Immunohistochemical demonstration of MAM-3 and MAM-6 antigens in normal human skin appendages and their tumors

Summary The expression of MAM-3 and MAM-6 antigens was immunohistochemically investigated on 110 tumors of human skin appendages. Forty-two samples from tumor-adjacent normal skin appendages were also studied. MAM-3 antigens, as detectable by monoclonal antibodies (MoAbs) 67D11, 115G3, and 115H10 were present in the inner layer cells but not in the outer layer cells of normal eccrine excretory ducts. Sporadic positivity was also found in cytoplasm of apocrine acini with 115G3, while 67D11 and 115H10 were negative. MAM-6 antigens, as detectable by the MoAbs 115D8, 115F5, 139H2, 140C1, and 126E7 were found in the secretory canaliculi of normal eccrine acini and within the apical lumina at the terminal portion of ducts. Apocrine acinar cells mainly exhibited an apical staining, but a focal supranuclear dot-like staining could also be observed. A foamy reaction pattern for MAM-6 was noted in mature sebocytes. However, none of the antigenic epitopes was expressed in normal squamous epithelium or hair follicles. In benign tumors, the staining patterns for both antigens, in general, resembled their distribution in the corresponding normal tissues. However, carcinomas orginating from sweat glands, sebaceous glands, and the pilar apparatus expressed both antigens in a more irregular and heterogeneous pattern. This might preferably be explained by the loss of those mechanisms controlling the antigen expression in mature, functional tissues. Conclusions from these immunohistochemical studies with regard to the histogenesis mainly of the malignant skin appendage tumors should be drawn with caution.     

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 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.     

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 ornithine decarboxylase in human skin

The localization of ornithine decarboxylase (ODC) in human skin and cultured keratinocytes was studied with an immuno-histochemical method. ODC was found in the epidermis, hair follicles, sweat glands and errector muscles. Irritation induced by stripping or UV-B irradiation did not change the staining pattern in the epidermis. In psoriasis, the staining was most marked at the tip of the epidermal rete ridges. In basal cell carcinoma, there was a homogeneous labelling of the tumor cells and, in squamous cell carcinoma, the labelling was strong but less homogeneous. Melanoma and dermal naevus also positively stained for ODC. Cultured human keratinocytes also showed ODC positive immunofluorescence. This technique detects the ODC antigen present rather than levels of ODC activity.     

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.     

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.     

In situ expression of messenger RNA of interleukin-1 and interleukin-6 in psoriasis: interleukin-6 involved in formation of psoriatic lesions

Summary Psoriasis is a disease of abnormal proliferation and differentiation of epidermal cells. Several cytokines released by keratinocytes are implicated as factors responsible for this pathological condition of the epidermis. In order to elucidate the role of these cytokines in psoriasis, messenger RNA (mRNA) expression of interleukin-1 (IL-1) and IL-6 in psoriatic epidermis was investigated using biotin-labelled complementary DNA (cDNA) of the cytokines. Messenger RNA of IL-1 was weakly detected in some normal healthy epidermis specimens and more strongly in all the perilesional uninvolved psoriatic epidermis specimens. It was also expressed in the transitional zone between uninvolved and fully developed psoriatic skin, but was not expressed in lesional skin. In contrast, IL-6 mRNA was rarely expressed in normal healthy epidermis, but was expressed in perilesional uninvolved psoriatic epidermis, in the transitional zone and in the fully developed lesional epidermis, with the maximum intensity in the transitional zone. Expression of mRNA of IL-6 receptor showed a similar tendency to that of IL-6. It was expressed in psoriatic epidermis, most strongly in the transitional zone, but not in normal healthy epidermis. IL-6 was demonstrated immunohistochemically in psoriatic epidermis, but IL-6 receptor was demonstrated only in the transitional zone. Thus IL-6 and its receptor expression correlated well with the formation of psoriatic lesions where IL-1 may initiate their expression. IL-6 may play an important role in the pathogenesis of psoriasis.     

Demonstration of interleukin-2, interleukin-4 and interleukin-6 in sera from patients with localized scleroderma

Localized scleroderma has been reported to be accompanied by immunological abnormalities related to B cells, but little is known about T-cell activation in this disease. In this study, serum levels of interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-6 (IL-6), which are known to be released by activated T cells, were determined using a sensitive enzyme-linked immunosorbent assay in 48 patients with localized scleroderma and 20 with systemic sclerosis, and in 20 healthy control subjects. IL-2, IL-4 and IL-6 were detected in serum from patients with localized scleroderma but not in that from healthy controls. The presence of antihistone antibodies correlated significantly with elevated IL-4 and IL-6 levels. Decreased serum levels of IL-2, IL-4 and IL-6 paralleled improvement in cutaneous sclerosis. Frequent detection of these lymphokines in serum from patients with localized scleroderma reflects T-cell activation in this disorder.     

Immunohistochemical localization of parathyroid hormone-related protein (PTHRP) in normal human skin

Human keratinocytes secrete large amounts of a parathyroid hormone-related peptide (PTHRP) in vitro. Because recent studies indicate that PTHRP could have a number of autocrine or paracrine functions in the skin, localization of this peptide in vivo is important. A monoclonal and two affinity-purified polyclonal antibodies were employed to locate PTHRP in normal human skin and cultivated human keratinocytes. PTHRP is present throughout the viable portion of the epidermis, in adnexal epithelial cells, and in all cultivated keratinocytes. These findings do not support the provocative suggestion that PTHRP is a marker for squamous differentiation.     

重组人IL－6对角朊细胞体外增殖分化影响的研究

为寻找一种对角朊细胞增殖分化具有调节作用的细胞因子，以人转化型角朊细胞株Ｃｏ－ｌｏ－１６细胞为模型，通过活细胞计数，３Ｈ－ＴｄＲ掺入法，细胞因子生物活性检测、电镜及免疫组化方法研究了重组人白细胞介素－６对角朊细胞体外增殖分化的影响。结果表明，ＩＬ－６对角朊细胞的体外增殖和ＤＮＡ合成具有双向调节作用；大剂量的ＩＬ－６能减少角朊细胞肿瘤坏死因子－α、ＩＬ－６的产生；对角朊细胞胞膜、线粒体有直接损伤作用；并抑制角朊细胞角蛋白表达。提示ＩＬ－６可作为角朊细胞体外增殖分化的免疫调节因子。     

高温高强度训练条件下氧化应激对血清白细胞介素-6的影响

目的研究高温、高强度和阳光中紫外线照射下训练士兵血清中白细胞介素-6（IL-6）的变化。方法选择训练组和对照组士兵共60人，于训练结束后，抽取外周血。采用酶联免疫吸附测定法检测对照组和训练组血清中IL-6、活性氧（ROS）、一氧化氮（NO）、丙二醇（MDA）、超氧化物歧化酶（SOD）的表达水平。结果对照组和训练组血清中IL-6、ROS、NO、SOD表达水平比较有统计学意义（P〈0．05）；对照组和训练组血清中MDA表达水平比较差异无统计学意义（P〉0．05）。结论高温、高强度和阳光中紫外线辐射下训练，机体产生氧化应激，血清中IL-6表达水平升高。