Metabolism of arachidonic acid by human epidermal cells depends upon maturational stage

Synthesis of 12- and/or 15-HETE by human epidermal cells was investigated after separating basal cells from suprabasal epidermal cell layers. We found that the main metabolite of 3H-arachidonic acid (3H-AA), formed by freshly prepared upper epidermal layers (stratum granulosum and spinosum), upon RP-HPLC co-eluted with authentic 3H-12-HETE. A 3H-15-HETE co-eluting peak selectively occurred in chromatograms obtained from supernatants of fractions containing basal cells. Supernatants of freshly prepared suspensions rich in basal keratinocytes appeared to contain 3H-15-HETE as their main 3H-AA metabolite, by far exceeding the recovered amounts of 3H-12-HETE. Moreover, keratinocytes cultured for 1 week or longer were found to produce predominantly a 3H-AA metabolite co-eluting with 3H-15-HETE. In supernatants of cultured cells, little if any 3H-12-HETE was detectable. Cultured human skin fibroblasts were not found to produce relevant amounts of HETE. Genuine tissue rich in basal cells, i.e., cells of hair follicles, were found to form twice as much 3H-15-HETE as 3H-12-HETE (3H-15-HETE/3H-12-HETE-ratio = 1.9 +/- 0.8; n = 7). Apparently, different epidermal layers are able to produce a characteristic pattern of 3H-AA metabolites. 3H-15-HETE generation seems to be a marker for proliferating keratinocytes, whereas 3H-12-HETE formation appears to be typical for differentiating suprabasal epidermal cells. Our results may explain the heretofore varying patterns of AA-metabolites by keratinocytes reported in the literature.     

蛋白激酶C抑制剂GF109203X诱导角质形成细胞去分化形成表皮干细胞的初步研究

目的探讨蛋白激酶C(PKC)抑制剂GF109203X诱导角质形成细胞去分化形成表皮干细胞的可能性。方法采用酶消化法结合Ⅳ型胶原快速贴壁法获得人原代表皮干细胞及角质形成细胞,倒置相差显微镜下观察细胞生长状况,免疫细胞化学染色法检测GF109203X诱导培养后角质形成细胞的表型及功能改变。同期分离培养的人在体表皮干细胞作为本次实验的阳性对照,原代角质形成细胞培养2 d后加等体积二甲基亚砜为阴性对照。结果表皮干细胞快速黏附,培养4 d后细胞呈圆形,形态规则,折光性强,明显克隆,β1整合素、CK19及CK14呈阳性表达,CK10阴性表达;已分化角质形成细胞不能快速黏附,培养4 d细胞呈类圆形,大小不一,折光性较差,无明显克隆,β1整合素、CK19及CK14呈阴性表达,CK10呈阳性表达。角质形成细胞经GF109203X诱导培养2 d后,实验组与阳性对照组细胞群β1整合素、CK19、CK14均呈阳性表达,但实验组较阳性对照组中细胞β1整合素、CK19、CK14阳性细胞数少,CK10均呈阴性表达;阴性对照组中细胞群β1整合素、CK19及CKl4呈阴性表达,CKl0呈阳性表达。结论 GF109203X能够诱导角质形成细胞去分化形成表皮干细胞。     

Isolation of human sebaceous glands and cultivation of sebaceous gland-derived cells as an in vitro model

An experimental technique is presented as an in vitro model for the study of human sebaceous gland-derived cells. Intact sebaceous glands were isolated from full-thickness human skin after incubation in dispase (2.4 U/ml) and in deoxyribonuclease (0.02%) by using microsurgical instruments under microscopical observation of the epidermal underface. Subsequently, the ducts of the glands were removed, the isolated gland lobules were seeded on a 3T3-cell feeder layer in Dulbecco's modified Eagle's medium and Ham's F 12 medium (3:1) supplemented with fetal calf serum (10%), L-glutamine, antibiotics, epidermal growth factor (10 ng/ml), hydrocortisone (0.4 microgram/ml), and cholera toxin (10(-9) M), and were then cultivated in a CO2-incubator at 37 degrees C. After 2-3 wk cell outgrowths resulting from the periphery of the gland lobules were obtained and dispersed cells were passaged for three subcultures with or without 3T3-cell feeder layer. The cultured cells preserved in vitro morphologic characteristics and differentiation patterns comparable to those described for normal human sebocytes in vivo, with a high rate of viable cells. Their labeling pattern with MoAb showed close similarities to the pattern reported for sebocytes in vivo but differences to the pattern of keratinocytes in vivo and in vitro. In their cytoplasm oil red and nile red stained droplets were detected, and the observed density and distribution evidenced in vitro lipogenesis. The technique presented here may provide a promising model for further experimental studies on sebaceous gland cell development and function.     

Metabolism of an aromatic retinoid Ro 10-9359 by cultured human epidermal keratinocytes

Summary An aromatic retinoid Ro 10-9359 is metabolized after absorption from intestine to form Ro 10-1670 which is an active therapeutic compound. The epidermal keratinocytes, a main target tissue of retinoid therapy in dermatology, was examined in the capacity to metabolize the retinoid. The culture of human epidermal keratinocytes was treated with 10^-6 M Ro 10-9359 and the metabolites released in the medium was analyzed by HPLC. The HPLC profile showed a distinct peak of Ro 10-1670. The human skin fibroblasts, HeLa cells, Chang liver cells and 3T3 cells were less active in metabolizing Ro 10-9359, and only a small amount of Ro 10-1670 was detected in the culture of human skin fibroblasts treated with 10^-6 M Ro 10-9359 for 2 days. When these cells were disrupted by a glass homogenizer, and incubated with Ro 10-9359, no Ro 10-1670 formation was detected.     

Culture and characterization of murine dendritic Thy-1+ epidermal cells

Although numerous advances have been made in characterizing the phenotype, ontogeny, ultrastructure, and cytochemistry of the murine Thy-1+ dendritic epidermal cell (Thy-1+ EC), elucidation of its functional qualities has been hampered by the difficulty in preparing pure populations of these cells. We therefore sought to obtain expanded, purified populations of Thy-1+ EC using culture techniques. Since Thy-1+ EC are bone marrow-derived, density gradient enriched populations of freshly harvested epidermal cells (FH-EC) were placed in culture under conditions known or suspected to promote mitogenesis among leukocyte subsets. FH-EC prepared from truncal skin of C3H/HeN mice (Thy-1.2+) were cultured at 37 degrees C in 5% CO2 in complete medium (CM) of Eagle's Hanks' amino acid with 10% fetal calf serum, nutrients, and antibiotics at 10(6) FH-EC/well in 24-well culture plates. CM was supplemented with one or more of the following: concanavalin A (Con-A), interleukin-1/epidermal cell-derived thymocyte-activating factor (IL-1/ETAF), IL-2, IL-3, gamma interferon, indomethacin (IM), and anti-Thy-1.2 antibody. Media with appropriate supplements were changed every 2-3 days. Freshly isolated, enriched FH-EC contained 7-20% Thy-1+ EC (defined as brightly fluorescing cells readily distinguishable from weakly fluorescing keratinocytes), which also stained with antibodies directed against asialo GM1, Ly 5.1, and vimentin but did not stain with antibodies to other T cell-, B cell- or macrophage phenotypic markers. Analysis of 10 separate cultures revealed a 3- to 10-fold expansion of nonkeratinocyte Thy-1+ cells after 21 +/- 4 days in culture in CM supplemented with Con-A and IM, and 70-100% of viable cells after expansion were Thy-1+. Phenotypic analysis of expanded cells revealed the emergence in 10 separate cultures of one of two mutually exclusive distinct populations: one Thy-1+, asialo GM1+, L3T4- (natural killer phenotype) and the other Thy-1+, asialo GM1-, L3T4+ (T helper phenotype). Experiments designed to explain the emergence of an L3T4+ population suggest that phenotypic modulation occurred in vitro.     

Dynamic expression of pemphigus and desmosomal antigens by cultured keratinocytes

Dynamic expression of pemphigus antigens by cultured human and mouse keratinocytes was compared with that of desmosome-associated molecules and cellular markers relating to epidermal differentiation. Plakoglobin was detected in localized areas of keratinocyte sheets in low Ca²⁺ (0·15mM) KGM medium. In minimum essential medium (MEM) containing 1·8 mM Ca²⁺. plakoglobin was expressed in the intercellular spaces (ICS) throughout the keratinocyte sheet. Desmoplakin I and II. which were present in the cytoplasm of keratinocytes in the low Ca²⁺ medium, moved to the cell surface after the medium was changed to MEM. Desmoglein 1 and pemphigus vulgaris (PV) antigens were observed in the ICS of both the monolayers and stratilied areas in the MEM. Pemphigus foliaceus (PF) antigens, frequently together with desmoglein 1, involucrin and keratins specific for the upper layer of the epidermis, were expressed by stratified keratinocytes but not the cells in the monolayers. The Western blotting study of the cultured keratinocyte extract showed 160- and 130-kDa bands positive for desmoglein 1 antigens and a 1 30-kDa band stained with PV sera. These findings suggest that although desmoglein 1 molecules bear PF antigenic sites, their expression pattern by cultured keratinocytes is closely related to that of PV rather than PF antigens. The PF antigenic sites may be formed on desmoglein 1 during epidermal differentiation.     

Expression of OKM5 antigen on human keratinocytes in vitro upon stimulation with gamma-interferon

Using murine monoclonal antibodies against human OKM5, OKM1 and HLA-DR antigens antigenic characteristics of freshly separated human epidermal cells (EC) and those of EC cultured in the presence of Interferon-gamma (IFN-gamma) were studied. After 8-12 days of culture, primarily OKM1- OKM5- HLA-DR- keratinocytes displayed OKM5 and HLA-DR antigens when exposed to IFN-gamma. Our data support the concept, that human keratinocytes may possess accessory cell functions.     

Optimization of murine keratinocyte culture for the production of graftable epidermal sheets.

The aim of the present study was to optimize murine epidermal cell cultures in order to obtain graftable sheets. Newborn (1-3 days old) Balb/c mice skin were used to optimize culture media and plating cell concentration, then epidermal sheet production, and grafting. Epidermal cells were plated at various concentrations in different culture media containing low (0.1 mM) or high (greater than 1 mM) Ca2+ levels. After a 3 day culture at the 10(4) cells/cm2 plating cell concentration, the percentage of differentiated cells was more than 80% in the high Ca2+ culture medium and less than 50% with bulky cells in the low Ca2+ culture medium. Under these conditions confluence was not obtained. At the 10(5) cells/cm2 seeding inoculum, the percentage of confluence increased to 95-100% during the first 72 h of culture in both high and low Ca2+ culture media. Three-day-old culture showed stratified multilayer epidermal sheets in the high calcium medium, and monolayer epidermal sheets were present in the low calcium medium after seeding keratinocytes in fibronectin precoated flasks. Seven days after plating, post confluent cultures were composed of a high percentage of differentiated cells (90%) with an increase in shedding cells in the medium. Considering the above morphological observations, sheets obtained with 10(5) cells/cm2 in MCDB-153 (A), DME-HAM (B) or GMEM (C) media after 3 days in culture were grafted. Twenty days after grafting, histological analysis of biopsies showed an epidermal structure and organization comparable to normal murine epidermis without hair follicles. Epidermal transplants showed a complete basement membrane, hemidesmosomes, and tonofilament bundles. Sheets obtained after seven day culture in all media showed lower coverage of the wound bed. These studies point out the importance of the plating cell and Ca2+ concentrations, and the culture time for murine keratinocyte confluence and differentiation to obtain graftable epidermal sheets.     

Supernatants from cultured human epidermal keratinocytes stimulate angiogenesis

Conditioned media from three different strains of human epidermal keratinocytes in culture were assayed on the chorioallantoic membrane of the chick embryo. Vascular responses were examined 4 days later stereo-microscopically and compared with controls—unconditioned medium and medium incubated with 3T3 cells. Specimens were also collected after 1,2,3 and 4 days for serial histological examination. Media from all three strains of keratinocytes stimulated statistically significant vascular growth relative to controls. Leukocytic infiltration was not demonstrated histologically at any time. In addition, quantitation of ectodermal epithelial hyperplasia in experimental groups did not reveal any significant difference when compared with controls. These results have demonstrated that epidermis may directly encourage vascular growth in the absence of significant inflammation.     

Effects of cholera toxin on proliferation of cultured human keratinocytes in relation to intracellular cyclic AMP levels

In the culture of epidermal keratinocytes, the cellular growth rate is reported to be accelerated by cholera toxin. The mechanism by which cholera toxin exerts biological effects is thought to result from changes in intracellular cyclic AMP concentrations. But in many reports cyclic AMP elevating agents appeared to inhibit growth of keratinocytes in culture. This study was done to clarify the discrepancy of this problem. Determination of cyclic AMP revealed that cholera toxin over a range of 10-14-10-8 M increased the intracellular concentration of cyclic AMP of cultured keratinocytes about 100-fold over the controls after incubation for 6 hr. When a small number (10(5)) of cells were inoculated in a 60 X 15 mm culture dish, cholera toxin strongly stimulated colony growth. When a relatively larger number (8 X 10(5)) of cells were inoculated in a dish, cholera toxin moderately accelerated cell division, and increased DNA and protein levels of the culture during early days of cultivation. But after about 20 days, of cultivation when the culture reached confluence, cholera toxin decreased both DNA and protein content in a culture dish. The cultures were pulse labeled with 3H-thyrmidine at 12 and 24 hr after the addition of 10-10 M cholera toxin, and its uptake into DNA was determined. In the early days of cultivation the uptake of 3H-thymidine increased after treatment with cholera toxin. But in the late days of cultivation, cholera toxin decreased the rate of 3H-thymidine incorporation into DNA. These results indicated that cholera toxin-cyclic AMP has effects on the proliferation of keratinocytes in culture biphasically according to cellular concentrations in culture.     

Pemphigus,pemphigoid, and epidermal upper-cytoplasmic antigens: Changes in expression in cultured human keratinocytes

Summary In an approach of epidermal differentiation, the expression of pemphigus, bullous pemphigoid, and upper-cytoplasmic epidermal antigens was studied in human keratinocytes in culture. The cells were cultured without feeder cells, dermal tissue, or collagen at an acid pH (5.6–5.8) similar to that of the surface of the skin in vivo. Cell suspensions from fresh trypsinized skin and primary, secondary, and tertiary cultures were tested by indirect immunofluorescence for the presence of each antigen using human sera from patients with pemphigus, bullous pemphigoid, and human sera with antibodies against upper-cytoplasmic antigens. Normal sera and cultured human normal fibroblasts and melanoma cells were used as controls. Pemphigus and pemphigoid antigens were found to be expressed, and synthesized by keratinocytes in vitro. The expression to upper-cytoplasmic antigens decreased with time in culture, and they were absent in secondary or tertiary cultures while expressed by 45–65% of cells prepared from fresh skin. Both upper-cytoplasmic and pemphigoid antigens can be used to type subpopulations of human epidermal cells; however, these findings suggest that epidermal differentiation in vitro differs from that which occurs in vivo.

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Abreviations BMZ basement zone - BP bullous pemphigoid - FCS fetal calf serum - MEM minimal essential media - IC inter cellular - IF immunofluorescence - PBS phosphate-buffered saline - PV pemphigus vulgaris - U-CYT upper-cytoplasmic Supported in part by a grant from the Fondation de l'Industrie Pharmaceutique pour la Recherche. Paris, France, by Research Grant CA 13844.07 from the USPHS, and by grant PCM 7911783 from the National Science Foundation, USA Offprint request to: Prof. J. Thivolet (address see above)     

Fibronectin-mediated keratinocyte migration and initiation of fibronectin receptor function in vitro

Cell suspensions of human keratinocytes, freshly isolated from skin specimens, did not express plasma fibronectin (pFN) receptor function in short-term assays for cell attachment and spreading on pFN-coated culture dishes and binding and phagocytosis of pFN-coated latex beads. These activities were expressed, however, by the cells harvested from primary keratinocyte cultures after 2-4 days of culture. Analysis of the cell types arising during primary culture, based on staining with antikeratin antibodies and bullous pemphigoid (BP) serum, revealed that about 90% of the originally isolated cell population consisted of keratinocytes (keratin-positive) and 30% were basal cells (BP antigen-positive). After 2 days of culture, 95% of the cells were keratinocytes and 70% were basal cells. In vitro initiation of pFN receptor function also was observed in cells harvested from epidermal explants. After 9 days in culture, the cells that migrated out of the explants also were active in short-term cell adhesion assays, while cells remaining in the central region of the explant had much less activity. In related studies, the role of pFN in epidermal cell migration was analyzed, and it was found that anti-pFN antibodies inhibited migration of keratinocytes out of epidermal explants. Addition of preimmune IgG, however, had no effect. It appears, therefore, that pFN is important in all aspects of keratinocyte adhesion, and the expression of pFN receptor function may be a critical activation step necessary for basal cell phagocytosis and migration during wound healing.     

Expression of lysosome-associated membrane protein 1 (Lamp-1) and galectins in human keratinocytes is regulated by differentiation

Lysosomes and their components are suspected to be involved in epidermal differentiation. In this study, lysosomal enzyme activities, expression of the lysosome-associated membrane protein 1 (Lamp-1) and expression of the epidermal galectins-1, -3 and -7 were investigated in human keratinocytes cultured at different cell densities (subconfluence, confluence and postconfluence) in order to induce differentiation. Detected by Western blot and immunofluorescence, Lamp-1 expression is transiently upregulated at culture confluence, but reduced at postconfluence. Northern blot analyses performed on subconfluent, confluent and post-confluent cultures of keratinocytes show that Lamp-1 mRNA expression is also upregulated at culture confluence, but downregulated at postconfluence. Measurements of lysosomal enzyme activities indicate a transient upregulation at culture confluence, whereas cathepsins B, C and L are particularly downregulated at postconfluence. Cell density and differentiation of epidermal cells also differentially regulates galectin expression in autocrine cultures. As the expression of galectin-1 mRNA is high in subconfluent cells, it is assumed to be associated with their proliferative state. On the other hand, as the mRNA levels for galectins-3 and -7 are notably upregulated at culture confluence (galectin-7) or at postconfluence (galectin-3), their expression is thought to be related to the differentiated state of keratinocytes. However, we collected evidence by confocal microscopy that galectin-3 and Lamp-1 do not colocalize in vitro in keratinocytes. Altogether, our results suggest that the upregulated Lamp-1 expression at confluence could be involved in keratinocyte differentiation, but apparently not through interaction with galectin-3.     

CD8+ T cell granzyme B activates keratinocyte endogenous IL-18

IL-18 is a pro-inflammatory cytokine of the IL-1 family involved in Th1/Th2 polarization. IL-18 is produced and stored as an inactive precursor (proIL-18) in several cells including keratinocytes, and thus appropriate processing is required to release its active form. In a previous study using recombinant protein, we demonstrated that granzyme B (GrB) cleaves proIL-18 into its active forms in a similar fashion as caspase-1 and human mast cell chymase. GrB released from cytotoxic T lymphocyte (CTL) and NK cells has roles in apoptosis and cytotoxic activity. In certain inflammatory skin diseases with epidermal cell death, the epidermal keratinocytes are targets of CTL and NK cells. However, IL-18 activation during the direct interaction of CTL/NK with keratinocytes has not been described so far. We investigated the interaction between CTL and keratinocytes, and IL-18 processing by CTL-derived GrB using cultured CD8+ T cells and keratinocyte cell line HaCaT. GrB(+)/caspase-1(?) CD8+ T cells cultivated from healthy human PBMC were co-cultured with interferon(IFN)-γ-treated HaCaT cells. The expression of GrB and caspase-1 in HaCaT cells was analyzed by flow cytometry and PCR. The IL-18 concentration in the culture supernatant was measured by specific ELISA. The interaction between HaCaT cells and CTL co-culture increased the number of cytoplasmic GrB-positive HaCaT cells with limited endogenous GrB mRNA expression. The concentration of mature IL-18 levels increased in the co-culture supernatant. GrB from CTLs acts double roles to keratinocytes: a IL-18 converting enzyme and pro-apoptotic factor in the skin inflammatory diseases.     

Effect of oxygen on the growth of human epidermal keratinocytes

We studied the growth of secondary cultures of neonatal human keratinocytes at oxygen concentrations between 1 and 89%. Keratinocytes were grown in MCDB medium with 5% fetal bovine serum, 10 ng/ml epidermal growth factor, 5 micrograms/ml insulin, 0.5 microgram/ml hydrocortisone, and 0.1 mM ethanolamine and phosphorylethanolamine. Medium in the flasks was equilibrated with gas mixtures containing 5% CO2, various percentages of oxygen from 0-95% and nitrogen to balance. Cells were seeded at 10(4) cells/cm2 in sealed flasks (25 cm2). These were incubated at 37 degrees C in incubators maintained at the experimental oxygen tensions. Cells grew best at PO2 (partial pressure of oxygen) 133 mm Hg (18% O2), with a mean population doubling time of 2.8 days. Growth was retarded by 60% at PO2 38 mm Hg (5% O2) and by 98% at PO2 7 mm Hg (1% O2). However, the oxygen tension that resulted in the best plating efficiency was at PO2 12 mm Hg (2% O2). When oxygen tensions were shifted to 78-133 mm Hg, cells seeded under low oxygen tensions began to proliferate. These data suggest that a better harvest of keratinocytes is obtained when cells are seeded under low oxygen tension and then shifted to ambient oxygen tensions. At high oxygen tensions, above 20%, growth was inhibited by 75% at PO2 241 mm Hg (34% O2) and 98% at PO2 374 mm Hg (52% O2). At PO2 637 mm Hg (89% O2) no cell growth occurred. These findings showed that high oxygen tensions, above 20%, have no beneficial effect on the growth of keratinocytes.     

Production of epidermal sheets in a serum free culture system: a further appraisal of the role of extracellular calcium.

Rhenwald and Green's technique is currently the standard method for growing stratifying epidermal cell cultures. The serum free system developed in Ham's laboratory (MCDB 153) was designed to grow keratinocyte monolayers in clonogenic conditions. Our aim was to optimize conditions in serum-free MCDB 153 for culturing epidermal sheets from adult normal skin, and to assess the effect of extracellular calcium and temperature on proliferation and differentiation of cultured keratinocytes. Sixteen strains derived from plastic surgery specimens (mean age of donors 37 years; range 5-89) were used. Primary cultures were seeded at an optimal density of 8 x 10(4) cells/cm2 in primary cultures and 10(4) cells/cm2 in secondary cultures in complete medium including EGF, insulin, hydrocortisone and bovine pituitary extract, supplemented with isoleucine, tyrosine, methionine, phenylalanine, tryptophane and histidine. Amino acid (AA) supplementation allows a 5.8-fold increase in cell counts at confluency and monolayers with densely packed cells are obtained. In AA supplemented cultures, confluency is obtained in 16 +/- 3 days in primary cultures and in 13 +/- 0.5 days at first passages. Switches to 1.1 mM calcium at first or second passages resulted in a significant increase in cell counts (P less than 0.001), when compared with AA supplemented low calcium cultures. Low temperature/low calcium cultures resulted in a 50% decrease in cell counts. Low temperature/high calcium cultures gave similar cell counts as the 37 degrees C controls. AA and calcium supplemented cultures were evaluated for differentiation markers: involucrin expression was increased, keratins 5, 6, 14, 17 were expressed, and the sheets were 6-10 layers thick by electron microscopy, with keratohyalin granules and cornified envelopes appearing at layers 3-6 (from basal layer). Dispase treatment allowed an easy detachment of these sheets. These results show that the culture medium MCDB 153 can be adapted without serum supplementation to batch culture of human adult keratinocytes to produce epidermal sheets suitable for grafting. They also indicate that extracellular calcium in physiological range of concentration is not a sufficient signal for growth arrest when other growth conditions are optimized.     

体外培养人表皮角朊细胞的生长曲线

本文报告了体外培养人表皮角朊细胞的生长情况.用覆以胶原膜的塑料培养皿进行培养,细胞接种后18~24小时贴壁,10~14天细胞能铺满培养皿.角朊细胞的生长曲线可分3段,延缓期,继而生长期,最后为停滞期.本文还讨论了人表皮角朊细胞培养的技术及体会.     

Enzymatic dissociation of keratinocytes from human skin biopsies for in vitro cell propagation

Four techniques for dissociation of skin biopsies were compared to identify the method of choice for optimal expansion of isolated keratinocytes. Equivalent biopsies were obtained from 4 healthy human subjects and each divided into four parts. One part was minced and placed in a trypsinizing flask containing 0.05% trypsin and 0.01% ethylenediaminetetraacetic acid (EDTA). Released cells were harvested hourly. With the other parts, the epidermis was separated from the dermis after treatment with 0.5 mg/nml thermolysin, 2.5 mg/ml Dispase, or 0.17% trypsin and the epidermal portions were minced and incubated for 1 h in trypsin:EDTA. The cells were cocultivated with irradiated 3T3 fibroblasts to study the keratinocytes proliferative capacity. Freshly isolated cells were immunostained with anti-vimentin antibodies or grown in fibroblast-supportive conditions to detect the presence of human dermal fibroblasts. The mean number of cells dissociated per cm2 biopsy was higher after trypsin:EDTA digestion of a dermis-containing biopsy using a trypsinizing flask (4.0x 10(6) cells/cm2) compared to a biopsy where dermis-epidermis had been separated by thermolysin (2.8x 10(6) cells/cm2), Dispase (2.3x 10(6) cells/cm2) or trypsin (1.1 x 10(6) cells/cm2). Between 0.5% and 4% of the cells dissociated from a dermis-containing biopsy were human fibroblasts. This comprised more than twice the number of fibroblasts obtained by using epidermal/dermal split techniques. The proliferative capacity in primary and secondary culture was higher in cells isolated by trypsin:EDTA incubation in the trypsinizing flask or after epidermal-dermal separation using thermolysin, suggesting that Dispase or trypsin may have a more detrimental effect on the isolated keratinocytes. Our results show that dissociating the cells by trypsin:EDTA incubation in a trypsinizing flask or after epidermal-dermal separation using thermolysin, are preferable methods for isolating keratinocytes from human skin.     

T‐lymphocyte‐induced,fas‐mediated apoptosis is associated with early keratinocyte differentiation

Please cite this paper as: T‐lymphocyte‐induced, fas‐mediated apoptosis is associated with early keratinocyte differentiation. Experimental Dermatology 2009. Abstract: The development of eczematous lesions is thought to be due in part to a breakdown in skin barrier function as a result of T lymphocytes (T cells) invading the skin causing epidermal keratinocyte apoptosis. In this study, we investigated the interaction of T cells and keratinocytes on apoptosis and terminal differentiation using an in vitro co‐culture system. Experiments were performed using the HaCaT keratinocyte cell line or normal human epidermal keratinocytes. Activated human peripheral blood‐derived T cells were found to induce Fas‐dependent keratinocyte apoptosis by up to sixfold. Increased Fas was associated with increased IFN‐γ. The T‐cell apoptotic signal was found to target preferentially keratinocytes in the very early stages of terminal differentiation, such as those with low levels of α6‐integrin expression, and result in subsequent increased caspase 3 activity. This observation was accompanied by a marked increase in keratinocyte ICAM‐1 expression and its ligand LFA‐1 on T cells. Our data suggest that T cells may initiate the onset of keratinocyte terminal differentiation making them more susceptible to Fas‐dependent cell death signals delivered by the T cells.