Immunoperoxidase and enzyme-histochemical demonstration of human skin tryptase in cutaneous mast cells in normal and mastocytoma skin

Summary Trypsin-like proteinase isolated from human skin was localized in cutaneous mast cells using immunoperoxidase and enzyme-histochemical techniques. Skin biopsy specimens were taken from four mastocytoma and four healthy patients. Immunoperoxidase staining was performed with protein A-sepharose purified rabbit polyclonal antibody raised against human skin tryptase and using aminoethylcarbazole as chromogen. The positively stained cells in the dermis were granular in character. Using peptide 4-methoxy-2-naphthylamide substrates (Bz-Arg-MNA, Z-Lys-Arg-MNA, Z-Gly-Arg-MNA, Z-Pro-Arg-MNA and Z-Gly-Pro-Arg-MNA) and Fast Garnet GBC as chromogen the red azo dye was found to precipitate in the cytoplasmic granules of the cutaneous mast cells. The enzymatic reaction was totally inhibited by diisopropyl fluorophosphate, leupeptin, and benzemidine. No marked inhibition was seen with soybean trypsin inhibitor and alpha-1-antitrypsin. The best substrate was Z-Gly-Pro-Arg-MNA giving the strongest red azo dye when incubation time was 15,30 or 60 min. These results show the localization of human skin tryptase in dermal mast cells and the usefullnes of Z-Gly-Pro-Arg-MNA as a suitable substrate tested for enzyme-histochemical localization of mast cells in healthy or mastocytoma 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 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.     

Distribution of metallothionein in normal and pathological human skin

The expression and distribution of metallothionein (MT) in frozen sections of normal and pathological human skin was studied using the monoclonal antibody L2E3 directed against MT derived from human fetal liver. Immunohistochemical staining of normal fetal and adult skin revealed strong reactivity in basal keratinocytes of epidermis and outer hair root sheath, hair matrix cells and the secretory coil, but not the exocrine portion of eccrine glands; myoepithelial cells around apocrine sweat glands were similarly stained. In epidermal hyperplasia, variable numbers of suprabasal keratinocytes were stained, whereas in interface dermatitis, interrupted staining was found in the basal layer. Weak or scattered staining was observed in squamous tumours, whereas basal cell carcinomas did not show consistent staining. The distribution of MT in normal skin was in line with the germinative role of basal keratinocytes and hair matrix cells, whereas its distribution in hyperplastic epidermis was in line with experimental animal data, and reflected the increase in the germinative pool in these conditions. It is concluded that monoclonal antibody L2E3 may serve as a valuable immunohistochemical marker in diagnostic cutaneous pathology since it labels basal keratinocytes selectively, and since it discriminates between eccrine and apocrine sweat glands.     

Vitronectin in normal human skin and in skin lesions]

Vitronectin (S-protein of complement, serum spreading factor) is a multifunctional glycoprotein present in human plasma and in the elastic tissue of various organs. It belongs to the group of adhesion proteins and is of importance in the terminal stages of both the coagulation and complement system and in fibrinolysis. In human skin it is localized on dermal elastic fibers and on pathologically altered elastic material (solar elastosis, pseudoxanthoma elasticum) as well as on keratin filament material such as keratin bodies in lichen planus or amyloid deposits in localized cutaneous amyloid. It is also found in the abnormally thickened cutaneous blood vessels in erythropoietic protoporphyria and porphyria cutanea tarda. Late-stage inhibition of the complement cascade in bullous disorders in which activation of the complement system is of pathogenetic significance may be an additional important function of vitronectin in skin diseases.     

Amyloid P components in normal human skin and skin with lesions

Amyloid P component (AP) is a glycoprotein which is found in tissue deposits of all types of amyloid and is identical to and derived from serum amyloid P component (SAP). SAP binds in a calcium-dependent fashion to various ligands, such as agarose, desoxyribonucleic acid, fibronectin, C4-binding protein, glycosaminoglycans and isolated amyloid fibrils. Tissue AP (TAP) is also a constituent of the normal human renal glomerular basement membrane and is, in adult humans, invariably associated with elastic fiber microfibrils in connective tissue throughout the body, including that of blood vessels. In normal human skin anti-AP antibody binding was localized to the microfibrils of oxytalan fibers in the papillary dermis and to the peripheral microfibrillar mantle of elaunin and mature elastic fibers in the reticular dermis. Since SAP binds to fibronectin and glycosaminoglycans, which in turn bind to collagen fibers, TAP on elastic fiber microfibrils may play an important role in the maintenance of the normal dermal architecture and in dermo-epidermal adhesion. Under pathological conditions, AP is found in all forms of cutaneous amyloidosis, including primary localized cutaneous amyloid (PLCA); it is also detectable on keratin bodies, which represent precursor structures for PLCA. The association of AP with elastic fiber microfibrils and amyloid fibrils and their close anatomical relationship in vivo may reflect the significance of AP in the deposition of cutaneous amyloid.     

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     

Quantitative analysis of bikunin-laden mast cells in follicular eruptions and chronic skin lesions of atopic dermatitis

Bikunin, an inhibitor of serine proteases, is widely distributed in human tissues, including the skin, and may inhibit tryptase and modulate allergic inflammation. The purpose of the present study was to compare follicular eruptions (FE), so-called atopic skin or perifollicular accentuation, with atopic dermatitis (AD) lesions (ADL) by immunohistochemical analysis using antibodies to bikunin and tryptase. Immunohistochemically, bikunin was colocalized with tryptase in dermal mast cells, and a small quantity of bikunin was also deposited in the intercellular spaces in FE and ADL. The number of bikunin-laden mast cells per 0.78 mm(2) of skin was 78.1+/-7.1 (mean+/-SEM, n=14) in FE, 25.4+/-2.3 (n=10) in normal skin from children and infants, 91.3+/-11.8 (n=10) in ADL, 25.6+/-4.8 (n=5) in nonlesional skin of AD, and 27.8+/-2.0 (n=13) in normal adult skin. The difference between FE and normal control skin from children and infants, between FE and nonlesional skin of AD, and between lesional and nonlesional skin of AD were significant. Based on the above findings and the occasional presence of spongiosis and lymphocyte infiltration, in FE moderate inflammation is apparent histopathologically even though little inflammation is apparent clinically.     

Conjugated avidin identifies cutaneous rodent and human mast cells

Avidin conjugated to the fluorescent dyes rhodamine or fluorescein binds to mast cell granules in rodent and human skin. Sequential staining of tissue mast cells first with conjugated avidin, and then with a metachromatic stain demonstrated that both techniques identify the same mast cell granules. Specificity for mast cells was confirmed by the absence of avidin-positive cells in the skin of mast cell-deficient (W/Wv) mice. Binding of conjugated avidin to mast cells was inhibited by pretreating tissue specimens with unconjugated avidin but not by pretreating conjugated avidin with biotin, indicating that avidin does not bind to biotin or a biotin-like molecule. Within murine dermis, unique patterns of mast cell distributions were observed, with a prominent perivascular localization in ear skin, and a complete absence of mast cells underlying the scales in tail skin. In tissue sections of guinea pig skin undergoing basophil hypersensitivity reactions and in murine and human skin specimens infiltrated with eosinophils, conjugated avidin selectively stained only dermal mast cells, demonstrating specificity for mast cells in sites of inflammation. Conjugated avidin also readily stained rat peritoneal mast cells, demonstrating its utility for identifying extracutaneous mast cells. Unlike the metachromatic stains, avidin binding to mast cells in tissues is not limited by methods of fixation or special embedding and cutting procedures. Thus, mast cell identification with conjugated avidin is a reliable, specific, and simple method with important clinical and investigative applications.     

Substance P-induced histamine release in human cutaneous mast cells

Substance P is an undecapeptide found in multiple sites throughout the central and peripheral nervous systems including small unmyelinated (type C) cutaneous nerve fibers. Previous studies demonstrated that antidromic stimulation results in substance P (SP) release from nerve endings, SP stimulates histamine release (HR) from rat mast cells in vitro, and intradermal SP in humans produces wheals identical to those induced by histamine. These studies suggest a possible role for SP as a link between neurologic events and cutaneous mast cell-mediated reactions. We therefore investigated SP-induced HR in an in vitro preparation of human skin mast cells. Human foreskin sections were incubated with varying concentrations of SP. Histamine was assayed using automated fluorimetry and release was calculated as a percentage of total tissue histamine. Substance P caused dose-dependent HR over a range from 10(-5) M (1.3%) to 5 X 10(-4) M (25.1%). Histamine release was optimal at 3 mM calcium and was blocked by pretreatment with calcium chelation. Naloxone failed to block HR. These studies suggest that HR from skin mast cells by SP may play a role in neural modulation of poorly understood inflammatory skin conditions.     

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.     

The presence of tryptase-positive and bikunin-negative mast cells in psoriatic skin lesions

Human mast cells are well known to produce a serine protease, tryptase, which appears to play a pathogenic role in various skin inflammations. It was previously reported that a rat homologue of bikunin may inhibit tryptase activity. Various type of cells (i.e. keratinocytes) are able to produce this protein inhibitor, it still remains unclear if bikunin is present in dermal inflammatory milieu, in which mast cells, through secretion of tryptase, play an inflammatory role. Therefore, the purpose of the present study was to exploit expression and production of bikunin in dermis and dermal constituents. We first compared the dermal mast cells in psoriatic lesions with those in lesional skin of atopic dermatitis or of chronic eczema by use of immunoelectron microscopy and immunohistochemical analyses using antibodies to bikunin and tryptase. Then, we tested what kinds of cytokines may regulate the de novo synthesis of bikunin. To do so, RNA was extracted from a human mastocytic cell line, HMC-1, reverse-transcribed, and semiquantitative RT-PCR was performed using primers specific for bikunin. With immunoelectron microscopy, bikunin was found to localize on the cell membrane, while tryptase was in the secretary granules of the mast cells. In psoriatic lesions, around 70% of dermal mast cells were positive for both tryptase and bikunin, and the remaining was mostly positive for tryptase, but the expression of bikunin was under the detection limit of the experimental setting. This observation was seen in only psoriatic lesions, even in almost cured lesions, while in atopic dermatitis or chronic eczema only mast cells doubly positive for bikuin and tryptase were seen. In HMC-1, bikunin was constitutively expressed at an mRNA level, which was upregulated by stimulation with interleukine-4, but was suppressed by interferon-γ. Bearing in mind the concept that in psoriasis local cytokine milieu is shifted toward a Th1 pattern (predominant secretion of interferon-γ), tryptase-positive, bikunin-negative mast cells may be induced.     

Human cutaneous mast cells–a study of fixative and staining reactions in normal skin

Routinely used formal saline fixation reduces the number of demonstrable mast cells in human skin by up to 30% compared with paired specimens fixed in Carnoys medium. Using metachromatic (toluidene blue), orthochromatic (alcian blue/safranin), enzymatic (chloroacetate esterase reaction) and immunofluorescence (berberine and fluorescein conjugated avidin) staining techniques, mast cells were demonstrated and quantified. Alcian blue/safranin and fluorescein-conjugated avidin were both superior to the other staining methods used. We recommend the use of Carnoys medium fixed tissue stained with either alcian blue/safranin or conjugated avidin for optimal visualization and assessment of mast cells in human skin.     

Retinol binding protein in normal human skin and in cutaneous disorders

In order to investigate the molecular basis of substrates, and the inhibitory specificity of tyrosinase, a large series of phenolic compounds have been analysed by using a High Performance Liquid Chromatographic-Scanning Spectrophotometric system. Depending on their chemical structure, phenolic compounds may act as substrates or as competitive inhibitors of tyrosinase. The ability to act as substrates requires the presence in the molecule of electron donor groups, while competitive inhibition on the contrary requires the presence of powerful electron acceptor groups. Certain phenolic compounds used as therapeutic agents or as food preservatives are chemically capable of acting as alternative substrates or competitive inhibitors of tyrosinase in vitro; their effect on melanocytes in vivo therefore merits investigation.     

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.