Studies on fibronectin in the skin. III. Indirect immunofluorescence studies in lichen planus.

Fibronectin is a glycoprotein which is responsible for a varity of functions in the human organism, such as mediation of contact between cells and between cells and fibres, opsonic qualities, interaction in the stabilization of fibrin, etc. Fibronectin is an important constituent of the ground substance having a special affinity to collagen. In indirect immunofluorescence studies its presence has been abundantly demonstrated in normal human skin, in collagen-rich structures such as the basement membranes, the papillary and reticular dermis, and in the vascular and neural structures, demonstrable by its characteristic staining patterns. Fibronectin is not found in the epidermis. In lichen planus, the distribution in unaffected skin is identical with that in normal skin, whereas in affected skin, changes in the pattern of fibronectin are found. The basement membrane zone becomes broader and hazy, later undergoing disintegration and destruction, concomitant with swelling and homogenization of the reticular distribution of fibronectin in the papillary dermis. Globular structures containing fibronectin are found in the basement membrane area, together with an intensified immunofluorescence in the vascular system. Fibronectin has certain adhesional properties and changes in the distribution of this glycoprotein may result in loss of tissue stability. The pathophysiological significance of the changes of fibronectin in lichen planus is, however, difficult to evaluate at present.     

Immunofluorescent patterns in the skin in Besnier's prurigo

The immunofluorescent patterns in the skin of eighteen patients with Besnier's prurigo were studied at different stages of eczematous lesions. The presence, distribution pattern, and numbers of immunoglobulin and complement-positive lymphocytes in twenty-nine biopsies are reported. In all cases IgE-positive lymphocytes were present in both clinically uninvolved and involved skin. IgG, IgM and IgE-positive lymphocytes showed a characteristic distribution pattern in the dermis. In the acute stages IgM deposition was prominent in the basement membrane zone and it is suggested that damage to the basement membrane results in diffusion of immunoglobulin and complement into the epidermis. These features were not present in a control group of normal skin and of other dermatoses.     

Immunofluorescent patterns in the skin in Besnier's prurigo. The eczema asthma syndrome.

The immunofluorescent patterns in the skin of eighteen patients with Besnier's prurigo were studied at different stages of eczematous lesions. The presence, distribution pattern, and numbers of immunoglobulin and complement-positive lymphocytes in twenty-nine biopsies are reported. In all cases IgE-positive lymphocytes were present in both clinically uninvolved and involved skin. IgG, IgM and IgE-positive lymphocytes showed a characteristic distribution pattern in the dermis. In the acute stages IgM deposition was prominent in the basement membrane zone and it is suggested that damage to the basement membrane results in diffusion of immunoglobulin and complement into the epidermis. These features were not present in a control group of normal skin and of other dermatoses.     

Distribution of hydrolytic activity catalyzes the biotransformation of prednisolone 21-acetate in human skin.

We investigated the distribution of hydrolytic enzymes which metabolize prednisolone 21-acetate (PNA) to prednisolone (PN) in human skin. Km (Michaelis-Menten constant) and Vmax (maximum rate) of hydrolytic enzyme in human skin was 25.1 microM and 0.46 nmol/min/mg protein, respectively. Specific activities of hydrolysis in dermis and epidermis were similar and, in consideration to their thickness, hydrolytic activity in epidermis was 12.1 times higher than in dermis. Moreover, the highest amount of metabolite (PN) was found at 80-120 microm from the skin surface by skin slicing. Therefore, hydrolytic activity which metabolized PNA was distributed in epidermis, especially in the basement membrane area; epidermis borders dermis in this area and the papillary plexus is reached just beneath the dermal papillae. These results suggest that the distribution of hydrolytic activity in human skin may prevent certain substances from entering the systemic circulation in their unhydrolyzed form.     

Studies on fibronectin in the skin

Fibronectin is an important constituent of normal human skin, mediating cell/cell and cell/fibre interactions. In affected skin in discoid and systemic LE, changes in the distribution of fibronectin in the dermo-epidermal junction and in the papillary dermis are observed with homogenization and degenerative changes, IF negative gaps and slit formation in the dermo-epidermal region, together with IF positive globular bodies and transport of fibronectin into the epidermis. Unaffected skin in LE demonstrates the pattern of fibronectin as found in normal human skin.     

Human dermal fibroblasts synthesize laminin

Laminin, a glycoprotein of approximately 900,000 daltons, is a major component of the basement membrane that separates the epidermis from dermis in human skin. Previous studies have shown that keratinocytes and other epithelial cells synthesize laminin and utilize it for attachment to other extracellular matrices such as heparan sulfate proteoglycan and basement membrane collagen. The relationships between phenotypically normal mesenchymal cells and laminin have been much less emphasized in the literature. In this study, we have used antibodies that specifically label the A and B chains of laminin (but not fibronectin or other unrelated proteins) by Western blot analysis to immunoprecipitate biosynthetically derived laminin from [35S] methionine labeled cultures of neonatal and adult human skin fibroblasts. To be sure that the precipitated bands were laminin and not fibronectin, which has a molecular size very close to that of the laminin B chains, experiments were performed in which fibronectin was removed from the radiolabeled proteins by first immunoprecipitating with antifibronectin antibody and then sequentially immunoprecipitating laminin from the fibronectin-depleted supernates with antilaminin antibody. These experiments definitively demonstrate that human dermal fibroblasts synthesize and secrete laminin.     

The importance of laminin 5 in the dermal-epidermal basement membrane

The skin consists of two main layers, epidermis and dermis, separated by the basement membrane. Epidermal-dermal communication through the basement membrane is important for skin homeostasis. The basement membrane contains specialized structures, called the anchoring complex, which ensure the stability of connection and communication between these two tissue compartments. The proteins within the anchoring complex provide links to both the intracellular cytoskeletal keratins in keratinocytes and connective tissue proteins of the dermis. One of the key components of the complex is laminin 5, which is essential to epidermal cell attachment. The biological function of laminin 5 has been investigated by using a skin equivalent model in vitro and during keratinocyte sheet grafting in vivo. As a major link between the epidermal basal cells and the papillary dermis, laminin 5 initiates hemidesmosome formation and provides stable attachment of the epidermis to the dermis. Laminin 5 also accelerates the assembly of basement membranes and may enhance the recovery of damaged skin. An intact basement membrane at the epidermal-dermal junction is essential to stability of the skin.     

Changes in the distribution of laminin alpha1 chain in psoriatic skin: immunohistochemical study using confocal laser scanning microscopy

BACKGROUND: Recent studies have demonstrated the presence in psoriatic skin of ultrastructural and molecular alterations in the basement membrane and an altered polarized distribution of the integrins. Previous studies have demonstrated the existence of some epithelial cell lines synthesizing only laminin beta and gamma chains that, in the absence of the laminin alpha chain, do not form a distinct basal lamina. OBJECTIVES: To investigate a possible reduction/absence of the laminin alpha 1 chain in keratinocytes in psoriatic skin and to correlate this with fibronectin distribution. METHODS: Using monoclonal antibodies against the laminin alpha1 chain or human plasma fibronectin and using confocal laser scanning microscopy, we evaluated the immunohistochemical expression of these two proteins in cutaneous biopsies from involved and uninvolved skin of the sacral region of 12 men with extensive chronic plaque psoriasis. Site-matched biopsies of normal skin from four men without psoriasis were used as controls. Results: In normal skin antilaminin alpha 1 chain antibodies stained the dermal-epidermal junction in a regular and continuous manner. In involved and uninvolved psoriatic skin large regions of discontinuous immunostaining were observed, mainly at the apex of the dermal papillae; in the same regions, clusters of keratinocytes appeared markedly reactive and fibronectin was overexpressed in the papillary dermis under the interruptions of the basement membrane. CONCLUSIONS: The present study defines the location of the laminin alpha1 chain in involved and uninvolved psoriatic skin and suggests a possible role of the alteration of this chain, together with T-cell lymphokines and fibronectin, in the dysregulation of cell morphological processes.     

Basement membrane components and galactosylhydroxylysyl glucosyltransferase in suction blisters of human skin

Basement membrane components and collagen biosynthesis were studied in suction blisters in human skin. The basement membrane components were characterized by immunofluorescence using specific antibodies to type IV collagen, laminin and fibronectin, and collagen biosynthesis was studied by assaying galactosylhydroxylysyl glucosylatransferase. In suction blisters, the separation of epidermis and dermis occurred above the lamina lucida, indicating that the basement membrane, composed of lamina lucida and lamina densa, forms a mechanically strong entity. During the regeneration phase of blisters, type Iv collagen and laminin were not observed in the old epidermal blister roof. This indicates that keratinocytes when separated from the underlying basement membrane or connective tissue do not synthesize laminin or type IV collagen. Galactosylhydroxylysyl glucosyltransferase activity could be demonstrated in blister fluid and was about the same as in serum when expressed on the basis of protein in fresh blisters. It increased by 2-3 fold during the repair of blisters, indicating that there was local production of this enzyme. Further studies revealed that pure epidermis contained galactosylyhdroxylysyl glucosyltransferase and hydroxyprolineand this suggests that epidermis may synthesize some collagen type which, according to these studies, is not type IV (basement memebrane) collagen.     

Basement membrane and human epidermal differentiation in vitro

To test the role of basement membrane in differentiation of human epidermis reconstituted on human dermis, we prepared dermis with and without basement membrane and cultured epidermal cells on these two dermal substrata. The absence of basement membrane components was confirmed by immunofluorescence staining for laminin and type IV collagen and by electron microscopy. A high degree of differentiation of reconstituted epidermis did not require basement membrane as shown by the development of basal, spinous, and granular cell layers, and synthesis of 58 and 65-67 kDa keratins when epidermis was attached directly to dermis. On the other hand, we found that the basement membrane regulated the adhesive interaction between the epidermis and dermis. On dermis with basement membrane, attached epidermal cells formed hemidesmosomes and mechanically stable bonding. In the absence of basement membrane, the epidermal cells did not form hemidesmosomes, and bonding between the epidermis and dermis was unstable. Moreover, dermis from which the basement membrane was removed was reorganized by the epidermal cell layer.     

Basement membrane and connective tissue proteins in early lesions of Kaposi's sarcoma associated with AIDS

Nearly one-third of all young homosexual men diagnosed as having acquired immune-deficiency syndrome (AIDS) develop a disseminated form of dermal Kaposi's sarcoma (KS). Although the histogenesis of KS cells is unclear, certain evidence suggests that the aberrant cells are of endothelial derivation. We have examined the presence and distribution of connective tissue-specific and basement membrane-specific macromolecules by indirect immunofluorescence and immunoperoxidase staining of frozen sections in early cutaneous lesions of KS from individuals with AIDS. The KS cells typically line the spaces between collagen bundles of the reticular dermis. When stained for the connective tissue-specific glycoprotein fibronectin, all Kaposi's sarcoma lesions showed an intense staining pattern, revealing a complex array of linear deposits of antigen that outlined the exterior surface of the collagen bundles. Antibodies to laminin and type IV collagen, both basement membrane-specific macromolecules, produced an intense staining pattern similar to that found with the anti-fibronectin antiserum, indicating that all 3 antigens are closely codistributed. In contrast, antibodies to type I collagen, the major collagen of the dermis, uniformly stained the collagen bundles in the KS lesions and in the normal control skin. Antiserum to factor VIII-associated antigen, an antigen specific to blood vascular endothelium, frequently stained the KS lesions but the staining pattern was diffuse and of variable intensity. The results suggest that KS cells are derived from the endothelium of the blood microvasculature and maintain their secretory phenotype of secreting basement membrane-specific macromolecules.     

Separation of epidermis from dermis with sodium thiocyanate.

After human skin is treated with 2 N sodium thiocyanate, epidermis is easily separated from dermis. The level of cleavage occurs at the lamina lucida of the basement membrane zone. Bullous pemphigoid antigen remains attached to the epidermis.     

Production of fibronectin by epithelium in a skin equivalent

Although human keratinocytes in vitro have been shown to produce fibronectin, whether keratinocytes can contribute fibronectin to the dermal-epidermal junction or wound matrix is unknown. In order to approach this problem experimentally, we used the "skin equivalent" model composed of a native collagen gel populated with cultured fibroblasts and covered by cultured keratinocytes. By using bovine fibroblasts to populate the gel, fetal bovine serum in the culture medium, and human keratinocytes to form the epithelium, we were able to be certain that any human fibronectin produced in the culture was synthesized by the keratinocytes. A monoclonal antibody to fibronectin was found to recognize human but not bovine fibronectin. When the skin equivalent was stained by indirect immunofluorescence with antifibronectin, fibronectin was visible as an intensely staining band at the dermal-epidermal junction. In sections in which the dermis and epidermis had separated, the staining was usually limited to the dermal aspect of the skin equivalent. The results indicate that epithelium can contribute fibronectin to the dermal-epidermal junction and suggest that dermal staining in skin sections may originate from the epidermis. Since the developing skin equivalent has a rapidly growing epithelium and simulates a healing wound, contribution of fibronectin by the epithelium, in addition to that possibly contributed by serum and fibroblasts, may be of importance in wound healing.     

Loss of hyaluronan in the basement membrane zone of the skin correlates to the degree of stiff hands in diabetic patients

Glycosaminoglycans are important components of all extracellular matrices. One of the glycosaminoglycans is hyaluronan, which is ubiquitously distributed throughout the connective tissue. Hyaluronan is especially abundant in the skin, in which it is of both structural and functional importance. This study describes the localization and distribution of hyaluronan in the skin of healthy individuals and of 23 patients with insulin-dependent diabetes mellitus and various degrees of limited joint mobility. In normal skin, hyaluronan staining was seen in all layers but most prominently in the papillary dermis and the basement membrane zone. In the skin from diabetic patients with normal or only moderately restricted mobility of the hands (limited joint mobility grades 0 and 1), the distribution of hyaluronan was similar to that of normal skin. In the skin of patients with severe restriction in joint mobility (limited joint mobility grade 2) the staining pattern was significantly different with weak hyaluronan staining in the papillary dermis and the basement membrane zone almost devoid of hyaluronan. Moreover, an increased epidermal thickness in the latter patients was evident as well as a pronounced hyaluronan staining compared with normal epidermis.     

Abnormal maturation pathway of keratinocytes in psoriatic skin

We compared the maturation pathway of normal and psoriatic epidermis using three different markers: (1) Involucrin, which is normally detected in the stratum granulosum in normal skin, was detected in all but the basal layer of involved psoriatic skin; (2) an antigen, recognized by the murine monoclonal antibody psi 3, was present in all but the basal layer of involved psoriatic skin but was absent from uninvolved and normal skin; (3) fibronectin, which normally localizes in the dermis and the epidermal-dermal junction, was also detected intra- and extracellularly in the psoriatic epidermis. These results indicate that the alterations in keratinocyte maturation found in psoriasis do not arise from a truncation of the normal maturation pathway but rather reflect the onset of an abnormal pathway of differentiation characterized by the expression of psi 3 antigen and fibronectin and the premature appearance of involucrin.     

A comparison of expression of surface-bound immunoglobulin E on antigen-presenting cells in cutaneous tissue between patients with allergic, irritant and atopic dermatitis

The expression of surface-bound immunoglobulin E by dendritic cells within cutaneous tissue has been compared in atopic and contact dermatitis. 45 patients were recruited into 4 groups using clinical criteria and patch testing to a standard series of allergens: atopic (12 cases), allergic contact dermatitis (14 cases), irritant contact dermatitis (10 cases) and the control group (9 cases); using clinical criteria and patch testing to a standard series of allergens. Skin biopsies from each patient were analysed by the indirect immunofluorescence technique. This differentiated 3 patterns of cutaneous IgE distribution: (i) no detectable cutaneous IgE; (ii) detection of IgE solely within the dermis; (iii) detection of IgE within both epidermis and dermis. Detection of IgE within the epidermis was always associated with the presence of IgE within the dermis. In each case, IgE was surface-bound by dendritic cells. Immunoglobulin E was detected within both epidermis and dermis in skin biopsies from 8 (66.7%) atopic patients and 2 (20%) patients with irritant contact dermatitis. No other cases demonstrated IgE deposition within both the epidermis and dermis. Atopic patients were significantly more likely to have detectable IgE deposition, within both epidermis and dermis, than patients with contact dermatitis (allergic and irritant groups combined, p = 0.0011) or controls (p = 0.0049). This finding suggests that the demonstration of IgE within both epidermis and dermis supports a diagnosis of atopic dermatitis. It would therefore be of value in differentiating between atopic and contact dermatitis, where clinical diagnosis is in doubt.     

Cytoplasmic vitamin A binding proteins in chick embryo dermis and epidermis

Excess vitamin A has striking morphologic and developmental effects on chick embryo skin. While cytoplasmic retinoic acid-binding protein (CRABP) was known to be abundant in chick embryo skin, neither quantitative values nor the distribution between dermis and epidermis have been established. We determined CRABP levels in collagenase-separated dermis and epidermis from 8-day-old embryos using specific binding of all-trans-[11-3H]retinoic acid in cytosols prepared from gram quantities of these tissues. The level of CRABP in dermis was twice the level in epidermis whether calculated on the basis of wet weight, cytosol protein, or DNA. When averaged over many preparations, 3 times as much dermis as epidermis was recovered from a single piece of skin. Therefore, the dermis contained 85% of the extremely high CRABP levels found in collagenase-treated skin, while epidermis contributed only 15%. Cytoplasmic retinol binding protein (CRBP) was also detected in chick embryo skin, but the binding was low and the levels in epidermis and dermis were not significantly different. The amount of CRABP in chick embryo skin (1600 pmol/g wet weight or 100 pmol/mg cytosol protein) is the highest level reported in any tissue and suggests an important role for vitamin A in the normal development and maturation of skin.     

Activation of heparanase by ultraviolet B irradiation leads to functional loss of basement membrane at the dermal–epidermal junction in human skin

Recently, we reported that heparanase plays important roles in barrier-disrupted skin, leading to increased interaction of growth factors between epidermis and dermis and facilitating various cutaneous changes, including epidermal hyperplasia and wrinkle formation. However, the role of heparanase in sun-exposed skin remains unknown. Here, we show that heparanase in human keratinocytes is activated by ultraviolet B (UVB) exposure and that heparan sulfate of perlecan is markedly degraded in UVB-irradiated human skin. The degradation of heparan sulfate resulted in a marked reduction of binding activity of the basement membrane for vascular endothelial growth factor, fibroblast growth factor-2 and -7 at the dermal–epidermal junction. Degradation of heparan sulfate was observed not only in acutely UVB-irradiated skin, but also in skin chronically exposed to sun. Interestingly, heparan sulfate was found to be degraded in sun-exposed skin, but not in sun-protected skin. These findings suggest that chronic UVB exposure activates heparanase, leading to degradation of heparan sulfate in the basement membrane and increased growth factor interaction between epidermis and dermis. These changes may facilitate photo-aging.     

Distribution of fibronectin and laminin in basal cell epitheliomas

The distribution of fibronectin (FN) and laminin (LM) in basal cell epithelioma was evaluated by indirect immunofluorescence. FN is a glycoprotein which promotes interaction between cells and the extracellular matrix, and is present at the dermal-epidermal junction (DEJ) and throughout the dermis, but absent in the normal epidermis. LM, a noncollagenous basement membrane protein, plays a role in epithelial adhesion to type IV collagen, and is normally present in the DEJ, but absent in the epidermis. The role of FN and LM in epithelial differentiation has not been established. Therefore, the distribution of FN and LM in a tumor of epithelial origin was studied by indirect immunofluorescence. Using affinity-purified antibodies to FN and LM, and the appropriate fluorescein-conjugated second antibodies, normal skin and 7 basal cell tumors were examined. By immunofluorescence, nests of malignant basal cells were surrounded by linear LM staining. FN immunofluorescence was intense throughout the connective tissue stroma of all tumors. Five tumors also showed FN staining within the nests of neoplastic cells, and 4 of these were also LM-positive in the tumor bulk. These immunofluorescent findings suggest that epidermal neoplasia can be associated with alterations in the distribution of FN and LM.     

Human Tattoo

Ultrathin serial sections of human biopsy specimens, taken at 24 hours, 1 month, and 1, 3, and 40 years post-tattooing were examined under the electron microscope. The ink particles found in cells were measured and compared with control ink particles embedded in agar. Freshly tattooed skin showed an inflammatory reaction followed by ultrastructural necrosis. The time of healing, about 1 month from introduction of ink to complete healing, has been divided into three phases: inflammatory reaction and necrosis, formation of basement membrane, and normal epidermis and dermis. Once the skin showed normal ultrastructure, ink particles were found only in dermal fibroblasts.