Ultrastructural morphometric analysis of human mast cells in normal skin and pathological cutaneous lesions

Electron micrographs of human mast cells in normal neonatal and adult skin and in cutaneous lesions of basal cell carcinoma (BCC), hemangioma and mastocytosis were assessed by morphometric analysis. Using this quantitative histologic approach, adult skin mast cells were found to be significantly larger (47.7 microns 2 +/- 2.4 SEM vs. 38.3 microns 2 +/- 1.8 SEM, p less than or equal to 0.001) and have larger granules (0.63 micron +/- .02 SEM vs. 0.53 micron +/- .02 SEM, p less than or equal to 0.001) than infant mast cells while both mast cell populations had comparable nuclear sizes (13.7 microns 2 +/- 0.9 SEM vs. 14.3 microns 2 +/- 0.8 SEM) and numbers of cytoplasmic granules (72 +/- 4.0 SEM vs. 66 +/- 4.0 SEM). Morphometric analysis of mast cell infiltrates in the adult skin lesions of BCC and hemangioma revealed that these cells were larger than neonatal mast cells but were similar to normal adult controls. Cutaneous mast cells from 2 mastocytosis patients, however, had significantly larger mean cell surface areas (78.0 microns 2 +/- 3.4 SEM and 70.6 microns 2 +/- 3.2 SEM, p less than or equal to 0.001), nuclear areas (20.8 microns 2 +/- 1.1 SEM and 21.3 microns 2 +/- 1.2 SEM p less than or equal to 0.001) and granule diameters (0.82 micron +/- 0.4 SEM and 0.83 micron +/- .03 SEM, p less than or equal to 0.001) when compared with mast cells in normal adult skin and in the other pathologic lesions. No difference in the total number of cytoplasmic granules was observed in the different mast cell populations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural and immunohistochemical characterization of basal cells in three-dimensional culture models of the skin

Keratinocytes were cultured on fibroblast-free dermal substitutes made of type I collagen film (collagen dermal substitute) and an extracellular matrix gel film (matrix dermal substitute), each of which was laid on a lyophilized type I collagen sponge. The morphology of the basal keratinocytes in these three-dimensional culture models of the skin was studied ultrastructurally and immunohistochemically to assess their differentiation to basal cells. The basal keratinocytes in the artificial epidermis cultured on the collagen dermal substitute showed poorly organized tonofibril networks and desmosomes. Neither the tonofibril-hemidesmosome complex nor the lamina densa were detected along the interface, where many cytoplasmic projections of basal keratinocytes were noted. There were no detectable antigens of type IV or VII collagen, LDA-1, or laminin in the interface. Bullous pemphigoid (BP) and 1-2B7B antigens and integrins were expressed along the cytoplasmic membrane and the projections of the basal keratinocytes. A high molecular weight keratin (keratin 1, 68 kDa, 34B4) was detected only in part of the uppermost layers of this artificial epidermis. In contrast, basal keratinocytes in the artificial epidermis on the matrix dermal substitute developed tonofibril networks radiating to desmosomes and hemidesmosomes, under which a primitive lamina densa was present. Basement membrane zone antigens, such as type IV and VII collagens, LDA-1 and laminin were noted along the interface as were 1-2B7B and BP antigens and integrins. Laminin and type VII collagen were also detected along or in the membrane of the endoplasmic reticulum of basal keratinocytes. The high molecular weight keratin was detected in the suprabasal layers of this artificial epidermis, but the basal keratinocytes lacked expression of this epitope as does epidermis in vivo. The artificial epidermis cultured on the matrix dermal substitute had a basal layer of keratinocytes that could be characterized as basal cells, forming junctional structures resembling those of epidermis in vivo.     

Identification and characterization of multiple forms of tryptase from human mast cells

Abstract Mast cell tryptase purified from human adult skin (AS), adult lung (AL) and newborn foreskin (NS) with a monoclonal antitryptase B2 immunoaffinity Sepharose column was further fractionated by HPLC using a Mono-S cation exchange column at pH 6.5. Tryptases exhibited two clearly separated major fractions, both of which also revealed at least two overlapping peaks. Native tryptase molecules from skin consisted of two diffuse protein bands in SDS-PAGE at about 31 and 35 kDa, whereas those from lung usually exhibited a predominant diffuse band at about 29 kDa. The forms of tryptases separated by Mono-S HPLC gave a different banding pattern in SDS-PAGE. Tryptase from NS exhibited chromatographic peaks that each showed M_r values approximately 1–3 kDa higher than those of tryptase from AS. By gel filtration, the M_r values for native major fractions of tryptases derived from AS and AL were 178 kDa and 141 kDa, respectively. After carbohydrate removal by glycanase, the observed differences in M_r values in SDS-PAGE reduced to two similar sharp bands of M_r approximately 28 kDa and 30 kDa for all tryptase preparations. AS and AL tryptases and their subfractions exhibited similar enzyme kinetic values and similar immunoreactivities in a tryptase immunoassay. Inactivation rates at physiologic ionic strength were similar for both AL and AS tryptases. The results show the enzymatic and antigenic similarity between lung and skin tryptases, and suggest that tryptase is stored mainly as β-tryptase in human mast cells. Tryptase immunoassay measures similarly both lung and skin tryptases and, thus, this assay is suitable for detection of mast cell activation, in contrast to assays for other proteinases of mast cells, e.g. chymase, cathepsin G and carboxypeptidase, that are present in MC_TC cells mainly in skin only. Received: 2 June 1998 / Received after revision: 12 October 1998 / Accepted: 16 October 1998     

Ultrastructural localization of lectin-binding sites in normal skin

The distribution of carbohydrate residues in keratinocytes of normal epidermis was studied. Normal skin was embedded in Lowicryl. Thin sections were incubated with concanavalin A (Con A), peanut agglutinin (PNA), wheat germ agglutinin (WGA), Ulex europaeus agglutinin I (UEA I), dolichos biflorus agglutinin (DBA), and soybean agglutinin (SBA). A positive reaction in the dermis, in the basal lamina (lamina densa, lamina lucida), intracellularly and within the plasma membrane including the desmosomes was obtained after incubation with Con A and WGA. PNA binding sites were found predominantly in the plasma membrane between the desmosomes. The labeling with Con A, WGA, and PNA was most pronounced in the upper stratum spinosum and granulosum. Incubation with UEA revealed heavy labeling of the keratohyalin granules and the cytoplasm of the corneocytes. Incubation with DBA and SBA revealed weak labeling of the keratinocytes. The study of the distribution of carbohydrate residues in normal epidermis is important, since alterations in this distribution might be linked to autoimmunity or malignant transformation.     

Ultrastructural features of mast cells in systemic mastocytosis.

A 3-year-old boy with systemic mastocytosis has been observed since the age of 4 months when he was first diagnosed as suffering from urticaria pigmentosa. Involvement of skin, liver, spleen and bones was observed. The electron microscopy of skin and liver revealed varied alterations in the morphology of mast cells. The most important findings were irregularly-shaped cells and unusual long and interdigitated cytoplasmic villi, with consequent aggregation of mast cells which was more prominent in the dermis. Proliferation and accumulation of mitochondria in one part of the cell and deeply indented nuclei were frequent. The problem, whether the morphological changes encountered--especially the complex interdigitation of villi--should be interpreted as a sign of expected neoplastic development, is discussed.     

Ultrastructural findings in tissue mast cells in urticaria pigmentosa

In a study on 10 patients suffering from urticaria pigmentosa, biopsies of skin lesions were electron microscopically investigated. In contrast to normal skin, the mast cells showed irregular, partly bilobed nuclei, prominent nucleoli, hypogranulation, and rather small granules; giant granules were rarely observed. In addition, we frequently found degranulation, and the development of microvilli was much more prominent than with normal mast cells. These deviations from normal skin represent at least in part functional alterations. However, some of the findings suggest that urticaria pigmentosa may be considered a neoplastic disease of the tissue mast cells, although clear evidence is still lacking. In any case, our findings show that mast cells in mastocytosis are not identical with those in normal skin.     

Phenotypic evaluation of cultured human mast and basophilic cells and of normal human skin mast cells

In order to evaluate various markers for human mast cells, two human mast/basophilic cell lines (HMC-1/KU812), cultured mast cells from the peripheral blood monocytic fraction and peripheral blood monocytes were compared with mast cells in tissue sections from normal skin, using histochemistry, enzyme histochemistry and immunohistochemistry. All reagents stained normal skin mast cells, with toluidine blue, tryptase reactivity and antibodies against the FcRI and the stem cell factor receptor (c-kit) being most active. The cell lines and mast cells cultured from peripheral blood were negative for avidin, safranin and chymase, strongly positive for c-kit and variably reactive with all other reagents. All antibodies except AA1 against tryptase also stained one or several epidermal and dermal cell types or blood monocytes. Histochemical stains (toluidine blue, avidin) and reagents for the enzymes tryptase and chymase are thus specific markers for mast cells. The frequent reactivity of antibodies against mast cells with other cell types indicates interesting functional and ontogenetic relationships between these cells.     

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.     

Matriptase expression in the normal and neoplastic mast cells

Matriptase, a type II transmembrane serine protease, is distributed in almost all normal human epithelium. Several studies have demonstrated that matriptase expression is correlated with tumor progression in epithelium-derived cancer cells. Mast cells, which originate from pluripotent hematopoietic cells in the bone marrow, can produce and store almost cellular-specific neutral serine proteases, such as tryptase and chymase, and are functionally involved in both the immediate hypersensitivity response and anaphylactic shock. Mast cells are significantly increased in several neoplasms, indicating that they most likely play a role in degrading the tissue matrix. Recently, trypsin has been revealed to activate the latent matriptase on the surface of several human cancer cell lines, suggesting that matriptase and trypsin cooperatively function in extracellular proteolysis. In our study, almost all mast cells in tissues throughout the body stained positive for matripase. Matripase was also found in neoplastic mast cells. To our knowledge, this is the first time that matriptase has been shown to be expressed by mast cells. Therefore, we suggest that this expression of matriptase may not only be useful as an additional marker for mast cells but also be involved in their physiopathological function.     

Absence of MHC class II antigen on mast cells at sites of inflammation in human skin

Abstract Since the presence of major histocompatibility complex (MHC) antigens has recently been reported on murine and human mast cells under various conditions, we have investigated their expression on mast cells in different types of cutaneous inflammation. Cryostat sections from lesional biopsies of patients with psoriasis, atopic eczema, chronic urticaria, lichen planus, bullous pemphigoid and urticaria pigmentosa were immunohistochemically stained with monoclonal antibodies against MHC class I and class II antigens using a double staining APAAP/toluidine blue methodology. While strongly positive staining with the antibody directed against MHC class I antigens was found on nearly all mast cells in normal skin and in inflammatory dermatoses, reactivity for HLA-DR and HLA-DQ antigens on mast cells could not be detected, except for less than 2% of cells with doubtful staining. Human mast cells therefore probably play no significant rôle as antigen-presenting cells in the conditions studied.     

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Action of adenosine triphosphate on mast cells in normal and denervated skin

Summary Treatmentin vitro with adenosine triphosphate (ATP) causes degranulation of dermal mast cells in the skin of the rat's external ear. The magnitude of this effect increases with the concentration of ATP applied. In normal skin there is a marked increase in the proportion of degranulated mast cells with 2.5·10^–6M ATP, while in skin denervated for 14 days this increase occurs with 1.25·10^–6 M ATP. Thus, denervation is followed by hypersensitivity of the mast cells to ATP. This result is discussed in conjunction with other evidence that ATP may be a neurotransmitter substance released into the skin by impulses travelling antidromically in sensory axons.

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Zusammenfassung In vitro-Behandlung mit Adenosintriphosphat (ATP) verursacht eine Degranulation der dermalen Mastzellen in der äußeren Ohrhaut der Ratte. Die Degranulation nimmt mit der verwendeten ATP-Konzentration zu. In normaler Haut ist die Zahl der degranulierten Mastzellen bei einer Konzentration von 2,5·10^–6 M ATP gesteigert, während in einer 14 Tage lang denervierten Haut diese Steigerung bei einer Konzentration von 1,25·10^–6 M ATP vorliegt. Die gesteigerte Hypersensibilität der Mastzellen auf ATP ist somit von der Denervation abhängig. Diese Beobachtung unterstützt die Vermutung, daß ATP möglicherweise eine Neurotransmittersubstanz ist, die durch sensible Axonimpulse in der Haut freigesetzt wird.

     

Indolent systemic mast cell disease: immunophenotypic characterization of bone marrow mast cells by flow cytometry

BACKGROUND: Mast cell disease has a low prevalence and is difficult to diagnose in the absence of the characteristic skin lesions that usually accompany the condition. Extracutaneous involvement is not easy to assess. There are reports in the recent literature on the use of tryptase as a reliable immunohistochemical marker as well as on the study of the immunophenotype of bone marrow mast cells. The latter is of great help for the diagnosis of systemic involvement as, after the skin, the bone marrow is the organ most commonly affected by the disease. OBJECTIVE: We describe two cases of indolent systemic mast cell disease (SMCD) where flow cytometry was used to identify immunophenotypical characteristics of bone marrow mast cells. Recent advances in the application of this technique prove it can be a good diagnostic tool for assessing systemic involvement of the disease. PATIENTS AND METHODS: Two adult subjects with indolent SMCD had multiple clinical symptoms. Cutaneous lesions were the clue to the diagnosis but, subsequently, in the presence of disturbing symptoms, involvement of other organ systems was confirmed. In both cases, the authors used flow cytometry techniques, as described by Escribano et al. (1998) to define the immunophenotype of bone marrow mast cells. RESULTS: Both patients were diagnosed with indolent SMCD with cutaneous and bone marrow involvement. Also, they presented visible clues to the presumptive bone, cardiovascular and nervous system involvement. Gastrointestinal manifestations were documented in one case. CONCLUSIONS: The use of flow cytometry on bone marrow samples from patients with mastocytosis reveals immunophenotypic differences that can serve to allow classification of these subjects in the category of indolent SMCD even though involvement of another organ system may not be thoroughly confirmed.     

Ultrastructural and immunohistochemical identification of malignant schwannoma of the skin

Histogenetic identification of poorly differentiated spindle cell tumours can be very difficult if only routine histology is used. In the case of a 64-year-old man suffering from a recurrent and spreading malignant tumour on his neck, the precise diagnosis of malignant schwannoma was not established until a skin node was examined by electron microscopy. The neurogenic nature of the neoplasm was additionally confirmed by demonstration of S 100 protein within most of the tumour cells. Ultrastructurally, the tumour was composed predominantly of atypical Schwann cells and, in varying number, of perineurial cells, endoneurial cells and some myofibroblasts exhibiting features enabling their differentiation. Some electron microscopical findings not previously reported in malignant schwannoma were detailed.     

Generation of multiple stable dermcidin-derived antimicrobial peptides in sweat of different body sites

Antimicrobial peptides (AMPs) are effector molecules of innate immunity. Dermcidin (DCD), a recently discovered AMP with broad-spectrum activity, is produced constitutively by the eccrine sweat glands and secreted into sweat. In this study, we investigated the proteolytic processing, site-specific expression, and stability of DCD peptides in eccrine sweat. Using surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and reversed-phase high-pressure liquid chromatography analysis, we identified in eccrine sweat 14 proteolytically processed DCD peptides. Semiquantitative SELDI-TOF-MS analysis indicated that processing of DCD-1L is individually different, but generates a few dominant peptides. At body sites with a high probability for contact with pathogenic microorganisms, a high amount of antimicrobial active DCD peptides was detected in sweat. Furthermore, we show that the secretion rate of DCD is constant during a period of prolonged sweating and that DCD peptides are stable in sweat over several hours. Other known AMPs like the human cathelicidin LL-37 and alpha- or beta-defensins were not detected in significant quantity in eccrine sweat. Owing to the durable and abundant presence, DCD-derived peptides contribute to the first line of defense by building a constant barrier that overlies the epithelial skin.     

Mast cells and atopic dermatitis. Stereological quantification of mast cells in atopic dermatitis and normal human skin

Stereological quantification of mast cell numbers was applied to sections of punch biopsies from lesional and nonlesional skin of atopic dermatitis patients and skin of healthy volunteers. We also investigated whether the method of staining and/or the fixative influenced the results of the determination of the mast cell profile numbers. The punch biopsies were taken from the same four locations in both atopic dermatitis patients and normal individuals. The locations were the scalp, neck and flexure of the elbow (lesional skin), and nates (nonlesional skin). Clinical scoring was carried out at the site of each biopsy. After fixation and plastic embedding, the biopsies were cut into 2 μm serial sections. Ten sections, 30 μm apart, from each biopsy were examined and stained alternately with either toluidine blue or Giemsa stain and mast cell profile numbers were determined. The study yielded the following results: (1) in atopic dermatitis lesional skin an increased number of mast cell profiles was found as compared with nonlesional skin, (2) comparing atopic dermatitis skin with normal skin, a significantly increased number of mast cell profiles per millimetre squared was found in specimens from the neck, (3) staining with toluidine blue yielded a lower number of mast cell profiles than Giemsa staining, (4) the use of Carnoy’s fixative resulted in a lower mast cell profile count than the use of formaldehyde, and (5) there was no statistically significant correlation between the clinical score and the number of mast cell profiles per millimetre squared. Using stereological techniques, this study indicated that mast cells might participate in the inflammatory process in skin leading to atopic dermatitis. Received: 17 April 1996