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The skin barrier is composed of a thin horny layer, which prevents water loss and intrusion of noxious factors, and a thicker, viable layer of epidermis, which is strongly attached to the underlying dermis. Serious impairment of the skin barrier may result from genetic diseases interfering with the attachment and/or terminal differentiation of keratinocytes. In epidermolysis bullosa, defects in the anchoring proteins of epidermis cause neonatal blistering and a life-long problem with widespread skin erosions. In congenital ichthyosis, various enzyme deficiencies (transglutaminase 1, steroid sulfatase, etc) or mutations in structural proteins (cytokeratins, plakophilin, etc) cause massive hyperkeratosis and/or inflammation resulting in chronic problems with the skin barrier. Our increasing knowledge of the etiology of these diseases has already facilitated diagnosis and genetic counseling. Hopefully this knowledge will also pave the way for new remedies, including cutaneous gene therapy for the most severe conditions.

To maintain a constant thickness of the stratum corneum the desquamation rate and the de novo production of corneocytes is delicately balanced. Using a plantar stratum corneum model we have obtained evidence that proteolysis is a central event in the desquamation process. A number of regulatory mechanisms for desquamation have been postulated based on our findings.

Stratum corneum chymotryptic enzyme may be important in desquamation. It has also been suggested that other proteases, especially stratum corneum tryptic enzyme, may be involved. Stratum corneum tryptic enzyme has been purified and its cDNA has been cloned. Results from expression analyses indicate that stratum corneum tryptic enzyme is as skin specific as stratum corneum chymotryptic enzyme. In this work we have produced and characterized antibodies specific for stratum corneum tryptic enzyme. We have also by means of biochemical, immunochemical, and immunohistochemical methods performed studies on stratum corneum tryptic enzyme in normal human epidermis. Antibodies against bacterial recombinant stratum corneum tryptic enzyme were produced and purified by affinity chromatography. Two types of antibodies were obtained: one reacting only with pro-stratum corneum tryptic enzyme and one specific for the catalytically active part of stratum corneum tryptic enzyme. Immunohistochemistry with the antibodies reacting with pro-stratum corneum tryptic enzyme showed a staining pattern similar to stratum corneum chymotryptic enzyme-specific antibodies, i.e., the expression was confined to cornifying epithelia with a need of desquamation-like processes. Extracts of tape strips with superficial human stratum corneum were found to contain precursors as well as active forms of stratum corneum tryptic enzyme and stratum corneum chymotryptic enzyme. The enzymes had maximal activity at pH 8, but both had considerable activity also at pH 5.5. The results were compatible for a role of stratum corneum tryptic enzyme in desquamation. Stratum corneum tryptic enzyme may act in concert with stratum corneum chymotryptic enzyme and/or function as a stratum corneum chymotryptic enzyme-activating enzyme. The presence in normal superficial stratum corneum of precursors as well as of active forms of stratum corneum chymotryptic enzyme and stratum corneum tryptic enzyme, and the activity of both enzymes over a broad range of pH-values, suggest some possible ways by which the desquamation may be regulated.

Human stratum corneum chymotryptic enzyme (SCCE) may play a central part in epidermal homeostasis. Its proposed function is to catalyze the degradation of intercellular structures, including desmosomes, in the stratum corneum as part of the desquamation process. In order to facilitate physiologic and pathophysiologic studies on SCCE we have looked for the corresponding murine enzyme. A cDNA obtained by reverse transcription-polymerase chain reaction with total RNA prepared from mouse tails as starting material was cloned, and the expression of the corresponding mRNA studied. The murine cDNA showed 77% homology to human SCCE cDNA. It had an open-reading frame encoding a protein comprising 249 amino acids with 82% amino acid sequence homology to human SCCE including the conserved sequences of the catalytic traid of mammalian serine proteases. The murine protein was deduced to have a 21 amino acid signal peptide and a four amino acid propeptide ending with a tryptic cleavage site, followed by a sequence motif identical to the N-terminal amino acid sequence of native active human SCCE. As in human SCCE the P2 position of the propeptide was occupied by an acidic amino acid residue, and the position corresponding to the suggested bottom of the primary substrate specificity pouch occupied by an asparagine residue. Analyses of mouse tissues by reverse transcriptase-polymerase chain reaction showed high expression in the skin, low expression in lung, kidney, brain, heart, and spleen, and no expression in liver or skeletal muscle. In situ hybridization of mouse skin showed expression in high suprabasal keratinocytes and in the luminal parts of hair follicles. Our results strongly suggest that we have cloned the murine analog of human SCCE cDNA.

Stratum corneum chymotryptic enzyme (SCCE) is a serine protease which may function in the turnover of the stratum corneum by means of degradation of intercellular adhesive structures between corneocytes. It is also potentially an epidermal activating enzyme for cytokines such as interleukin-1beta. The aim of this work was to study the expression of SCCE in psoriatic epidermis by means of immunohistochemistry, and to elucidate the nature of the SCCE present in psoriatic scales by means of biochemical analyses. In comparison to normal skin the number of cell layers expressing SCCE in psoriatic lesions was consistently increased. In nonlesional psoriatic skin the pattern of SCCE expression varied. It was similar to the pattern in normal skin in some biopsies, whereas in other biopsies evidence of an increased expression of SCCE was found. By means of zymography and immunoblotting, extracts of psoriatic scales were found to contain active SCCE as well as enzymatically inactive SCCE precursor. Also the effects of inhibitors on the activity towards a chromogenic protease substrate in the extracts after partial purification by gel exclusion chromatography were compatible with the presence of enzymatically active SCCE. We conclude that the expression of SCCE in psoriasis may be upregulated, and that the conversion of inactive SCCE-precursor to active SCCE occurs in the psoriatic lesion. The possible role of SCCE in the pathophysiology of psoriasis remains to be elucidated, but should be considered in future studies.

Stratum corneum chymotryptic enzyme (SCCE) may be involved in desquamation, a process necessary for maintaining a normal anatomy at all sites where there is continuous turnover of cornified epithelia. Using immunohistochemistry and in situ hybridization, we have, in this work, analysed SCCE expression in the sebaceous follicle. We found expression of SCCE in luminal parts of the pilary canal, common sebaceous ducts and proximal sebaceous ducts. In addition, SCCE was seen in cells apparently situated within the distal parts of the glandular lobules. Co-expression of SCCE and keratin 10 was seen only in the pilary canal and the common sebaceous ducts. The results give further support for SCCE being involved in desquamation-like processes. The association with cornification seems to be more general for SCCE than for keratin 10. The possible role of SCCE in diseases involving disturbances in the turnover of cornified cells in the sebaceous follicle, such as acne vulgaris, is a question for future studies.

The major interleukin 1 beta (IL-1 beta) species from human plantar stratum corneum was purified and found to have an N-terminal amino acid sequence homologous to a stretch of the human IL-1 beta precursor, starting with His115. Whereas SDS-polyacrylamide gel electrophoresis followed by immunoblotting revealed only one component in plantar stratum corneum with IL-1 beta-like immunoreactivity, and with an apparent molecular mass around 18 kDa, isoelectric focusing under non-denaturing conditions showed one major component with isoelectric point around 6.1 and two minor components isoelectric at pH 6.3 and 6.9, respectively. Digestion of recombinant human IL-1 beta precursor with chymotrypsin, producing a C-terminal fragment with N-terminal Yal114, yielded a component with IL-1 beta-like immunoreactivity isoelectric at pH 6.3. Recombinant bacterial variants of human IL-1 beta with N-terminal amino acids corresponding to Val114, His115 and Ala117 were isoelectric at pH 6.3, 6.1 and 6.9, respectively. Cloning and subsequent nucleotide sequencing of IL-1 beta precursor cDNA from a human keratinocyte line showed total identify with the sequence previously published for the human monocyte IL-1 beta precursor. The authors conclude that the IL-1 beta species present in plantar stratum corneum have isoelectric points determined by their respective amino acid sequences, and that there is a mechanism for IL-1 beta activation in human epidermis not involving interleukin 1 beta convertase.

Calcium plays an important role in the regulation of cellular differentiation and desquamation of epidermal keratinocytes. In this study, we examined the calcium distribution in reconstructed epidermis in an attempt to understand the physiology of keratinocyte differentiation and desquamation in vitro. Ion capture cytochemistry (the potassium oxalate-pyroantimonate method) was employed to localize ionic calcium in reconstructed epidermis generated under three different culture conditions (in serum-containing medium, serum-free medium, and serum-free medium supplemented with retinoic acid), allowing a comparison of the physiology of incompletely and well-differentiated keratinocytes. The reconstructed epidermis generated in serum-containing medium showed features of incomplete differentiation, and compared with the native skin, a high calcium content within incompletely differentiated cells in the stratum corneum. Use of serum-free medium containing vitamin and lipid supplements led to a marked improvement of the stratum corneum ultrastructure and penetration pathway across the stratum corneum, indicating improved barrier formation of the reconstructed epidermis. In parallel, the calcium distribution pattern was normalized showing the highest levels of calcium in the stratum granulosum and low levels in the inner stratum corneum. Addition of retinoic acid to the serum-free medium resulted in an altered keratinocyte differentiation and re-appearance of large quantities of calcium precipitates in the stratum corneum. Proton probe X-ray microanalysis was applied to investigate the calcium distribution quantitatively in native and reconstructed epidermis generated in serum-free medium, and verified the calcium distribution demonstrated by the precipitation technique. Regardless of the presence or absence of calcium in the stratum corneum, all examined culture systems exhibited insufficient desquamation, which correlates with the finding that stratum corneum chymotryptic enzyme was present predominantly as an inactive precursor. This study demonstrates that improvement of the stratum corneum barrier properties in vitro is concurrent with the normalization of the epidermal calcium gradient, whereas deregulation of terminal differentiation correlates with an accumulation of calcium ions within incompletely differentiated corneocytes.

We have recently presented evidence that human plantar stratum corneum and psoriatic scales contain biologically active interleukin-1beta (IL-1beta) which has been activated in a process not involving interleukin-1beta-converting-enzyme. The aim of the present study was to compare this form of native IL-1beta with recombinant mature human IL-1beta as regards activity and the effects of inhibitors. In an assay based on the ability of IL-1 to induce the expression of E-selectin in cultured endothelial cells, the maximal activity of IL-1beta partially purified from plantar stratum corneum and recombinant IL-1beta was approximately the same. The specific activity was slightly higher for recombinant IL-1beta, although this difference was within one order of magnitude. An antibody to IL-1beta caused total inhibition of both forms of IL-1beta with no significant differences in the dose-response curves for the antibody. Immunochemical analyses and experiments with neutralising antibodies specific for IL-1alpha and tumor necrosis factor-alpha (TNF-alpha) verified that the observed activity in the partially purified preparation was due to IL-1beta, and not to IL-1alpha or TNF-alpha. There were no significant differences between the two forms of IL-1beta as regards the inhibitory effects of recombinant IL-1 receptor antagonist. Partially purified IL-1beta from plantar stratum corneum and from psoriatic scales were both highly active in the D10 proliferation assay. This activity could be totally inhibited with an IL-1beta specific antibody. This work thus confirms the presence of biologically active IL-1beta in plantar stratum corneum and psoriatic scales. Alternatively activated IL-1beta in the epidermis should be considered in future studies on skin biology and pathophysiology.

The aims of the present work were to elucidate the biochemical properties of interleukin-1 beta (IL-1 beta) in psoriatic scales to get information on the processing of epidermal IL-1 beta in psoriasis, and to elucidate whether the IL-1 beta in psoriatic scales possesses biological activity. By means of ion exchange chromatography, IL-1 beta in extracts of psoriatic scales was purified to a stage where it could be analyzed with electrophoretic methods and immunoblotting. Compared to mature recombinant human IL-1 beta (Ala 117 IL-1 beta), IL-1 beta in psoriatic scales had a slightly higher apparent molecular mass and a more acidic isoelectric point, as revealed by two-dimensional electrophoresis under denaturing conditions. Isoelectric focusing under non-denaturing conditions of IL-1 beta partially purified from psoriatic scales, or from non-inflamed plantar stratum corneum (Nylander Lundqvist, E., Bäck, O. and Egelrud, T., J. Immunol. 1996. 157: 1699), and of mature IL-1 beta, followed by immunoblotting with IL-1 beta-specific antibodies, showed that psoriatic scales contained two components with IL-1 beta-like immunoreactivity which were isoelectric at pH 6.1 and 6.3, respectively. These components could also be detected in extracts of plantar stratum corneum, which also contained small amounts of an IL-1 beta-like component isoelectric at pH 6.9. Mature IL-1 beta was isoelectric at pH 6.9. No IL-1 beta-like biological activity could be detected in crude extracts of psoriatic scales. These extracts also contained high amounts of IL-1 receptor antagonist. Partially purified preparations of IL-1 beta from psoriatic scales, in which an apparently total separation of IL-1 beta and IL-1 receptor antagonist had been achieved, could induce expression of E-selectin in human umbilical vein endothelial cells. This activity was inhibited by antibodies specific for IL-1 beta, but not by antibodies specific for IL-1 alpha. It is concluded that psoriatic scales contain biologically active IL-1 beta, which has been processed by a mechanism which may be similar to that present in non-inflamed plantar stratum corneum, and which does not involve IL-1 beta converting enzyme.

Interleukin 1 beta (IL-1 beta) is produced as a biologically inactive 31 kD precursor, which is converted to the active 18 kD form by proteolytic processing. Keratinocytes express IL-1 beta but not the active form of the specific IL-1 beta converting enzyme (ICE). We have recently presented evidence that IL-1 beta activation in human epidermis occurs via an alternative mechanism involving hitherto unknown proteases. We asked whether stratum corneum chymotryptic enzyme (SCCE), which is a serine protease specifically expressed in keratinizing squamous epithelia, can act as an IL-1 beta activator in vitro. Recombinant human pro-IL-1 beta was incubated with recombinant SCCE, and the reaction products were characterized as regards molecular size and ability to induce expression of E-Selectin in human umbilical cord endothelial cells. SCCE caused degradation of pro-IL-1 beta and the accumulation of a component with electrophoretic mobility slightly lower than recombination mature IL-1 beta. Whereas incubation mixtures with pro-IL-1 beta which had been incubated in the absence of SCCE, or with SCCE, which had been incubated in the absence of pro-Il-1 beta, did not induce expression above baseline levels of E-Selectin, pro-Il1 beta which had been incubated with SCCE induced a significant increase in E-Selectin expression. This effect could be abolished by neutralizing antibodies to IL-1 beta, but not by antibodies to IL-1 alpha. In addition to IL-1 beta activation, SCCE also prepared to be able to catalyze a further degradation of IL-1 beta, leading to a loss of biological activity. We conclude that SCCE is a potential candidate for being responsible for IL-1 beta activation in human epidermis.

Stratum corneum chymotryptic enzyme (SCCE) may function in the degradation of intercellular cohesive structures in the stratum corneum preceding desquamation. Previous results have suggested that SCCE may be specifically expressed in squamous epithelia undergoing terminal differentiation and keratinization. The aim of the present work was to further elucidate the association between SCCE expression and terminal differentiation in squamous epithelia. Using immunohistochemical methods, we have examined the expression of SCCE in two diseases of human oral mucosa, which produce a pathological keratinization of the epithelium at sites which are normally non-keratinized. Affinity-purified polyclonal rabbit antibodies raised against recombinant SCCE and monoclonal antibodies against the differentiation-specific keratins nos. 10 and 13 were used on formaldehyde-fixed and paraffin-embedded biopsies. Whereas there was essentially no expression of SCCE in normal, non-keratinized buccal mucosa, there was a strong expression in suprabasal cells in orthokeratotic and parakeratotic areas of the lesions of oral lichen planus (an inflammatory disease) and benign oral keratosis (a non-inflammatory disease). There was a close association between the expression of SCCE and keratin no. 10, i.e. a keratin which is specifically expressed in cornifying squamous epithelia. The results suggest that SCCE expression may be a true marker of terminal differentiation in squamous epithelia and give further evidence for a role of SCCE in the formation and/or turnover of the stratum corneum.

Palmar and plantar epidermis is characterized by specific features such as the development of a striking lucidum, a very thick stratum corneum, prominent rete ridges and the unique expression of keratin K9. Using organotypic cocultures of keratinocytes and fibroblasts, we investigated to which extent the specific phenotype of palmar keratinocytes is maintained in vitro and under systemic host influences after transplantation onto nude mice. In vitro, palmar keratinocytes developed a thick epithelium with a prominent, although parakeratotic stratum corneum showing no significant differences in proliferation and differentiation in coculture with either palmar or nonpalmoplantar fibroblasts. All differentiation markers including keratohyaline and membrane coating granules as well as keratin K9 were also found, but at reduced levels and with slightly altered localization. In transplants, substantial normalization towards the palmar phenotype occurred. In 3-week-old grafts, a homeostatic state was reached, as illustrated by a constant thickness of the stratum Malpighii, presence of keratin K10 throughout the entire suprabasal compartment, increased numbers of K9- and filaggrin-positive cells, and reduction of keratins K16 and K17. At the ultrastructural level, numerous membrane coating granules and an enlargement of keratohyaline granules were seen accordingly, and immunofluorescence showed intense continuous lining of the dermo-epidermal junction by laminin, type IV collagen and integrin alpha 6. The high percentage of bromodesoxyuridine-positive cells, mainly in the basal compartment, underlined the hyproproliferative state, comparable to palmoplantar epidermis. In conclusion, (i) palmar keratinocytes can preserve the potential to express their specific phenotype upon transfer to culture conditions, and (ii) this intrinsic property is not significantly modulated by the type of cocultured fibroblasts. This suggests that fibroblasts act primarily by sustaining keratinocyte proliferation which is permissive for the fully differentiated phenotype.

Stratum corneum chymotryptic enzyme (SCCE) is a recently discovered human serine proteinase that may be specific for keratinizing squamous epithelia. SCCE has properties compatible with a function in the degradation of intercellular cohesive structures during stratum corneum turnover and desquamation. SCCE is expressed in suprabasal keratinocytes. In this study, we demonstrate the subcellular localization of SCCE in the upper granular layer, in the stratum corneum of normal non-palmoplantar skin, and in cohesive parts of hypertrophic plantar stratum corneum, using immunoelectron microscopy of ultrathin cryosections labeled with SCCE-specific monoclonal antibodies detected with gold-labeled secondary antibodies. A narrow zone close to the transition between the granular and cornified layers showed positive SCCE staining after fixation. By means of immunoelectron microscopy, SCCE was found in association with structures resembling intracellular lamellar bodies in the uppermost granular cells and in similar structures undergoing extrusion to the extracellular space between the uppermost granular cells and the lowermost cornified cells. In the stratum corneum, the detected SCCE was confined to the extracellular space and was found in association with intact and partially degraded desmosomes, as well as in the parts of the extracellular space devoid of desmosomes. We conclude that SCCE may be stored in lamellar bodies in the stratum granulosum and transported via these structures to the stratum corneum extracellular space. The results further support the idea that the physiologic function of SCCE may be to catalyze the degradation of desmosomes in the stratum corneum during remodeling of the deeper layers of this tissue, and at a later stage serve as a prerequisite for desquamation.

The aetiology of many skin diseases is to be sought in the multiplicity of interactions between mechanisms generally present in the body, such as the inflammatory response and immune systems, and mechanisms present in the skin. In the case of organ-specific skin diseases, it is reasonable to suppose the underlying disturbance to affect structures and molecular processes unique to the skin. The stratum corneum represents the culmination of epidermal differentiation and consists of very resistant and cohesive cells surrounded by an intracellular space filled with lamellar lipids, thus forming a highly effective and resistant physico-chemical barrier between the interior of the body and the external environment. As the stratum corneum is continuously produced, a prerequisite of its well-regulated thickness is an equally well regulated shedding of cells from the skin surface. This is preceded by proteolytic degradation of intracellular cohesive structures--i.e. desmosomes. We have recently discovered a new serine proteinase, stratum corneum chymotryptic enzyme (SCCE), which may be involved in this process. SCCE has been purified, cloned and expressed in mammalian cells.

In recent work we have shown that a serine proteinase, stratum corneum chymotryptic enzyme, with properties compatible with a role in desquamation in vitro as well as in vivo, is generally present in human stratum corneum. The enzymologic properties of the stratum corneum chymotryptic enzyme in a KCl extract of dissociated plantar corneocytes were compared with those of other known chymotryptic serine proteinases. Stratum corneum chymotryptic enzyme was found to differ significantly from bovine chymotrypsin, human cathepsin G, and human mast cell chymases in regard to inhibitor profile and substrate specificity. Stratum corneum chymotryptic enzyme was further purified from KCl extracts of dissociated plantar corneocytes by affinity chromatography on gels with covalently linked soybean trypsin inhibitor. The purified preparation contained one major component with apparent molecular weight 25 kD and one minor component with slightly higher apparent molecular weight as revealed by Coomassie staining after electrophoresis in polyacrylamide gels with sodium dodecyl sulphate of samples that had not been reduced. Both these components were associated with chymotrypsin-like activity as revealed by zymography in polyacrylamide gels with co-polymerized casein. On zymography gels, the purified preparation was also found to contain minor amounts of components with trypsin-like activity. The major purified protein had an apparent molecular weight of around 28 kD after reduction and full denaturation and was shown to contain carbohydrate.

Epicutaneous patch testing of a battery of 35 dental test substances was carried out in 24 patients with visible lichenoid oral mucosal lesions and in 24 patients with burning mouth syndrome (BMS) without any visible lesions. Reactions to mercury ammonium chloride were found in 33% (8/24) of the patients with visible lesions compared to 0% (0/24) of the patients with BMS. The difference was statistically significant. In 7 of the 8 patients who reacted to mercury, total or partial regression of the lesions was observed after removal of dental amalgam. Reactions to nickel sulfate were found in 21% (5/24) of the patients with BMS compared to 3% (1/24) of the patients with lichenoid lesions. This difference was also statistically significant. Nickel is a rare component in dental restorations, but the oral mucosa is daily exposed to nickel through food and water intake. Removal of nickel from the environment of the patient can therefore be hard to accomplish.

Plantar stratum corneum was examined by means of transmission electron microscopy after conventional osmium fixation and after fixation with ruthenium tetroxide. The latter fixative was used in order to reveal the possible existence of lamelarly ordered lipids in the intercellular space, as has previously been demonstrated for non-palmo-plantar stratum corneum. A major part of the plantar stratum corneum intercellular space was occupied by extracellular parts of desmosomes. In specimens fixed with ruthenium tetroxide the intercellular space not occupied by desmosomes was found to contain multiple alternating electron dense and electron lucid bands, suggestive of membraneous structures. This pattern appeared to be similar to that previously described for non-palmo-plantar stratum corneum. It is suggested that the intercellular lipids of palmo-plantar stratum corneum may be qualitatively similar to the intercellular lipids of non-palmo-plantar stratum corneum. The lower lipid content, expressed as weight per unit weight of tissue, in palmo-plantar stratum corneum as compared to non-palmo-plantar stratum corneum may be related to the fact that a larger portion of the intercellular space of the former tissue is occupied by desmosomes. The relatively high water permeability of palmo-plantar stratum corneum implies that desmosomes, i.e. non-lipid regions of the intercellular space, may have a high water permeability and hence could establish a hydrophilic route through the stratum corneum.

A chymotrypsin-like proteinase that may be involved in the desquamation process in plantar stratum corneum has recently been partially characterized. The aim of the present study was to elucidate whether a similar proteinase is also present in non-palmo-plantar stratum corneum. Stratum corneum was obtained by tape stripping of volar forearm skin after the skin surface had been painted with colourless nail varnish. The adherent tissue was released from the tape strips by acetone treatment, then extracted with diethyl ether and dried. Extracts of this acetone-ether powder were analyzed with respect to proteolytic activity by means of electrophoresis under non-reducing conditions in polyacrylamide gels containing sodium dodecyl sulphate and casein. The extracts were found to contain one major chymotrypsin-like proteinase with an apparent molecular weight of around 25 kDa, and several minor proteinases with trypsin-like activity. The 25 kDa proteinase was active at pH 5.5-8, and could be inhibited by aprotinin, chymostatin and zinc ion, but not by leupeptin. No difference could be found between the 25 kDa enzyme in forearm stratum corneum and the recently described chymotrypsin-like enzyme in dissociated plantar stratum corneum cells as regards electrophoretic mobility, pH dependency, and inhibitor profile. The fact that the enzyme could degrade casein at pH 5.5 and that it appears to be present in stratum corneum in general suggests that it may play a role in the desquamation process under in vivo conditions. The tentative name "stratum corneum chymotryptic enzyme" is proposed for this newly discovered proteinase.

We have recently shown that cell cohesion in plantar stratum corneum is mediated to a significant extent by protein structures, and that endogenous proteolysis plays an important role in desquamation in this tissue. This paper is a report of our investigations into whether similar mechanisms for cell cohesion and desquamation can be found in non-palmo-plantar stratum corneum. Biopsies of non-palmo-plantar human skin were incubated at 37 degrees C, pH 8, in a buffer with and without additions of detergents (a mixture of N,N-dimethyldodecylamine oxide and sodium dodecyl sulphate), ethylene diamine tetraacetate (EDTA), and the proteinase inhibitor aprotinin. Released cells were examined by phase contrast microscopy and counted. The incubated biopsies were examined by light microscopy. As has been previously shown by others, we found that in the presence of detergents there was a dissociation of stratum corneum cells. This dissociation was stimulated by EDTA and inhibited by aprotinin. After 36 h of incubation the entire stratum corneum and, on some parts of the biopsies, the stratum granulosum had dissociated. There was no evidence of cell dissociation in the spinous or basal epidermal layers. We conclude that the detergent-induced cell dissociation in non-palmo-plantar human stratum corneum is dependent on the action of proteinases present in the tissue on protein structures. These structures may be of significant importance for non-palmo-plantar stratum corneum cell cohesion.

We have recently described a process leading to a unipolar cell shedding from pieces of plantar stratum corneum incubated in vitro, which seems to be dependent on the activity of a serine proteinase. This process has been studied further. Electron microscopy studies suggest that cell dissociation is preceded by a degradation of the intercellular parts of desmosomes. An antiserum was raised against the transmembrane protein desmoglein I (DG I) of bovine desmosomes. In extracts of layers of plantar stratum corneum with strong intercellular cohesion, this antiserum reacted with a protein of the same apparent molecular weight as bovine DG I. In dissociated cells this DG I-like protein could not be detected; instead components with molecular weights lower than DG I which reacted with the antiserum were found. During incubation of pieces of plantar stratum corneum, under conditions leading to unipolar cell shedding, there was a progressive decrease in the amounts of the DG I-like protein, and the appearance of the lower molecular weight components with DG I-like immunoreactivity. This apparent degradation of the DG I-like protein was inhibited by aprotinin, chymostatin, and zinc ion, but not by leupeptin. The results suggest that proteolytic degradation of desmosomes may be an important part of the process leading to cell dissociation in plantar stratum corneum in vitro, and that desmosomes may play an important role in plantar stratum corneum cell cohesion.

Granular deposits of IgA in radioactively labeled thin slices of papillary dermis from 4-mm punch biopsies of clinically normal skin from patients with dermatitis herpetiformis were solubilized with sodium dodecyl sulfate (SDS) or peptic digestion at pH 4.5. Solubilized IgA-like material was isolated by immunoprecipitation and analyzed by electrophoresis in one and two dimensions in polyacrylamide gels containing SDS followed by autoradiography. SDS extracts contained IgA-like components corresponding to monomers, dimers, as well as higher polymers of IgA. A fraction of the SDS-soluble material behaved like IgA upon immunoprecipitation but could not be deaggregated to alpha- and light chains by reduction and alkylation, suggesting that it was present as irreversible aggregates with itself or with other proteins. Peptic digestion at pH 4.5 released fragments which were precipitated by antibodies to human alpha-chains and had molecular weights similar to the proteolytic fragments corresponding to F(ab)2 and Fab formed by peptic digestion of human monomeric IgA under the same conditions.

Pieces of plantar stratum corneum were incubated with trypsin. This resulted in cell dissociation. The only observable ultrastructural change caused by trypsin was a degradation of desmosomal plates between dissociating cells. This suggests that desmosomes are of primary importance in plantar stratum corneum cell adhesion.

A technique has been devised to isolate the thin papillary part of the dermis from punch biopsies. Papillary dermis has been treated with various proteolytic enzymes in order to release or solubilize the granular IgA deposits from the papillary dermis of patients with dermatitis herpetiformis. Incubation of the thin skin preparations with pepsin caused a disappearance of the specific fluorescence with antibodies to human IgA. After peptic digestion small amounts of IgA could be detected in the supernatants. There was some evidence that this amount was larger for preparations from patients with dermatitis herpetiformis than from controls. Corresponding procedures with trypsin, collagenase, or elastase had no detectable effect on the IgA deposits. The experiments with elastase seemed to give support for previous reports on association between the granular IgA deposits and the microfibrils of elastic fibers.

Cell shedding from plantar stratum corneum was studied in vitro. Cells were shed only from the surface that had faced outwards in vivo. A quantitative measure of the cell release was obtained by determining the amount of protein that could be extracted from released and sedimented cells with 1 M sodium hydroxide. The cell release was optimal at pH 7-9 but was significant also at pH 6. The rate of cell release increased with increasing temperature, but was decreased abruptly at temperatures above 50 degrees C. The cell dissociation could be inhibited by aprotinin (Trasylol) and soybean trypsin inhibitor. Thus, it is evident that the unipolar cell dissociation in this system is mediated by an enzymatically catalyzed process, most likely with the involvement of a serine protease with an alkaline pH-optimum. The in vitro cell release shows properties indicating that it may be mediated by mechanisms also active in vivo.

The effects of lipoprotein lipase, phospholipase A2 and phospholipase C on chylomicron phosphatidylcholine and triacylglycerol were studied with rat lymph chylomicrons containing phosphatidylcholine labeled with [14C]oleic acid. Lipoprotein lipase purified from bovine milk readily hydrolyzed chylomicron phosphatidylcholine to lysophosphatidylcholine and fatty acid, and triacylglycerol to monoacylglycerol, fatty acid and glycerol. The rates of hydrolysis of phosphatidylcholine and triacylglycerol increased with enzyme concentration, and both decreased when fatty-acid binding sites on albumin in the incubation medium were limited. The proportion and amount of phosphatidylcholine hydrolyzed was always less than that of triacylglycerol. Analyses of hydrolytic products showed that lipoprotein lipase cleaved the 1-acyl ester bond of phosphatidylcholine. The findings indicate that lipoprotein lipase can account for some of the phospholipase A1 activity found in postheparin plasma. Phospholipase A2 and phospholipase C hydrolyzed chylomicron phosphatidylcholine, greater than 92% in 10 min, but not triacylglycerol. The resultant phosphatidylcholine-deficient chylomicrons, which could be concentrated by ultra-centrifugation and resuspended in incubation medium, were readily depleted of triacylglycerol when incubated with lipoprotein lipase. The findings indicate that phosphatidylcholine can be removed from the surface film of chylomicrons without disrupting the particles or blocking the action of lipoprotein lipase on the core triacylglycerol.

The cytokeratin composition of palmo-plantar epidermis from sites with different degrees of mechanically induced thickening of the stratum corneum was analysed. The urea-soluble proteins of the stratum corneum were analysed by two-dimensional electrophoresis. Viable epidermal layers were analysed by immunofluorescence microscopy with polyclonal and monoclonal antibodies. A mouse monoclonal antibody specific for cytokeratin no. 9 was prepared for the study. Significant amounts of low molecular weight cytokeratins were found in suprabasal layers at sites with the most pronounced thickening of the stratum corneum. This was taken as evidence that palmo-plantar epidermis responds to mechanical stress with hyperproliferation. At sites where stratum corneum thickness is most increased this hyperproliferation appears to involve two different populations of cells--one capable of expressing high molecular weight, differentiation-related cytokeratins in the suprabasal epidermal layers, and one population that does not express these cytokeratins. At sites with intermediate epidermal hyperplasticity the high molecular weight cytokeratins were predominant in all suprabasal cells.

Precursor-product relationships among keratin polypeptides (cytokeratins) were studied in living and cornified layers of palmar-plantar epidermis. Serial 10 microns horizontal freeze-cut sections of punch biopsies were analysed by means of light microscopy and one-dimensional electrophoresis in a system that permitted identification of all major keratin polypeptides with apparent molecular weights over 60 kDa. Peptides that appeared to be related were compared by means of peptide mapping after partial proteolysis. The results suggest that both the basic-neutral (type II) cytokeratin no. 1 and the acidic (type I) cytokeratin no. 9 undergo two distinct modification steps with subsequent decreases in apparent molecular weight during keratinization. For both polypeptides the first modification appeared to take place and run to completion in close relation to the transition between the uppermost living epidermal layers and the lowest cornified layers. The second conversions of cytokeratins nos. 1 and 9 both appeared to take place within the stratum corneum but differed in two respects: 1) they appeared to start at different tissue sites; 2) whereas the second modification step appeared to comprise all cytokeratin 1 molecules, only a fraction of the cytokeratin 9 molecules passed through this step. These variations suggest that the different modification steps may be produced by different mechanisms that are regulated separately. It is concluded that the processing of cytokeratins during keratinization may be more complex than has previously been realized.

A rabbit antiserum prepared against the serum-stimulated lipase (lipoprotein lipase) from bovine milk crossreacted with serum-stimulated lipases from human milk and from human postheparin plasma, but not with bile salt-stimulated lipase from human milk or with salt-resistant lipase from human postheparin plasma. Thus, the serum-stimulated lipase in bovine milk has immunological determinants in common with the serum-stimulated lipases in human milk and in human postheparin plasma. The time-courses for the appearance of serum-stimulated lipase and salt-resistant lipase activities in human plasma after heparin injection were different. The two activities were separated by heparin-Sepharose chromatography. After treatment of postheparin plasma with the antiserum only the salt-resistant lipase activity could be eluted from the column. Thus, these two enzyme activities in postheparin plasma reside in two different enzyme molecules.

An antiserum raised against the bovine desmosomal protein desmoglein I (DGI), Mr approximately 160 kDa, was used in an immunochemical analysis of human plantar epidermis. Different layers of the tissue were prepared by means of horizontal freeze sectioning. Loosely attached surface layers were obtained by means of scraping of the skin surface with a scalpel. Tissue extracts were analysed by means of sodium dodecylsulphate polyacrylamide gel electrophoresis followed by immunoblotting. Significant amounts of a component with Mr approximately 160 kDa, reactive with the DG I-antiserum, were found in all layers except the loosely attached surface layers. In these layers the antiserum detected a component with Mr approximately 80 kDa, not found in other layers. This component may be a degradation product of DG I. Since DG I belongs to the group of transmembrane desmosomal proteins that is believed to constitute the link between the intracellular parts of desmosomes of opposing cells, it is concluded that desmosomes may play an important role in plantar stratum corneum cell cohesion, and that degradation of desmosomes may be an important step in desquamation in plantar epidermis.