALP/elafin is an inducible proteinase inhibitor in human epidermal keratinocytes.

Skin-derived antileukoproteinase (SKALP), otherwise known as elafin, is a recently discovered epidermal proteinase inhibitor with specificity for polymorphonuclear leukocyte (PMN)-derived elastase and proteinase-3; in addition to the proteinase-inhibiting domain, SKALP contains several transglutaminase substrate motifs. SKALP is virtually absent in normal human epidermis but is found in a number of inflammatory skin diseases, including psoriasis. Here we report the induction and processing of SKALP in vivo and in vitro. SKALP expression in vivo could be demonstrated following injury in normal human epidermis, using histology, western blotting, northern blotting and a functional assay. In vitro, SKALP expression was studied in conventional submerged keratinocyte culture systems and in keratinocytes cultured in an air-liquid interface model. Induction of SKALP activity in epidermis could be measured as early as 16 hours after skin injury; immunohistological examination showed that SKALP expression was confined to the outer layers of the stratum spinosum and the stratum granulosum. Northern blot analysis revealed a 0.8 kb transcript, both in vivo (psoriatic skin, injured skin) and in vitro (cultured keratinocytes). Western blot analysis showed that the major SKALP form in vivo was a low molecular mass fragment, containing the antiproteinase domain. In all cultures that were positive for SKALP, larger (8-10 kDa) forms of SKALP, containing the N-terminal transglutaminase substrate motifs in addition to the antiproteinase domain, were found. SKALP expression in cultured cells was found to be dependent on the system used. In a submerged culture system, SKALP could be induced by fetal calf serum.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

SKALP/elafin: an elastase inhibitor from cultured human keratinocytes. Purification, cDNA sequence, and evidence for transglutaminase cross-linking.

SKALP/Elafin is a proteinase inhibitor found in psoriatic epidermis as a short polypeptide of 6 kDa. Here we present evidence that this protein is synthesized as a larger precursor molecule with distinct biological features. Purification and NH2-terminal sequencing of SKALP/elafin from cultured human keratinocytes and the cloning of its cDNA revealed the existence of a mature protein, which upon cleavage of a hydrophobic signal sequence of 22 amino acids has a calculated molecular mass of 9.9 kDa (95 amino acids). In addition to the known proteinase inhibitor domain, the mature protein contains a domain with 4 repeats which are homologous to putative transglutaminase substrate motifs. We were able to demonstrate on Western blots that immunoreactive SKALP is present in high molecular weight proteins extracted from psoriatic skin. This suggests that SKALP is covalently attached to epidermal proteins. In addition it was found that both the complete SKALP molecule and a synthetic peptide of the NH2-terminal portion of SKALP could be used as a transglutaminase substrate. We therefore speculate that SKALP/elafin, secreted by epidermal keratinocytes in inflamed skin, exists both as a free 6-kDa form and as an immobilized 9.9-kDa form covalently attached to the cornified envelopes by transglutaminase cross-linking.