Serum-stimulated lipases (lipoprotein lipases). Immunological crossreaction between the bovine and the human enzymes.

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.

Hydrolysis of chylomicron phosphatidylcholine in vitro by lipoprotein lipase, phospholipase A2 and phospholipase C.

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.

Endogeneously regulated site-specific differentiation of human palmar skin keratinocytes in organotypic cocultures and in nude mouse transplants.

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.

Purification and preliminary characterization of stratum corneum chymotryptic enzyme: a proteinase that may be involved in desquamation.

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.

Cell shedding from human plantar skin in vitro: evidence of its dependence on endogenous proteolysis.

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.

Dermatitis herpetiformis: preparation of papillary dermis and the effect of proteolytic enzymes on the IgA deposits.

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.

Dermatitis herpetiformis: biochemical properties of the granular deposits of IgA in papillary dermis. Characterization of SDS-soluble IgA-like material and potentially antigen-binding IgA fragments released by pepsin.

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.

The dependence of detergent-induced cell dissociation in non-palmo-plantar stratum corneum on endogenous proteolysis.

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.

Evidence that cell shedding from plantar stratum corneum in vitro involves endogenous proteolysis of the desmosomal protein desmoglein I.

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.

Evidence that stratum corneum chymotryptic enzyme is transported to the stratum corneum extracellular space via lamellar bodies.

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.