Ultrastructural localization of lectin-binding sites in normal human eccrine and apocrine glands

The distribution of carbohydrate residues in eccrine and apocrine glands of normal human skin was studied using a post-embedding technique with Lowicryl K4M. Thin sections were incubated with Ulex europaeus agglutinin I (UEA I), wheat germ agglutinin (WGA), peanut agglutinin (PNA), concanavalin A (Con A), soybean agglutinin (SBA), and dolichos biflorus agglutinin (DBA). All lectins except for PNA showed labeling of the plasma membranes of dark cells, clear cells, and apocrine cells. The granules of the eccrine gland were labeled with all lectins except for DBA. The mitochondrial granules of the apocrine gland were not labeled with any lectin, whereas the lysosomal granules showed a positive reaction with all lectins except for PNA. After incubation with PNA, in eccrine glands the granules were the only structure labeled, whereas in apocrine glands the luminal side of the plasma membrane and cytoplasmic vesicles beneath it were the only structures labeled.     

Ultrastructural localization of actin in normal human skin

Summary Normal human skin was embedded in Lowicryl K4M. Actin microfilaments were localized by applyinga postembedding immunogold technique using the monoclonal anti-actin antibody HHF35. Actin microfilaments are part of the cytoskeleton in muscle and nonmuscle cells. Together with myosin they produce contraction. The antibody labelled myofilaments in smooth muscle arrector pili cells, myoepithelial cells and pericytes. In sweat gland cells the microvilli system, a zone beneath the cytoplasma membrane correponding to the adhesion belt region, and apocrine decapitation formations showed labelling.     

Ultrastructural localization of anionic sites in the rat skin demonstrated with cationic probes

For the purpose of demonstrating ultrastructurally the distribution of anionic sites in the rat skin, we applied two polyethyleneimines of different molecular weights and ruthenium red by the perfusion or immersion method. Using each cationic probe, sites were observed as electron dense particles along both the epidermal and interstitial sides of the lamina densa in the dermo-epidermal junction and the interstitial side of the periendothelial lamina densa in the dermal capillary. The ordered distribution of the anionic sites may function to transport fluid and macromolecular substances through these particular compartments.     

Ultrastructural localization of HMB-45 binding sites

Three malignant melanomas, two melanoma metastases, two junctional dysplastic nevi, and normal skin were embedded in Lowicryl. Ultrathin sections were incubated with HMB-45 and a gold-labeled anti-mouse antibody. Gold particles indicating the presence of HMB-45 were found in melanosomes Stage 1 and 2 and in the non-melanized portion of melanosomes Stage 3. Melanosomes Stage 4 and melanosome complexes in keratinocytes, as well as in melanophages, were consistently negative. No specific labelling with HMB-45 was seen in eccrine glands of normal skin.     

Ultrastructural localization of lectin binding sites in human melanocytes

Summary The ultrastructural localization of carbohydrate residues in human melanocytes of normal epidermis and of one compound naevus was studied. The following lectins were used in a post-embedding technique: 1. peanut agglutinin (PNA), which reacts specifically with N-acetyl-galactosamine; 2. Concanavalia ensiformis (Con A) indicating -d-glucose and -d-mannose binding sites; 3. Ulex europaeus agglutinin (UEA I) specific for -l-fucose; 4. Wheat germ agglutinin (WGA), reacting specifically with N-acetyl-glucosamine and neuraminic acid (sialic acid); and 5. Limax flavus agglutinin (LFA), also specific for sialic acid (Neu5Ac--2,3-Gal and Neu5Ac--2,6-Gal). When incubated with WGA, Con A and LFA strong labelling was seen within the cytoplasm and in the plasma membrane of melanocytes, whereas incubations with PNA and UEA I revealed an occasional gold particle only. The determination of the distribution of carbohydrate residues in normal melanocytes is a prerequisite for future studies of abnormal melanocytes.     

Plasminogen binding sites in normal human skin

Plasminogen is detected in the basal cell layer of the epidermis, keratinocytes can generate plasminogen activators and it is suggested that the generation of plasmin may facilitate keratinocyte division, migration and differentiation. In this study we have investigated the characteristics of plasminogen binding sites in normal human epidermis. It was found that 6-aminohexanoic acid and benzamidine displaced endogenous epidermal plasminogen from the basal layer suggesting that endogenous plasminogen binds initially via the kringle 5 aminohexyl (AH) site. Plasminogen binding sites in epidermis were further investigated by displacing endogenous plasminogen and incubating sections with exogenously added glu-plasminogen, lys-plasminogen and plasmin or the isolated plasminogen fragments kringles 1-3, kringle 4 and kringle 5L. The results suggest that the uptake of plasminogen involves primary interaction with the kringle 5AH site and a secondary interaction with lysine binding sites of kringles 1-3. Cell binding is not dependent upon additional reactions of the plasmin active centre.     

Ultrastructural localization of calcium in psoriatic and normal human epidermis

With ion capture cytochemistry, we previously demonstrated the distribution of calcium ions in murine epidermis, a pattern consistent with a role for this ion in the regulation of epidermal differentiation. Because of the known proliferation and differentiation defects in psoriasis, we compared the calcium distribution of involved vs uninvolved psoriatic lesions and normal human epidermis. Whereas normal human and uninvolved psoriatic epidermis revealed increased calcium-containing precipitates in the uppermost stratum granulosum, in contrast the basal layer of psoriatic lesions contained less extracellular calcium, a condition that favored enhanced proliferation. Moreover, all psoriatic suprabasal cell layers displayed heavier than normal concentrations of calcium, indicating loss of the normal calcium gradient that programs terminal differentiation. This abnormal profile may account for the differentiation defects (eg, parakeratosis) that occur in psoriasis. Finally, psoriatic lesions displayed retained ionic Ca in intercellular domains of the upper stratum granulosum with absence of normal intercellular bilayers, findings that may underlie the abnormal desquamation and permeability barrier in psoriasis.     

Immunohistochemical localization of lysyl oxidase in normal human skin

Lysyl oxidase (EC 1.4.3.13), a copper-dependent enzyme which catalyses the formation of aldehyde cross-links, and acts primarily on collagen and elastin, is known to be increased during wound healing and in fibrotic disorders including liver cirrhosis and atherosclerosis, and to be decreased in some hereditary connective tissue diseases and in malignant cell lines. A recent study showed that lysyl oxidase might possess tumour suppressor activity as an antioncogene for ras. Little is known about the localization of this enzyme in human skin. In this study, we determined immunohistochemically the localization of lysyl oxidase in normal skin of young and elderly subjects obtained from sun-exposed and unexposed regions of the body. All skin samples tested had similar distributions of lysyl oxidase. The enzyme was present both extracellularly and intracellularly. Extracellularly, a few granular aggregates of immunoreactants were observed along collagen and elastic fibres. These granules were more common in the adventitial portion of the dermis than in the reticular portion. Of all sun-exposed and unexposed regions studied, the skin of the face displayed the greatest amount of extracellular immunoreactants. Immunopositive granules were observed intracellularly in fibroblasts, vascular endothelial cells, sweat glands, sebaceous glands, arrector pili muscles and some keratinocytes. These findings provide evidence that, as suggested in recent reports, lysyl oxidase may have a variety of intracellular functions.     

Ultrastructural alteration of tape-stripped normal human skin after photodynamic therapy

The effect of photodynamic therapy on tape-stripped normal human skin was explored ultrastructurally. Back skin areas of 3 healthy Caucasian volunteers, 2 men and 1 woman, were tape-stripped 10 consecutive times followed by topical treatment with 5-aminolevulinic acid (20%, w/w) for 4 hours under occlusion (Tegaderm). Then the areas were irradiated for 30 minutes with Waldman PDT 1200 lamp (570-650 nm) with a total dose of 70 J/cm(2). Full-thickness punch biopsies were taken immediately after irradiation, and at 3 and 24 hours for electron microscopy examination. Photodynamic therapy caused morphological alterations mainly in the epidermis. Keratinocytes became oedematous and tonofilament bundles were split, but desmosomes remained normal. Many keratinocytes contained large intracellular vacuoles and extremely electronlucent cytoplasm implying cell damage. Although the majority of Langerhans cells were unaffected isolated Langerhans cells became apoptotic at 3 hours. The melanocytes preserved their normal morphology. The epidermal alterations recovered 24 hours after the irradiation. Inflammatory cell infiltrates were evident at 3 and 24 hours but no other dermal changes were observed. In conclusion, photodynamic therapy with 5-aminolevulinic acid affects mainly keratinocytes and can trigger apoptosis in Langerhans cells while melanocytes appear refractory, at least morphologically, to photodynamic therapy.     

Autoradiographic localization of endothelin-1 binding sites in porcine skin.

Autoradiographic techniques and 125I-labeled endothelin-1 were used to study the distribution of endothelin-1 binding sites in porcine skin. Specific endothelin-1 binding sites were localized to blood vessels (capillaries, deep cutaneous vascular plexus, arteries, and arterioles), the deep dermal and connective tissue sheath of hair follicles, sebaceous and sweat glands, and arrector pili muscle. Specific binding was inhibited by endothelin-2 and endothelin-3 as well as endothelin-1. Non-specific binding was found in the epidermis and the medulla of hair follicles. No binding was found in connective tissue or fat. These vascular binding sites may represent endothelin receptors, in keeping with the known cutaneous vasoconstrictor actions of the peptide. If all binding sites are receptors, the results suggest that endothelin could also regulate the function of sweat glands and may have trophic effects in the skin.     

Immunohistochemical localization of type V collagen in normal human skin

Summary Tissue distribution of type V collagen in normal human skin was studied using an indirect immunofluorescent technique to determine whether type V collagen is present in the interstitium or in the basement membrane. Type V collagen was isolated from the human placenta by pepsin digestion and was purified with fractioning salt precipitations. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that type V collagen contained 1(V) and 2(V) chains, but not the 3(V) chain. Specificity of the rabbit antibodies to type V collagen was assessed using enzyme-linked immunosorbent assay (ELISA) and an immunoblotting method. Antibodies showed no cross-reactivity to other collagens, laminin, and fibronectin. With an indirect immunofluorescent technique, type V collagen was found to be widely distributed throughout the dermis. Intense fluorescent staining was noted in the papillary dermis and adnexal dermis surrounding hair follicles and eccrine glands. The basement membrane of the dermoepidermal junction, skin appendages, and capillaries was not stained. By indirect immunoperoxidase double staining, type V collagen was not found to be deposited on type IV collagen present in the basement membrane. Immunoelectron microscopic studies showed that type V collagen was not located in the basal lamina. These results suggest that type V collagen is distributed in the interstitium, but not in the basement membrane of normal human skin.     

Immunohistochemical localization of lipocortins in normal and psoriatic human skin

The distribution of lipocortin I, a steroid-induced inhibitory protein of phospholipase A₂, was examined in normal and psoriatic human skin. Using immunoblotting analysis with specific antibody against human lipocortin I purified from human placenta, lipocortin I was detected as a 37 kDa protein in cultured epidermal cells, whole skin and epidermis. In the dermis and stratum corneum, lipocortin I was only weakly detectable by Western blotting. In contrast to normal skin, much less lipocortin I was detected by Western blotting analysis in psoriatic skin. Using immunoperoxidase immunohistochemical analysis, lipocortin I was demonstrated in the cytoplasm of keratinocytes in the upper and middle layers of the epidermis and in some infiltrating cells in the dermis in normal skin. In involved psoriatic skin, by contrast, lipocortin I was almost undetectable in the epidermis, although it was demonstrated in some infiltrating cells in the dermis. No immunostaining of lipocortin I was observed in the stratum corneum of normal or psoriatic skin. These results, together with the finding that phospholipase A₂ activity is higher in psoriatic epidermis than in normal epidermis, suggest that lipocortin I plays an important role in the regulation of differentiation and proliferation of epidermal keratinocytes.     

Ultrastructural localization of gap junction protein connexin 43 in normal human skin, basal cell carcinoma, and squamous cell carcinoma

The expression and localization of connexin 43 (Cx43) were investigated in normal human epidermis, pilosebaceous apparatus, basal cell carcinoma, and squamous cell carcinoma by immunofluorescence as well as by immunoelectron microscopy. In the normal epidermis the immunofluorescence was weak in the basal layer, increased in spinous layer and negative in the horny layer. In the sebaceous gland, peripheral lobular cells showed weak cell membrane dotted pattern. Cell membrane and cytoplasmic fluorescence was strong in the central lobular cells. In the lower hair follicle, the cortex, inner and outer root sheath cells showed cell membrane fluorecence. As cortical cells underwent keratinization, they lost Cx43 epitopes. Basal cell carcinoma and squamous cell carcinoma were poorly stained, and eccrine and apocrine glands were unstained. In immunoelectron microscopy, close membrane appositions of typical gap junctions were often observed in the spinous layers of the epidermis and the immunolabeling for Cx43 was seen along the gap junction structures. Circular and long gap junctions often were found in follicular root sheaths and sebaceous glands. Gold particles labeling Cx43 in these gap junctions were found on the gap junctions or localized in the cytoplasm near the gap junction membranes. Basal cell carcinoma and squamous cell carcinoma had a small number of small gap junctions, and gold particles were not only localized to gap junctions but scattered in the cytoplasm. No gap junctions were labeled in eccrine and apocrine glands. These findings confirmed that 1) long, curved or circular membrane appositions found in hair follicle and sebaceous gland are true gap junctions, 2) immature cells such as epidermal basal cells, peripheral germinative cells of sebaceous gland and basal and squamous cell carcinoma cells do not have fully developed gap junctions, and 3) Cx43 or its precursors are present in the cytoplasm as well as on poorly developed gap junctions in these immature cells. Immunofluorescence findings generally corresponded to ultrastructural distribution and structural maturity of gap junctions.     

Immunohistochemical localization of cathepsin L and cystatin A in normal skin and skin tumors

Cathepsin L, a cysteine proteinase, and cystatin A, an inhibitor of cysteine proteinases, are thought to regulate the invasion and metastasis of malignant cells. In this study, the expression of cathepsin L and cystatin A in skin tumors was investigated immunohistochemically in order to examine the relationship between these two enzymes in the pathophysiology of malignant cells. Formalin-fixed and paraffin embedded specimens from normal skin, seborrheic keratoses, and squamous cell carcinomas were reacted with polyclonal antibodies against rat cathepsin L or cystatin a which cross-react to human cathepsin L and cystatin A, respectively. The consequent immunostaining of these enzymes was observed to be strong in normal skin (4 cases) and seborrheic keratosis (6 cases). In well-differentiated squamous cell carcinoma (SCC) (9 cases), staining for cathepsin L and cystatin A was moderately positive in differentiated tumor cells and negative in undifferentiated SCC (5 cases). The degree of staining of these enzymes was inversely correlated with the differentiation of the malignant cells. These results suggest that the immunohistochemical analysis of cathepsin L and cystatin A is a useful indicator for an aspect of malignancy in human epidermal keratinocytes.     

Ultrastructural and immunohistochemical characterization of normal mast cells at multiple body sites

This article reviews the ultrastructural and immunohistochemical features of normal human mast cells (MC) at multiple tissue sites. Current literature indicates that granules containing discrete scrolls (scroll-rich morphology) are frequent in MC from bowel mucosa and lung, locations where the majority of MC show only tryptase immunoreactivity (MCT). In contrast, most MC from skin, breast parenchyma, axillary lymph nodes, and bowel submucosa are characterized by scroll-poor morphology (that is, granules are rimmed by incomplete scrolls forming parallel lamellae and containing central, amorphous granular material or grating/lattice-like structures) and show both tryptase and chymase immunoreactivity (MCTC). MC having granules with both scroll-rich and scroll-poor features can occur in all tissue sites, and an occasional MC, especially in lung and bowel, may show only chymase immunoreactivity (MCC). Chymase immunoreactivity in MC also is closely associated with avidin binding and carboxypeptidase reactivity. We conclude that there is ultrastructural and immunophenotypic diversity among normal human MC, although certain forms predominate in specific tissue environments. In skin, breast tissue, axillary lymph nodes, and bowel submucosa MC tend to have scroll-poor granules and stain for avidin, chymase, tryptase, and carboxypeptidase, whereas, in lung and bowel mucosa MC granules tend to be scroll-rich and stain only for tryptase with currently available reagents.