Human Dermal Cells Research Articles

Isolation and characterization of the glycoprotein fraction with a proliferation-promoting activity on human and hamster cells in vitro from Aloe vera gel.

Fractions of leaf gel from Aloe barbadensis Mill. were prepared by gel permeation using DEAE Sephadex A-25, Sepharose 6B, and Sephadex G-50 columns. These were then tested by in vitro assays for proliferation of human normal dermal and baby hamster kidney cells. The glycoprotein fraction promoted cell growth, while the neutral polysaccharide fraction did not show any growth stimulation. Moreover, the polar-colored glycoprotein fraction strongly inhibited the in vitro assays. An active glycoprotein fraction (protein 82%, carbohydrate 11%) examined on polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE showed a single band. Its molecular weight was 29 kD on a Sephadex G-50 column and its isoelectric point was pH 6.8. Immunoblotting after SDS-PAGE showed that the glycoprotein was composed of two subunits (14 kD). Deglycosylation of glycoprotein (Pg21-2b fraction) by trifluoromethanesulphonic acid provided a protein band with a molecular weight of 13 kD on SDS-PAGE. The colored glycoprotein fraction was shown on SDS-PAGE to be a mixture with a molecular weight of 18 kD-15 kD. It was later hydrolyzed with 10% H2SO4 to produce phenolic substances.

The identification of proteoglycan-associated mRNAs in human dental pulp cells

Characterization of the dental pulp proteoglycans has been largely confined to the glycosaminoglycan component of the proteoglycan molecule, while the protein core has received little attention. This study was conducted to identify mRNAs of previously well-characterized proteoglycans—biglycan, and versican—and link protein in dental pulp cells. Dermal fibroblasts were used as a positive control. Oligonucleotide probes were constructed based on published sequences for the four proteins from human tissues. Total RNA was isolated from cultured human pulp and dermal cells, separated according to size by formaldehyde gel electrophoresis and subsequently transferred to a nylon filter. Northern hybridizations using the oligonucleotide probes revealed the expression of biglycan, decorin, versican and link protein mRNAs. Biglycan and decorin are small proteoglycans that have a regulatory effect on collagen fibrillogenesis. Assuming expression of link protein and versican in vivo, the larger proteoglycans in the dental pulp are capable of forming large proteoglycan aggregates.

Expression, but lack of calcium mobilization by high-affinity IgE Fc epsilon receptor I on human epidermal and dermal Langerhans cells.

In atopic dermatitis (AD) patients, IgE molecules are demonstrated on the surface of Langerhans cells (LC). Fc epsilon RI molecules, which are present on the surface of LC in AD patients as well as normal individuals, are responsible for this binding. In this study, we have investigated phenotypic and functional characteristics of Fc epsilon RI on epidermal and dermal cell populations. Epidermal and dermal cell suspensions were prepared enzymatically with dispase followed by either trypsin or collagenase treatment, respectively. Peripheral blood basophils were negatively selected by excluding other leukocytes with surface marker staining. Consistent with previous reports, both peripheral blood basophils and epidermal LC were positively stained with anti Fc epsilon RI monoclonal antibody. In addition, an Fc epsilon RI positive population was demonstrated among dermal HLA-DR positive cells. These cells express significant amounts of HLA-DR molecules (DRHi) and co-express CD 1 a molecules, which identifies them as LC-like dendritic APC of the dermis. No other Fc epsilon RI positive population was found in the other dermal DRMid or DR- populations, except for a minor DRLo population, presumably mast cells. To analyze whether these Fc epsilon RI molecules are signal transducing for LC, intracellular calcium mobilization after crosslinking of Fc epsilon RI was measured with flow cytometry. Following crosslinking, peripheral blood basophils clearly increased intracellular calcium. On the other hand, neither normal epidermal LC nor dermal DRHiCD1a + cells changed their intracellular calcium level after Fc epsilon RI crosslinking. These data indicate that normal epidermal and dermal LC, but not basophils, are resistant to calcium flux following Fc epsilon RI engagement.

CD88 antibodies specifically bind to C5aR on dermal CD117+ and CD14+ cells and react with a desmosomal antigen in human skin.

The expression of the C5aR (CD88) on human epidermal and dermal cells was studied with five anti-C5aR mAb directed to the N-terminal domain of the receptor. All mAb bound to suspended dermal CD117+ mast cells and to dermal CD14+ cells. The binding to CD14+ and CD117+ cells could be blocked by rC5a and by peptide EX-1 representing amino acid residues 1-31 of the C5aR. In acetone-fixed frozen or in paraformaldehyde-fixed, paraffin-embedded tissue, we detected a binding of the Abs to dermal perivascular cells and, additionally, to keratinocytes and dermal epithelial cells that could be blocked by EX-1. Immunoelectromicroscopy revealed a binding of anti-C5aR mAb to desmosomal regions in human epidermis. However, the following results indicate that CD88 mAb cross-react with epithelium in a specific way: 1) the binding to suspended epidermal cells and to the epidermal cell line HaCat could be blocked by EX-1 but not by rC5a; 2) FITC-labeled C5a bound to CD117+ and to CD14+ cells but not to epidermal cells; 3) C5a led to transient calcium fluxes in CD14+ and CD117+ dermal but not in epidermal cells; 4) C5aR mRNA was detectable by reverse transcription PCR in granulocytes but not in keratinocytes or in HaCat. Our results show that CD88 mAb are good tools for the investigation of the C5aR on hemopoietic cells. Results with epithelial cells should be considered with caution, as the binding of CD88 mAb that were raised to a synthetic peptide sequence may be due to a cross-reactivity.

Identification of a Human Dermal Macrophage Population Responsible for Constitutive Restraint of Primary Dermal Fibroblast Proliferation

Primary human dermal cell suspensions prepared from the papillary dermis of keratomed skin strips were used to investigate the effect of indigenous dermal macrophages (HLA-DR+, CD11c+ CD11b+ CD1c- phagolysosome+) upon dermal fibroblast proliferation. Rapid dermal fibroblast expansion was induced upon immunomagnetic bead removal of CD11b+ or CD11c+ cells as well as by removal of more inclusive subsets contained within the DR+ population, but the removal of mast cells, endothelial cells, and CD1c+ dermal Langerhans cells from dermal cell suspensions failed to result in proliferation of the remaining cell subsets. Removal of 1B10+ fibroblasts from macrophage depleted (CD11b-) dermal cell suspensions essentially abrogated the unrestrained proliferation of the CD11b- dermal cells. Flow cytometric cell cycle analysis of cultured macrophage-depleted dermal cells confirmed that the unrestrained proliferating cells contain procollagen I+ as well as procollagen I- dermal fibroblasts. Inhibition of primary fibroblast expansion by adding a supernatant from unfractionated dermal cells suggested that a growth-inhibitory soluble activity of >30,000 kDa dominates the cytokine mixture released by unfractionated fresh dermal cells ex vivo. Inhibitory activity counterbalanced positive fibroblast growth- stimulatory cytokines released by dermal cells because neutralizing antibodies to insulin-like growth factor 1 and interleukin-1 beta resulted in decreased CD11b- dermal cell fibroblast proliferation. These data indicated an important role for dermal macrophages of the DR+ CD11b+ CD11c+ DC1c- phenotype in the normal homeostatic restraint of primary human dermal fibroblast proliferation.

Organotypic and epidermal-dermal co-cultures of normal human keratinocytes and dermal cells: Regulation of transforming growth factor α, β1 and β2 mRNA levels

Epidermal-dermal cell-cell interactions are recognized to influence keratinocyte proliferation in vivo as well as in vitro. To study the underlying molecular mechanisms of epithelial-mesenchymal interactions epidermal-dermal cell co-cultures and organotypic cultures were used. Steady-state mRNA levels are described for transforming growth factors (TGF) α, β1 and β2, factors known to stimulate or inhibit the epidermal proliferation rate. In epidermal-dermal monolayer co-cultures TGF α hybridization signals were absent. TGF β1 mRNA was expressed in all cell types (keratinocytes, fibroblasts and microvascular endothelial cells), yet not regulated. In contrast, TGF β2 mRNA was significantly induced in mesenchymal cells when they were co-cultured with keratinocytes. In organotypic cultures epidermal proliferation is dependent on the presence of fibroblasts within the gel. TGF β1 was expressed at low levels in all cell types whereas TGF β2 transcripts were not detectable at all. TGF α mRNA was present in keratinocytes at high levels, independent of epidermal cell proliferation or added epidermal growth factor. These results indicate complex regulative mechanisms for TGF α, β1 and β2 at the mRNA level. However, post-transcriptional steps are involved in the activation of TGF β1 and 2 and also have to be considered.

Heterogeneous populations of class II MHC+ cells in human dermal cell suspensions. Identification of a small subset responsible for potent dermal antigen-presenting cell activity with features analogous to Langerhans cells.

Little is known regarding the identification, classification, and function of class II MHC+ dendritic cells in the perivasculature of human connective tissues, such as the dermis. We developed a method for preparing papillary dermal cell suspensions from human keratome strips. Among the class II MHC+ populations of the dermis identified using triple color flow cytometry, cells of monocyte/macrophage lineage (CD45+ CD1- CD11b+ CD11clo-mid CD32+ CD36+ or - CD11a-) and mesenchymal cells of non-bone marrow origin (CD45-) were identified and characterized. Another distinct class II MHC+ subset was identified, which expressed a number of features analogous to epidermal Langerhans cells (LC) and other dendritic APC. These were a numerically minor population comprising only 2.7% +/- 1% (n = 7) of dermal cells. Like LC, they express HLA-DR, CD45, CD1a (albeit at a lower level of expression), CD1c, and CD32 and lack constitutive CD11a or ICAM-1. In contrast to LC, this dermal CD1a+CD1c+ subset expresses CD1b, CD11b, a higher level of CD11c, and intracytoplasmic factor XIIIa. Alloantigen presentation by unfractionated dermal cells was reduced by prior removal of this CD1b+ subset to the same degree achieved by removal of the entire DR+ population (20% of dermal cells), indicating that this was the critical DR+ subset. Cocultures of CD4+ T lymphocytes with cells sorted by flow cytometry into CD1c+DR+, CD1c-DR+ and DR- dermal cell subsets positively identified the CD1c+DR+ population as the most potent of potential APC subsets in human dermis. Thus, in distinction to other dermal macrophage and mesenchymal subsets with elongate morphology, the CD1aloCD1b,c+CD11c(hi)CD11b+CD32+DR+ population in human dermis is highly analogous to cells of LC/dendritic APC lineage in its phenotype and in its exclusive ability to potently present Ag to T lymphocytes. These studies identify and characterize the APC subset most potent in inducing activation of T cells initially entering the perivasculature of human dermis to be of LC/dendritic APC, and not tissue macrophage, lineage.

Quantitative analysis of human dermal cell suspensions after UV injury: A small dermal monocyte/macrophage subset undergoes marked expansion whereas potent APC's of Langerhans/dendritic cell lineage are depleted

Quantitative analysis of human dermal cell suspensions after UV injury: A small dermal monocyte/macrophage subset undergoes marked expansion whereas potent APC's of Langerhans/dendritic cell lineage are depleted

6 Cytokine stimulated human dermal microvascular endothelial cells (HDMEC) are a major source for interleukin 6

6 Cytokine stimulated human dermal microvascular endothelial cells (HDMEC) are a major source for interleukin 6

Human dermal mast cells contain and release tumor necrosis factor alpha, which induces endothelial leukocyte adhesion molecule 1.

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine that mediates endothelial leukocyte interactions by inducing expression of adhesion molecules. In this report, we demonstrate that human dermal mast cells contain sizeable stores of immunoreactive and biologically active TNF-alpha within granules, which can be released rapidly into the extracellular space upon degranulation. Among normal human dermal cells, mast cells are the predominant cell type that expresses both TNF-alpha protein and TNF-alpha mRNA. Moreover, induction of endothelial leukocyte adhesion molecule 1 expression is a direct consequence of release of mast cell-derived TNF-alpha. These findings establish a role for human mast cells as "gatekeepers" of the dermal microvasculature and indicate that mast cell products other than vasoactive amines influence endothelium in a proinflammatory fashion.

STIMULATION OF ENDOTHELIAL CELL GROWTH BY SERA FROM DIABETIC PATIENTS WITH RETINOPATHY

An in-vitro proliferation assay has shown that sera from patients with diabetic retinopathy, particularly those with the proliferative form, are two to four times more effective than sera from non-diabetics at stimulating 3H-thymidine incorporation into both human umbilical vein and human omental microvascular endothelial cells, but not at stimulating incorporation into human dermal fibroblasts or 3T3 cells. The factor(s) is heat stable and of molecular weight < 15 000, and its presence is unrelated to metabolic control. It is not present in patients with other forms of diabetic vascular disease, which suggests that it is not related to carbohydrate or lipid metabolism. These results provide evidence against the hypothesis that metabolic disturbances are central to the development of diabetic microvascular disease, and raise possibilities of novel forms of therapeutic intervention.

Formalin sensitivity and differential staining of mast cells in human dermis

The characteristics of the human dermal mast cell population with respect to formalin fixation sensitivity, toluidine blue staining and alcian blue/safranin staining were studied. Thirty-seven specimens of normal human skin were bisected. One half was fixed in 10% neutral buffered formalin and the other in Carnoy's fixative. Sections were cut and stained with either toluidine blue or alcian blue/safranin. Significantly more mast cells were visualized with alcian blue/safranin than with toluidine blue. With both stains, only approximately 50% of the mast cells observed in the Carnoy's fixed tissue could be visualized in the formalin-fixed tissue. Alcian blue/safranin staining revealed three patterns of mast cell granule staining: mast cells containing only alcian blue-positive granules, mast cells containing only safranin-positive granules, and mast cells containing a mixture of alcian blue-positive granules and safranin-positive granules. Mast cells containing only alcian blue-positive granules constituted the majority of the dermal mast cell population and 73% of these mast cells were formalin-sensitive. Mast cells containing only safranin-positive granules and those containing a mixture of alcian blue-positive granules and safranin-positive granules showed no evidence of formalin sensitivity. The human dermal mast cell population, therefore, displays heterogeneity with respect to formalin fixation sensitivity and alcian blue/safranin staining. Dermal mast cells were visualized in significantly greater numbers in skin from the head compared with that from the body or limbs.

Human dermal neuroendocrine cells (so-called dermal merkel cells): A further observation

Human dermal neuroendocrine cells (so-called dermal merkel cells): A further observation

Differing levels of excision repair in human fetal dermis and brain cells.

Abstract— The levels of DNA excision repair, as measured by unscheduled DNA synthesis (UDS) and the UV‐endonuclease sensitive site assay, were compared in cells derived from human fetal brain and dermal tissues. The level of UDS induced following ultraviolet (UV) irradiation was found to be lower (approx. 60%) in the fetal brain cells than in fetal dermal cells. It was determined, using the UV‐endonuclease sensitive site assay to confirm the UDS observation, that 50% of the dimers induced by UV in fetal dermal cells were repaired in 8h, while only 15% were removed in the fetal brain cells during the same period of time. Even after 24 h, only 44% of the dimers induced by UV in the fetal brain cells were repaired, while 65% were removed in the dermal cells. These data suggest that cultured human fetal brain cells exhibit lower levels of excision repair compared to cultured human fetal dermal cells.