Ultrastructural localization of the core protein of a basement membrane-specific chondroitin sulfate proteoglycan in adult rat skin

Summary Basement membranes are complex extracellular matrices present at epithelial/mesenchymal interfaces of tissues. The dermal-epidermal junction has been shown to contain numerous components, some of the most well known being laminin, types IV and VII collagens, heparan sulfate proteoglycan, fibronectin, and entactin/nidogen. In this paper we show, using core protein-specific antibodies, the presence of a newly described basement membrane-specific chondroitin sulfate proteoglycan at the epithelial/ mesenchymal interface of adult rat skin. Ultrastructurally, this antigen was proven to reside primarily within the basal lamina, apparently concentrated in the lamina densa. In addition, some of the proteoglycan was also present beneath the lamina densa, associated with the reticular lamina collagen fibrils.     

Reconstruction of basement membrane in skin equivalent; role of laminin-1

Abstract To reconstruct the basement membrane in a skin equivalent, the epidermodermal interface was coated with porcine type IV collagen and mouse laminin-1 at various ratios before keratinocyte seeding. Laminin-1, a component of the basement membrane, induced massive infiltration of keratinocytes into the dermal equivalent, while type IV collagen induced discrete demarcation between dermal and epidermal compartments without any infiltrating cells. Immunohistochemical staining indicated that the laminin-induced infiltrating cells expressed endogenous type IV collagens at the cell periphery, which were not incorporated into the basement membrane structure. The infiltrating cells did not express fibronectin receptor α₅β₁ integrin but showed MMP-9 secretion and cell surface associated MMP-2. However, when laminin-1 was preincubated with type IV collagen, laminin-1-induced keratinocyte infiltration as well as MMP-9 induction were almost completely suppressed to basal levels. Therefore, replenishment of the type IV collagen lattice seemed to cause laminin-stimulated cells to anchor to the lattice, in a similar manner to the basal cells on the basement membrane of normal skin. Our study suggests that the molar ratio of basement membrane components may determine the behavior of basal cells within the wound healing microenvironment, which is probably regulated either by extracellular matrix deposition or degradation. Received: 24 October 2000 / Revised: 9 February 2001 / Accepted: 31 March 2001     

The effects of epidermal keratinocytes and dermal fibroblasts on the formation of cutaneous basement membrane in three-dimensional culture systems

The cutaneous basement membrane (BM) plays an important role in normal and pathological conditions. However, few studies have addressed the formation of the cutaneous BM using three-dimensional culture systems. In this study, to elucidate the effects of human epidermal keratinocytes and dermal fibroblasts on the formation of the cutaneous BM, keratinocytes were cultured on several dermal substrates in the presence or absence of fibroblasts at the air–liquid interface. After 2 weeks of culture, immunohistochemical stainings for the components of the BM and electron microscopic studies of the BM zone (BMZ) were performed. In cultures of keratinocytes alone on dead reticular dermis or collagen gel without fibroblasts, 4 integrin chain, laminin, type IV and VII collagens were all expressed. However, ultrastructurally, BMZ was not formed. In cultures of keratinocytes on fibroblast-populated collagen matrix, laminin, and type IV and VII collagens were expressed more strongly than in the absence of fibroblasts. In addition, elements of the BMZ such as hemidesmosomes, lamina lucida, lamina densa and anchoring fibrils were formed, although it was still incomplete. In the culture of keratinocytes alone on de-epidermized dermis (DED) (surface up), 4 integrin chain, laminin, and type IV and VII collagens were strongly expressed. Also, the BMZ appeared similar to that in normal skin. In cocultures of keratinocytes and fibroblasts on DED or cultures of keratinocytes on DED combined with fibroblast-populated collagen matrix, type IV collagen was expressed more strongly than in cultures of keratinocytes alone. Ultrastructurally, similar findings to those of cultures of keratinocytes alone on DED were seen. Interestingly, when keratinocytes and fibroblasts were cocultured on DED, some fibroblasts were seen in the upper dermis as a result of migration into the dermis through partial loss of the lamina densa. These results show that keratinocytes produce most of the components of the BM such as laminin, and type IV and VII collagens. In addition, fibroblasts stimulate the expression of the components of the BM and the formation of a BMZ, suggesting that fibroblasts may produce laminin, and type IV and VII collagens or influence the effects of keratinocytes on the formation of the BM through a keratinocyte–fibroblast interaction.This investigation was supported by a grant (04-2001-027) from the Seoul National University Hospital Research Fund and partly by the Pacific Corporation.     

Study of basement membrane formation in dermal-epidermal recombinants in vitro

Summary Two different dermal-epidermal recombinants were prepared in vitro and used to study the synthesis and formation of basement membrane. The first was obtained by culturing keratinocytes on the surface of a collagen lattice populated by fibroblasts. The second was prepared by coculture of both keratinocytes and fibroblasts in a collagen lattice. After 6 weeks of culture, the basal lamina was observed with electron microscopy only if keratinocytes were cultivated on top of the collagen lattice populated by fibroblasts. In the second model, however, type IV collagen, laminin, and pemphigoid bullosa antigen could be detected by immunofluorescence as well as synthesis of type IV collagen in the culture, but no basement membrane was observed by electron microscopy. These data demonstrate that, in vitro, basement membrane formation depends not only on the presence of the macromolecular components but also on the culture conditions.     

Loricrin immunoreactivity in human skin: localization to specific granules (L-granules) in acrosyringia

Loricrin is a major component of the cornified cell envelope and is also expressed in the granular layer of human epidermis. In newborn mouse epidermis, loricrin accumulates in small round granules (L-granules) in the granular layer before being incorporated into the cornified cell envelope, but the expression of L-granules has not yet been demonstrated in human skin. In the present study we used postembedding immunoelectron microscopy to examine loricrin expression in normal human skin. We observed diffuse loricrin staining in the uppermost granular cell layer in interappendageal epidermis, that was not associated with any granular structures. In the cornified cells, most of the labelling was on the inner face of cornified cell envelopes. By contrast, in the upper segment of acrosyringia there were small granules (L-granules) that specifically labelled for loricrin. In the outer ductal cells, L-granules with a highly osmiophilic electron density were apparent in the nucleus as well as in the cytoplasm. In the inner ductal cells, L-granules were round or oval, less electron-dense and larger than the outer ductal L-granules, and present only in the cytoplasm. Some of the outer ductal L-granules and most of the inner ductal L-granules formed composite granules with filaggrin immunoreactive granules. These results suggest that in human interappendageal epidermis, loricrin might be rapidly incorporated into the cornified cell envelope without prior accumulation in any type of granule. By contrast, in acrosyringia loricrin accumulates in granules, perhaps reflecting increased synthesis or slower processing. The significance of the different morphological features of the L-granules in inner and outer acrosyringial cells remains to be clarified.     

Ultrastructural localization of Fras1 in the sublamina densa of embryonic epithelial basement membranes

Fras1 is the first identified member of a protein family comprising Fras1 and the related extracellular matrix proteins Frem1, Frem2 and Frem3. Mutations in Fras1, Frem1 and Frem2 have been associated with the bleb phenotype in mouse, whereas mutations in the human orthologs FRAS1 and FREM2 have been implicated in the pathogenesis of the human Fraser syndrome. Bleb mutant mice are characterized by embryonic sub-epidermal blistering, unilateral or bilateral renal agenesis or dysgenesis, cutaneous syndactyly and fused eyelids. As revealed by immunofluorescence, Fras1 co-localizes with the markers of epithelial basement membranes and is ultrastructurally detected underneath the lamina densa of embryonic mouse epithelia. Since the loss of Fras1 mainly affects the cohesiveness of the embryonic skin basement membrane with its underlying mesenchyme, we compared here the ultrastructural localization of Fras1 in the dermal–epidermal junction and in the basement membrane of other embryonic epithelia that do not show any overt phenotype using preembedding immunocytochemistry. Fras1 immunoreactivity was detected in all epithelia examined, within the sublamina densa adjacent to stromal tissue, as clustered gold/silver enhanced depositions, usually attached to anchoring fibrils. Interestingly, clusters corresponding to Fras1 were frequently detected in close proximity to mesenchymal cells, indicating that Fras1 could serve as a direct link between the sublamina densa and mesenchyme. The localization of Fras1 is consistent with previous results indicating that Fras1 exerts its function below the lamina densa and that Fras1 displays the same localization pattern in all epithelial basement membranes.     

Hemidesmosome ontogeny in digit skin of the human fetus

Hemidesmosomes are junctional complexes involved in the attachment of epidermal basal keratinocytes to the basement membrane. To try to understand better the sequence of events in the morphogenesis of hemidesmosomes, we undertook an ultrastructural analysis of hemidesmosome formation in fetal and neonatal digit skin. Hemidesmosomes, defined as membrane-associated densities or plaques, were counted and scored for three morphological characteristics: (1) the presence of a sub-basal dense plate, (2) association with anchoring filaments within the lamina lucida and (3) contacts with intermediate filaments. No hemidesmosomes were evident at 7 weeks' gestational age. Between 9 and 15 weeks the number of hemidesmosomes increased by about fourfold (from 20.6±3.8 (SD) to 95.5±8.4 per 40 μm of basal cell plasma membrane;P<0.01). The association of hemidesmosomes with intermediate filaments and anchoring filaments also increased after 15 weeks (P<0.05). Early attachment plaques first appeared as triangular focal densities on the basal plasma membrane with the appearance of sub-basal dense plates, which later became both larger and more electron dense. By 15 weeks, an inner plaque could be distinguished from the outer plaque, which coincided with a closer association with intermediate filaments. Hemidesmosomes appeared fully developed by 15 weeks' gestation. This study illustrates the structural relationship of hemidesmosomes to both intra- and extracellular filaments, suggesting close functional interactions. The complexity of the hemidesmo-some plaque is also revealed early during development.     

Epitope mapping of the laminin molecule in murine skin basement membrane zone: demonstration of spatial differences in ultrastructural localization.

Results of studies performed to date with polyclonal antilaminin antibodies have been conflicting as to the ultrastructural localization of this glycoprotein in skin basement membrane zone (BMZ). Whereas initial reports suggested its presence solely within the lamina lucida (LL), others have suggested that laminin is instead an exclusive component of the lamina densa (LD). In an attempt to more critically address this issue, we have examined both intact and partially separated (via 1 M NaCl) murine skin BMZ by indirect immunoelectron microscopy via a two-step immunoperoxidase technique on unfixed cryopreserved tissue, utilizing nine well-characterized monoclonal antibodies with binding specificity for laminin. Localization of the sites of the epitopes recognized by these antibodies on isolated laminin molecules was previously determined by rotary shadowing and by biochemical analyses on enzymatic fragments of laminin. Whereas at least faint immunoreactants were detected in both regions with eight of nine antibodies, predominant staining was noted within the LL with three of eight and within (and even sparsely below) the LD in three of eight. One antibody bound solely to the LL; another bound equally within both regions. Although some overlap was noted, it appears that the epitope on the distal portion of the long arm of the laminin molecule resides primarily within the skin LD, whereas epitopes on more central portions of the short arms are present within the LL or within both LL and LD. The findings of stratification of laminin epitopes within skin BMZ supports a similar recent observation in mouse kidney and suggests that portions of the laminin molecule span both LD and LL, and that there may be a non-random spatial orientation for the laminin molecule within murine skin BMZ.     

Immunoelectron microscopic localization of fibronectin in cultured human keratinocytes

We investigated the ultrastructural localization of fibronectin (FN) in cultured human keratinocytes using an improved method of peroxidase-immunoelectron microscopy. This method enabled us to visualize the precise localization of FN within the cells while preserving the morphology. FN was localized in the protein synthetic and secretory organelles, including the rough-surfaced endoplasmic reticulum, Golgi complex, multivesicular bodies and perinuclear space. It was also detected in the extracellular space, on small regions of the villous projections of cell membranes at the site of secretion and at cell-substratum contact sites. These findings confirm that human keratinocytes synthesize, secrete and deposit FN in the pericellular matrix.     

先天性大疱性表皮松解症基底膜带分子的研究

目的：通过透射电镜和免疫荧光研究先天性大疱性表皮松解症患者的基底膜带分子。方法：分析7个组织病理表现为表皮下疱的先天性大疱性表皮松解症患者的透射电镜和免疫荧光表现。结果：电镜检查1例为基底膜透明板裂隙,即JEB,其余6例为基底膜下裂隙,即DEB,间接免疫荧光检查此例JEB是BPAG2缺陷。结合临床资料诊断为泛发型萎缩性良性大疱性表皮松解症（generalized atrophic benign EB,GABEB),这是我国首例报告;6例DEB患者透射电镜和免疫荧光检查辅助诊断DDEB和RDEB。结论：先天性大疱性表皮松解症是一组疾病,电镜和免疫荧光检查对于分型和明确诊断是必需的,间接免疫荧光检查还可以指导进一步的基因诊断。     

Human skin basement membrane-associated heparan sulphate proteoglycan: distinctive differences in ultrastructural localization as a function of developmental age

Recent studies have demonstrated that skin basement membrane components are expressed within the dermo-epidermal junction in an orderly sequence during human foetal development. We have investigated the ultrastructural localization of basement membrane-related antigens in human foetal skin at different developmental ages using two monoclonal antibodies to a well-characterized basement membrane-associated heparan sulphate proteoglycan. A series of foetal skin specimens (range, 54-142 gestational days) were examined using an immunoperoxidase immunoelectron microscopic technique. In specimens representing very early developmental ages, very diffuse immunoreaction products were detected. However, by approximately 76 gestational days, some accentuation of heparan sulphate proteoglycan was noted along the lamina densa, and by 142 gestational days, the distribution of heparan sulphate proteoglycan was identical to that observed in neonatal and adult human skin. These findings demonstrate that active remodelling of the dermo-epidermal junction occurs during at least the first two trimesters, and affects not only basement membrane-associated structures but also specific antigens.     

Behaviour of laminin 1 and type IV collagen in uninvolved psoriatic skin. Immunohistochemical study using confocal laser scanning microscopy

Previous studies have demonstrated the presence in psoriatic lesions of ultrastructural and molecular alterations of the basement membrane and an altered polarized distribution of the integrins; this latter alteration has also been observed in uninvolved skin. The aim of the present study was to determine, by means of immunolocalization with monoclonal antibodies directed against laminin 1 and type IV collagen and using confocal scanning laser microscopy, whether there are also alterations of the main components of the basement membrane in uninvolved skin. The findings showed a discontinuous and fragmented staining of laminin 1 and a normal distribution of type IV collagen. Taking into account both these results and the results of studies on epithelial cell lines, the authors hypothesize the existence of a functional deficit in psoriatic keratinocytes affecting the synthesis of the ·1 subunit of laminin. This deficit would explain: (1) the incapacity to produce mature trimeric laminin; (2) the altered assembly into a distinct basal lamina; (3) the loss of keratinocyte adhesion to the basement membrane; (4) alterations in the polarized distribution of the integrins; and (5) the consequent total or partial block of the cell signals regulating the processes of cytomorphosis. Already present in uninvolved skin, and enhanced by various irritative stimuli, this situation could be decisive for the appearance of psoriatic lesions. Received: 18 October 1995     

Basement membrane zone as a target for human neutrophil elastase in psoriasis

Summary Human neutrophil elastase was found, by indirect immunofluorescence using rabbit anti-elastase antiserum, to be bound to basement membrane of psoriatic plaques in vivo. The enzyme was also identified inside the migrating neutrophils in the reticular dermis and dermal papillae, as well as outside the cells in micro-abscesses in psoriatic skin. In vitro incubation of normal skin with human neutrophil elastase resulted in the destruction of hemidesmosomes and separation of the epidermis from the dermis above localizations of bullous pemphigoid antigen. These findings are direct evidence that human neutrophil elastase could play a role in psoriasis in in vivo destruction of the epidermal — dermal junction.     

Ultrastructural lupus band test in the skin of MRL mice

Summary The ultrastructural localization of immunoglobulin deposits in the dermoepidermal junction of MRL/I mice was examined using immunoperoxidase staining. All specimens that showed positive findings at the basement membrane zone of the skin on light microscopy had reaction products at the subbasal laminar area when observed by electron microscopy. In the skin that had no distinct eruptions or which was distant from the lesions, the reaction products were distributed mostly just beneath the basal lamina in fine to course granular deposits. In the lesional skin, they formed a rather uniformly granular pattern among the irregularly arranged collagen fibrils and increased fine fibrilar amorphous material under the highly infolded basal lamina. Some of the reaction products were located just on the collagen fibrils and some appeared to be related to the free basal lamina. From the perspective of immunoelectron microscopy, the skin eruptions of MRL/I mice may serve as a good dermatological model of human systemic lupus erythematosus.This is the seventh part of a series of papers entitled Pathogenesis of Lupus Dermatoses in Autoimmune Mice     

Autoradiographic study of glycoprotein synthesis during embryonic epidermogenesis of the mouse in vitro

Summary Forelimb buds of mouse embryos (day 11+3 h of development) were cultured for 6 days. During this period, the epidermis developed from a two-layered stage (basal and periderm cell layer) to a multilayered squamous epithelium with stratum granulosum and stratum corneum. To investigate the glycoprotein synthesis during epidermogenesis in vitro, the limb buds were labeled for 30 min or 4 h with ³H-fucose at successive stages of development.Until the development of the stratum intermedium, no different labeling of the individual layers of the epidermis and the periderm was observed. Shortly before the beginning of the stratum granulosum, a strong labeling of the outer region of the intermediate layer, where the shape of the cells had changed from cuboid to flat, was visible. When the stratum granulosum developed, even in this layer and in the upper stratum spinosum, strong glycoprotein synthesis was observed, indicated by strong labeling with ³H-fucose. In the developing stratum corneum, no labeling of this layer was detectable 30 min or 4 h after ³H-fucose application. Labeling of peridermal cells was weak in the early stages of development and ceased in the period of stratum granulosum formation, indicating a stop of glycoprotein synthesis in this embryonal cell layer before its disappearance.