Cultivation of human dermal microvascular endothelial cells in vitro: immunocytochemical and ultrastructural characterization and effect of treatment with three synthetic retinoids.

A new reliable and reproducible technique to culture endothelial cells from the small vessels and capillaries of human skin is introduced, and proliferation and differentiation of the growing cells are characterized. The endothelial origin of the culture cells was confirmed by light- and electron microscopy and by labelling with Ulex europaeus Agglutinin I and an antibody against Factor VIII-related antigen. Further immunocytochemical characterization showed that 92-100% of the cells were positive for beta 2-microglobulin and the entire cell population expressed vimentin, whereas cytokeratins, desmin, HLA-DR antigen, Leu 6 and S 100 protein, could not be detected. As vascular endothelium is a common site of inflammation and retinoids have been shown to be of good clinical efficacy in some chronic inflammatory skin diseases, we investigated the influence of etretinate, etretin and isotretinoin on proliferation and antigen expression of our culture cells. All retinoids applied inhibited proliferation of endothelial cells in a dose- and time-dependent manner whereas they induced neither HLA-DR nor intercellular adhesion molecule-1 (ICAM-1). Furthermore, none of the retinoids applied influenced the gamma-interferon-induced expression of these surface antigens on endothelial cells. Our results suggest that the action of retinoids in skin inflammation is not mediated by modulation of HLA-DR or ICAM-1. The cell culture technique described here is an interesting and reliable model for studying the influence of drugs on endothelial cell growth and differentiation in vitro.

Effects of rIFN alpha, beta, and gamma on the morphology, proliferation, and cell surface antigen expression of human dermal microvascular endothelial cells in vitro.

The influence of recombinant human interferon alpha 2a (rIFN alpha), recombinant human interferon beta 1 (rIFN beta), and recombinant human interferon gamma (rIFN gamma) on human dermal microvascular endothelial cells (HDMEC) cultured in vitro was studied in various rIFN concentrations (0.1 IU/ml-10(4) IU/ml) over 2, 3, 4, 6, 8, and 10 d. Cell morphology and ultrastructure, cell proliferation, expression of class II alloantigens (HLA-DR and HLA-DQ), and intercellular adhesion molecule-1 (ICAM-1) were investigated using an in vitro technique established in our laboratory. All rIFN tested induced alterations of typical HDMEC morphology; the cells became spindle-shaped and fibroblastoid, although they maintained their endothelial cell marker expression. Also, all IFN dose- and time-dependently inhibited the proliferation of HDMEC in vitro (rIFN alpha greater than beta greater than gamma), whereby rIFN alpha exerted the strongest growth-inhibitory effect. Alkaline phosphatase anti-alkaline phosphatase (APAAP) immunocytochemistry of the cultured cells showed dose- and time-dependent stimulation of ICAM-1 and class II antigen expression only by rIFN gamma (HLA-DR greater than HLA-DQ), rIFN alpha and beta did not exert any immunomodulatory activity on HDMEC in vitro. These results indicate that HDMEC are an important target for the action of IFN. Besides growth inhibition, it seems that rIFN gamma in particular may be involved in the modulation of leucocyte adhesion and trafficking by altering the immunophenotype of the endothelial cell population.

Effects of recombinant tumor necrosis factor-alpha on cultured microvascular endothelial cells derived from human dermis.

We investigated the effects of recombinant human tumor necrosis factor-alpha (TNF) on cell proliferation and on expression of MHC class II antigens and intercellular adhesion molecule ICAM-1 in human dermal microvascular endothelial cells (HDMEC) derived from human foreskin. Second-passage HDMEC were treated with 0.1-10,000 U/ml TNF for up to 6 d, and cell growth was assessed by cell counts and a recently developed fluorogenic assay using 4-methylumbelliferyl heptanoate as a substrate. APAAP immunocytochemistry was performed using monoclonal antibodies against HLA-DR, HLA-DQ, and ICAM-1. TNF did not markedly inhibit the growth of HDMEC but induced expression of HLA-DR (1,000 U/ml and more) and of ICAM-1 (1 U/ml and more). Combination with interferon-gamma led to synergistic ICAM-1 induction. These results demonstrate a profound effect of TNF on the activation of dermal microvascular endothelial cells and suggest a major role of TNF in the mediation of leucocyte adhesion to endothelial cells of the skin microvasculature with possible implications for the initiation and maintenance of inflammatory skin processes.

[Preliminary findings on lectin binding in human follicular epithelium]. / Vorläufige Befunde zur Bindung von Lektinen im humanen follikulären Epithelium.

Glycoconjugates in the cell membranes of the outer and the inner root sheath of human anagen hair follicles were histochemically characterized by means of the following lectins: UEA-I, DBA, PNA, WGA, SBA, RCA, and Con A (avidin-biotin technique, counter-staining with methyl green). We observed intense labeling of the outer root sheath with UEA-I, DBA, Con A, and PNA, whereas the other markers were only weakly positive. VEA-I, DBA and SBA labeling was confiried to the upper two thirds of the outer root sheath. As to the inner root sheath, we found intense labeling with UEA-I, WGA, RCA, and SBA; all other lectins were only weakly represented. Our results suggest that the cell membranes of the outer and inner root sheaths of human hair follicles are characterized by the prevalence of different glycoconjugates; moreover, labeling with UEA-I, SBA, and DBA is obviously related to the process of differentiation within the follicle.

[Kyrle disease in juvenile diabetes mellitus and chronic renal failure]. / Morbus Kyrle bei juvenilem Diabetes mellitus und chronischer Niereninsuffizienz.

We report on a 32-year-old female patient with chronic diabetes mellitus, type I, and chronic renal failure, who developed the typical clinical picture of hyperkeratosis follicularis et parafollicularis in cuteum penetrans (Kyrle's disease) within one year. Histological examination revealed a defective epidermal differentiation with hyper- and parakeratosis as well as premature keratinization as early as in the epidermal basal cell layer. Studies on lectin binding showed that the glycosylation process was impaired in both the epidermis and the basement membrane zone of the lesional skin. In addition, electron microscopic investigation revealed diabetic microangiopathy of the dermal vessels as well as marked ultrastructural alterations of the dermo-epidermal basal lamina. These findings confirm the association of diabetes mellitus with Kyrle's disease previously described; they make us suggest that Kyrle's disease might be characterized by a defective differentiation of the epidermis and the dermo-epidermal junction--due to some alteration of the underlying glycosylation processes--rather than by a local disorder of keratinization. Regarding the clinical manifestation of the disease, both diabetes mellitus and chronic renal failure may play a part as precipitating factors.

[Presence and distribution of cytokeratins and filaggrin in human anagen follicles]. / Vorkommen und Verteilung von Zytokeratinen und Filaggrin in humanen Anagenfollikeln.

The presence and distribution of several types of cytokeratins and of filaggrin in human anagen hair follicles were investigated using the following monoclonal antibodies (McAB): KL 1, CK 8.60, CK 8.12, PKK-2, CK 18, CK 19 and anti-filaggrin McAb. Constant and characteristic patterns of McAb labelling were obtained in distinct compartments of the hair follicle, except for CK 18, which failed to label any of the follicular cells. Anti-filaggrin McAb was regularly bound to the Henle's layer of the internal root sheath, showing for the first time the presence of "filament aggregating protein" in the human hair follicle.

Disseminated Kaposi's sarcoma in AIDS: histogenesis-related populations and influence of long-term treatment with rIFN-alpha A.

Lesions (n = 19) of cutaneous Kaposi's sarcoma in different stages of development were obtained from 13 patients with acquired immunodeficiency syndrome (AIDS), and studied by light and electron microscopy. Six additional biopsies from 4 patients treated with recombinant alpha A interferon were obtained after treatment. Varying amounts of two proliferating cell populations were found: (1) Large cells showing cytologic and histochemical characteristics of endothelial cells. They were seen in close proximity to normal vessels, forming new vascular structures and large aggregates found in papular and nodular lesions. (2) Smaller spindle-shaped cells, probably of pericytic origin. They appeared in bundles and fascicles in the papillary dermis of the cutaneous Kaposi's sarcoma lesions and, in part, gave origin to thin-walled, bizarre-shaped vessels that show incomplete lumina proliferating from the upper to the deep dermis and are surrounded by extravasate erythrocytes and siderophages. After long-term systemic treatment with recombinant alpha A interferon, the endothelial type of tumor cell aggregates mostly disappeared, whereas most of the spindle-shaped pericytic-like cells were still present. Our findings lead us to suggest that some cellular product may, as a promoter factor, induce the proliferation and growth of endothelial cells. This factor may be blocked by alpha A interferon and cause regression of endothelial cell proliferation observed in AIDS patients undergoing long-term systemic therapy.

Growth of human hair follicle keratinocytes in vitro. Ultrastructural features of a new model.

A simple experimental technique was developed to provide an in vitro model for the study of human follicular keratinocytes. Anagen-phase human hairs were plucked from the scalp of healthy individuals; the follicles were separated, plated on coverslips coated with collagen G, and cultivated in McCoy 5A Medium in a CO2-incubator at 37 degrees C. Light and electron microscopy after 1, 2, 3, and 6 weeks showed selective and progressive cell growth with keratinocyte differentiation, producing multilayered cultures of cells joined with fully developed desmosomes. Three distinct patterns of differentiation, leading to the formation of an incomplete horny layer, were seen. The particular arrangement of tonofilaments, the considerable amounts of cytoplasmic glycogen, and the absence of malpighian differentiation were ultrastructural indicators of the follicular origin of the cultured cell population, which most likely grew from the outer root sheath of the hair. This technique may provide a promising model on which to base further studies of hair biologic processes and hair growth.

Azelaic acid vs. placebo: effects on normal human keratinocytes and melanocytes. Electron microscopic evaluation after long-term application in vivo.

The effects of topically applied 20% azelaic acid (AA) on normal human epidermis were investigated vs. placebo in a double blind study by electron microscopy in 15 volunteers. After 3 months of local application twice daily, the pattern of epidermal keratinization was found altered in skin treated with AA. In particular, the number and thickness of tonofilament bundles and the number of keratohyaline granules seemed decreased; the remaining granules were smaller, occasionally showing irregular electron densities. The perinuclear endoplasmic reticulum and the cytoplasmic cisternae were enlarged and swollen mitochondria were regularly observed in most malpighian keratinocytes. Thorough quantitative evaluation of the number and distribution of melanocytes by a MOP-videoplan computer system showed no differences between verum and placebo sites, although, the mean number of melanocytes had increased in both, as compared to the untreated controls taken before onset of therapy. No significant qualitative changes of the normal melanocytes were found. These findings indicate that azelaic acid may influence the differentiation of normal human keratinocytes by reducing the synthesis of keratin precursors and may, therefore, act as a mild antikeratinizing agent, whereas, the pigmentary system in normal human epidermis does not show any specific change after 3 months of treatment with AA.