The 3G5 antigen is expressed in dermal mast cells but not pericytes

Background:  It has been shown that the 3G5 antigen recognized by monoclonal antibody 3G5 (mAb 3G5) is a useful marker of pericytes in normal human skin. However, most 3G5 antigen-expressing cells in capillary vessels were stained negatively for α-smooth muscle actin (α-SMA), a prominent pericyte marker. This study was designed to determine whether the expression of the 3G5 antigen is restricted to specific stages of pericyte development, or if it is expressed in other cells rather than pericytes in capillary vessels.
Methods:  3G5 antigen-expressing cells were detected in normal human skin, granulating tissues from decubitus ulcers and inflammatory psoriatic skin with extensive angiogenesis using double immunofluorescent staining with mAb 3G5 and monoclonal antibodies (mAbs) to various pericyte markers, tryptase and chymase. Furthermore, using immunoelectron microscopy, 3G5 antigen-expressing cells were observed in the granulating tissues.
Results:  The immunoelectron microscopic findings and double immunofluorescent staining (using mAb 3G5 and either anti-tryptase or anti-chymase mAbs) showed that 3G5 antigen-expressing cells were mast cells in normal skin, granulating tissues and psoriatic skin.
Conclusions:  The results indicated that 3G5 antigen is a marker of mast cells, but not of pericytes in normal and diseased skin.     

Human dermal pericytes express 3G5 ganglioside – A new approach for microvessel histology in the skin

BACKGROUND: Pericytes cover the abluminal surface of microvessels and play an important role in capillary regulation and pathology. Studies on pericytes have been hindered by the lack of specific markers with which to facilitate microscopic identification of this cell type. Expression of the cell surface 3G5 ganglioside antigen has been reported in cultured retinal and cardiac pericytes. The objective of this study was to determine the usefulness of monoclonal antibody 3G5 as a pericyte marker in human skin. METHODS: Cryosections of 21 skin biopsies were examined by direct fluorescence technique with anti-3G5, anti-von Willebrand factor, anti-alpha-smooth muscle actin or DNA fluorochrome. RESULTS: In human dermis, 3G5 expression is limited to pericytes discriminating this cell type from all other cells including smooth muscle cells, myofibroblasts and myoepithelial cells. We found a pericyte: endothelial cell ratio of 1:12.4 (+/-7.1), and a difference of alpha-smooth muscle actin expression between the subpapillary plexus and the microvessels of the Stratum reticulare. CONCLUSIONS: 3G5 mAB is an excellent and so far the only reported tool for identification of dermal pericytes by fluorescent light microscopy. Moreover, this is the first report of the application of 3G5 technique to the microvasculature in tissue sections at the light microscopic level.     

Isolated basal keratinocytes express pemphigoid gestationis antigen

Basal keratinocytes were isolated from epidermal cell suspensions prepared by trypsinization of normal human skin. Cells were identified as basal cells by their adherence to collagen and confirmed as basal cells by the presence of pemphigoid antigen. Using an indirect immunofluorescence assay, cells were found to express pemphigoid gestationis-related antigen. Sera from patients with pemphigoid gestationis reacted in one of two immunofluorescence patterns: either polar, in a pattern similar to that observed with bullous pemphigoid serum, or with uniform staining around the cell periphery. Pemphigoid gestationis-related antigen is expressed by isolated basal keratinocytes and is resistant to trypsinization. The heterogeneity of immunofluorescence patterns may correspond to the heterogeneity of antigen recognition by different patients with pemphigoid gestationis.     

Nail bed keratinocytes express an antigen of the carcinoembryonic antigen family

Using a panel of monoclonal and polyclonal antibodies raised against human carcinoembryonic antigen (CEA) and CEA-related molecules, we detected strong expression of an antigen, with immunoreactivity consistent with non-specific cross-reacting antigen (NCA) (CD66c), in all of 26 normal human nail specimens obtained from various fingers and toes. In longitudinal sections, strong and constant expression of the NCA-like antigen was seen on keratinocytes distributed in the upper epithelial cell layers of the nail bed, while in transverse sections the expression was limited to the major central portions of the nail bed, but only where longitudinal epidermal ridges were observed. In the hyponychium and the ventral aspect of the proximal nail fold, the expression was weak or lacking. No expression was seen in the nail matrix, the nail plate, or the dorsal aspect of the proximal nail fold. The same results were obtained for all of the 26 nails studied. This report is the first to demonstrate that an antigen of the CEA family, with NCA-like immunoreactivity, is expressed in a specific subpopulation of keratinocytes in the nail bed. The specific expression pattern suggests that the antigen may play a part in adhesion of the nail plate to the nail bed.     

Melanocytes adhere to and synthesize laminin-5 in vitro

Melanocytes arise from the neural crest, migrate to the skin, and can be detected in the basal layer of the epidermis in skin biopsies of human fetuses as early as 11 weeks gestational age. During post-natal life, melanocytes reside at the basal layer of the epidermis, but the ligands to which they attach are unknown. Laminin-5 is a component of anchoring filaments of the lamina lucida of the epidermal basement membrane. In this report we show that human melanocytes adhere to purified laminin-5 to a level comparable with normal human keratinocytes. Blocking antibodies to the 165 kDa subunit of laminin-5 significantly inhibited fetal and neonatal melanocyte attachment to the surface of salt-split skin, which exposes laminin-5 on its surface, suggesting that laminin-5 is a ligand for melanocyte attachment to the basement membrane in vivo. Western blotting of concentrated culture supernatant of fetal and neonatal melanocytes with anti-laminin-5 antibodies demonstrated a single immunoreactive band of the expected size of laminin-5. In contrast, 3 human metastatic melanoma cell lines did not produce laminin-5. Immunofluorescence microscopy with antibodies to each of the three chains of laminin-5 confirmed the presence of laminin-5 in a peri-cellular distribution around melanocytes, but not melanoma cells. Our results suggest that laminin-5 may be a ligand for normal human melanocytes in the basement membrane, and that loss of laminin-5 production by melanoma cells may be a marker for malignant transformation.     

Merkel cells do not express bullous pemphigoid antigen

Merkel cells (MC) are epithelial cells expressing cytokeratin-type intermediate filaments. They often are localized within the basal cell layer of the epidermis. Since basal cell layer keratinocytes synthetize basal membrane components, it was of interest to investigate whether or not MC could also do so. We used both double-labeling immunofluorescence and immune electron microscopy techniques with a panel of antibodies allowing the identification of MC as well as the staining of basal membrane zone components (including bullous pemphigoid antigen, laminin, type IV collagen and epidermolysis bullosa antigen). A specific loss of BP antigen expression was observed below all MC directly in contact with BMZ. This suggests that, although being an epithelial cell and in contrast to basal keratinocytes, MC does not secrete BP antigens.     

Melanocytes freshly isolated from normal human skin express the cell membrane receptor for the adhesive glycoprotein thrombospondin

Summary Thrombospondin (TSP) is an adhesive protein with multiple binding sites, which Is able to mediate several cell-to-cell and cell-to-matrix interactions, particularly through its cell membrane receptor (TSP-R). Because human keratinocytes are able to synthesize and express TSP, and as TSP is also localized at the dermal–epidermal junction in normal human skin, we questioned whether epidermal cells are able to bind available TSP, that is to express TSP-R. To investigate this, we employed gold immunoelectron microscopy on epidermal cells freshly isolated from normal human skin; the TSP-R was detected by OKM5 monoclonal antibody. Epidermal cells showing ultrastructural characteristics of melanocytes were gold-stained on their plasma membrane, whereas keratinocytes. Langerhans cells and lymphocytes were unstained. Although functional studies are clearly necessary to clarify the role(s) played by the TSP-R on the cell surface of melanocytes, it is tempting to speculate that the TSP-R may be important for melanocyte adhesion to the dermal–epidermal junction and to keratinocytes. Such adhesion may not only subserve the steric localization of melanocytes. hut also have important implications for those functional activities of melanocytes which have been shown to require close contact between these cells and adjacent keratinocytes and/or basement membrane components.     

Granular-cell tumours of the skin do not express carcino-embryonic antigen

Granular-cell tumour (GCT) of the skin is an uncommon tumour of disputed histogenesis, that has been subjected to several immunohistochemical studies. The controversy existing in the literature concerning the expression of carcinoembryonic antigen (CEA) by GCT prompted us to study a series of 17 cases of cutaneous GCT by using an avidin-biotin-immunoperoxidase technique on routinely-processed tissue sections. No CEA activity was detected in any of the tumours screened. The reasons for this controversy are discussed.     

Human keratinocytes express functional Toll-like receptor 3, 4, 5, and 9

Keratinocytes are continuously in contact with external stimuli and have the capacity to produce several soluble mediators. Pathogen-associated molecular patterns (PAMPs) are recognized, among others, by Toll-like receptors (TLRs). The functional responses of keratinocytes to different PAMPs have not yet been fully established. Here we show that keratinocytes constitutively express TLR1, 2, 3, 4, 5, 6, 9, and 10 mRNA, but not TLR7 and 8. Stimulation of keratinocytes with TLR3, 4, 5, and 9 ligands resulted in differential immune-associated responses. Tumor necrosis factor-alpha, CXC chemokine ligand 8 (CXCL8), CCL2, and C chemokine ligand 20 (CCL20) release was enhanced in response to all PAMPs tested, in a time- and dose-dependent manner. Only TLR9 ligand CpG-oligodeoxynucleotides (ODNs) and TLR3 ligand poly-I:C could additionally induce type I IFNs. CCL27 production was selectively induced by poly-I:C and flagellin, whereas CXCL9 and CXCL10 were exclusively induced by CpG-ODNs and/or poly-I:C. Upregulation of ICAM-1, HLA-DR, HLA-ABC, FasR, and CD40 was mainly observed in response to poly-I:C, flagellin, and lipopolysaccharide. Furthermore, PAMP triggering resulted in the phosphorylation of phosphorylated-IkappaB alpha and in the nucleus translocation of NF-kappaB p65. Altogether, these findings stress an unexpectedly multifaceted role of keratinocytes in innate immunity as evident by their differential, TLR-mediated responses to PAMPs associated with different classes of pathogens.     

Atypical cells in lymphomatoid papulosis express the Hodgkin cell-associated antigen Ki-1

Lymphomatoid papulosis (LyP) is characterized by the presence of large multinucleated cells resembling Reed-Sternberg (RS) cells. Evidence of antigenic similarity between these two cell types has been sought by immunohistologic labeling of 10 biopsies from cases of LyP with monoclonal antibodies against Ki-1 and other RS and Hodgkin (H) cell-associated antigens. In all cases studied, a proportion of the large atypical cells expressed the Ki-1 antigen. On the contrary, in 20 biopsies of benign skin lesions or cutaneous T-cell lymphomas, Ki-1-positive cells were absent or only occasionally present. Furthermore the large atypical cells of LyP also expressed antigens (e.g., T3, T4, HLA-DR, IL-2 receptors) which we have previously demonstrated on RS cells in the majority of cases of Hodgkin's disease (HD). These findings, in conjunction with the observation that Ki-1 antigen expression can be induced on peripheral blood lymphocytes following exposure to phytohemagglutinin or HTLV I, provide evidence that the Ki-1 positive cells in LyP represent activated T cells as RS cells do in many cases of HD.     

Human plasmacytoid dendritic cells express receptors for anaphylatoxins C3a and C5a and are chemoattracted to C3a and C5a

The presence of plasmacytoid dendritic cells (pDC) was recently demonstrated in lesions of inflammatory skin diseases. Since anaphylatoxins or their precursors were also found in such lesions, we investigated a possible interaction between pDC and anaphylatoxins C3a and C5a. pDC precursors isolated from peripheral blood did not express the receptors for C3a and C5a, complement C3a receptor (C3aR) and complement C3a receptor (C5aR). If these pDC precursors were cultured with IL-3, the resultant immature pDC expressed both receptors. Expression of C3aR and C5aR could also be demonstrated on pDC in lesions of cutaneous lupus erythematosus and allergic contact dermatitis. Such pDC were immature since they lacked the expression of the maturation marker CD83. Blood-derived pDC matured with CpG oligonucleotides downregulated the receptors. Immature pDC responded to C3a and C5a (but not C3adesArg) stimulation with increased F-actin polymerization and chemotactic migration. In contrast, interferon alpha production, surface molecule expression, and T-cell stimulatory capacity were not significantly modulated by C3a or C5a. Thus, immature pDC represent another type of antigen-presenting cell that express C3aR and C5aR, and respond to anaphylatoxins with chemotaxis. This might be relevant in the direction of pDC to cutaneous lesions of inflammation, for example, in lupus erythematosus or contact dermatitis.     

Leprosy and hepatitis B surface antigen

An ELISA technique has been developed to detect HBsAg in the sera of leprosy patients. Out of ninety-two serum samples taken from untreated leprosy patients, 10 samples were positive for HBsAg. The ELISA used in the present investigation is a low cost, reliable and sensitive marker of HBsAg. It is better than lesser sensitive (haemagglutination and counterimmunoelectrophoresis), costly and hazardous (radioimmunoassay) techniques and is therefore recommended for routine use.     

1,25二羟基维生素D3对毛囊外毛根鞘无色素黑素细胞激活的实验研究

目的研究1,25二羟基维生素D3(1,25-Dihydroxyvitamin D3,VID)对毛囊外毛根鞘无色素黑素细胞(amelanotic melanocytes,AMMC)的激活作用。方法以高、中、低3种不同浓度的1,25(OH)2D3作用于培养的人毛囊外毛根鞘AMMC,倒置显微镜观察细胞形态的变化,然后收集细胞,测定细胞酪氨酸酶活性和黑素含量,透射电镜观察药物作用前后AMMC内黑素小体的变化,最后通过Western blot方法半定量分析药物作用前后AMMC内酪氨酸酶(tyrosinase,TYR)、酪氨酸酶相关蛋白1(tyrosinase related protein-1,TRP-1)和酪氨酸酶相关蛋白2(tyrosinase relatedprotein-2,TRP-2)表达的变化。结果 VID能增加AMMC内酪氨酸酶活性,促进AMMC生成黑素,电镜结果显示药物作用后的AMMC内出现大量Ⅲ、Ⅳ期黑素小体。Western blot结果显示VID可以促进AMMC中TYR和TRP-1的表达,但对TRP-2的表达无明显影响。结论 VID通过促进黑素生成相关酶TYR和TRP-1的表达激活了AMMC。     

Human keratinocytes express fractalkine/CX3CL1

BACKGROUND: fractalkine/CX3CL1 is a unique chemokine that has properties of both chemoattractants and adhesion molecules. The major source of this chemokine in the skin is still controversial. OBJECTIVE: studies were undertaken to determine the expression of fractalkine in human skin. METHODS: RT-PCR, Western blotting, and immunostaining were performed with normal human epidermal keratinocytes (NHEK) and HaCaT cells, human keratinocyte cell line, for the presence of fractalkine. Biopsy specimens of normal and diseased skin were also investigated. RESULTS: we identified that NHEK and HaCaT cells expressed fractalkine mRNA and protein. The combination of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma upregulated their expression by NHEK. Immunohistochemistry demonstrated fractalkine expression in keratinocytes in lichen planus and psoriasis vulgaris. RT-PCR also showed that lesional skin of psoriatic patients expressed higher levels of fractalkine mRNA than non-lesional skin from the same patients. CONCLUSION: these results suggests that keratinocytes strongly express fractalkine in lichen planus and psoriasis vulgaris and that the fractalkine-CXC3CR1 system in the diseased skin can be a target for the treatment.