Coculture of human keratinocytes and melanocytes: differentiated melanocytes are physiologically organized in the basal layer of the cultured epithelium

Human epidermal keratinocytes differentiate in vitro into a stratified epithelium suitable for grafting on burned patients. In this paper, we show that differentiated melanocytes are present in the cultured epithelium. In particular, we have found that i) melanocytes proliferate in the same culture conditions that allow keratinocyte growth, ii) during the culture the ratio between keratinocytes and melanocytes tends to remain constant, iii) melanocytes organize into the basal layer of the cultured epithelium independently of the presence of dermis, develop dendritic arborizations with melanosome-containing processes and transfer melanosomes into keratinocyte cytoplasm.     

Quinones are reduced by 6-tetrahydrobiopterin in human keratinocytes, melanocytes, and melanoma cells

Quinones are potentially dangerous substances generated from quinols via the intermediates semiquinone and hydrogen peroxide. Low semiquinone radical concentrations are acting as radical scavengers while high concentrations produce reactive oxygen species and quinones, leading to oxidative stress, apoptosis, and/or DNA damage. Recently it was recognised that thioredoxin reductase/thioredoxin (TR/T) reduces both p- and o-quinones. In this report we examine additional reduction mechanisms for p- and o-quinones generated from hydroquinone (HQ) and coenzyme Q10 and by 17beta-estradiol by the common cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)). Our results confirmed that TR reduces the p-quinone 1,4 benzoquinone and coenzyme Q10-quinone back to HQ and coenzyme Q10-quinol, respectively, while 6BH(4) has the capacity to reduce coenzyme Q10-quinone and the o-quinone produced from 17beta-estradiol. 6BH(4) is present in the cytosol and in the nucleus of epidermal melanocytes and keratinocytes as well as melanoma cells and colocalises with TR/T. Therefore we conclude that both mechanisms are major players in the prevention of quinone-mediated oxidative stress and DNA damage.     

Basic fibroblast growth factor and insulin-like growth factor I are strong mitogens for cultured mouse keratinocytes

Basic fibroblast growth factor (bFGF), but not acidic fibroblast growth factor (aFGF), was found to be mitogenic for cultured mouse keratinocytes. A six-to-nine fold increase in 3H-thymidine (3H-dT) incorporation into the acid insoluble pool and a similar increase of the labeling index can be measured when bFGF, at a concentration between 1 and 10 ng/ml, is added to keratinocytes arrested in serum-free and growth factor-free medium with a Ca++-concentration below 0.1 mM. The half-maximal response is observed between 0.2 and 0.7 ng/ml. In the same culture system, insulin-like growth factor I/somatomedin C (IGF-I) and insulin act as mitogens. IGF-I shows half-maximal stimulation with 2-3 ng/ml, insulin with 100-500 ng/ml. Basic FGF, IGF-I and insulin can be classified as strong stimulators of DNA synthesis in mouse keratinocytes. In this regard they are comparable to epidermal growth factor, which shows a half-maximal stimulation at a concentration between 1.5-2 ng/ml. These results show that in addition to mesenchymal cells, FGF is a growth factor not only for neuroectodermal cells, but ectodermal cells in general. They further support the idea that the growth promoting effect of insulin on keratinocytes may be mediated by the IGF-I receptor.     

Mitogenic, melanogenic, and cAMP responses of cultured neonatal human melanocytes to commonly used mitogens.

The following studies have been undertaken to compare and correlate the effects of 12-O-tetradecanoylphorbol acetate (TPA), basic fibroblast growth factor (bFGF), cholera toxin (CT), and isobutyl methylxanthine (IBMX) on neonatal human melanocyte (NHM) proliferation, tyrosinase activity, and cyclic adenosine monophosphate (cAMP) concentration. NHM proliferated at a maximal rate in medium containing 8 nM TPA, 200 ng/ml CT, and 10(-4) M IBMX. TPA alone did not result in optimal melanocyte proliferation, and, as previously shown, its mitogenic effect was greatly enhanced by the addition of CT and IBMX individually or concomitantly. Human recombinant (hr) bFGF could replace TPA in the NHM growth medium. Maximal proliferation was achieved using 3 ng/ml hrbFGF, 20 ng/ml CT, and 10(-4) M IBMX. The mitogenic effect of 1.2 ng/ml hrbFGF was potentiated in the concomitant but not individual presence of CT and IBMX. TPA alone in the absence of CT and IBMX caused a dose-dependent stimulation of tyrosinase activity. Maximal tyrosinase activity was obtained in the presence of 0.8 nM TPA, 20 ng/ml CT, and 10(-4) M IBMX. Unlike TPA, hrbFGF alone resulted in inhibition of tyrosinase activity. In the presence of hrbFGF, tyrosinase activity was potentiated by CT and IBMX, but not by CT alone. Neither TPA nor hrbFGF alone could increase intracellular cAMP levels. The effects of CT and IBMX on intracellular cAMP concentration were enhanced to a greater extent by TPA than by hrbFGF. Under our experimental conditions, in the presence of hrbFGF, CT but not IBMX resulted in a dose-dependent increase in cAMP concentration. Further studies on NHM will be aimed at determining the exact role of protein kinase C (PKC) in regulating proliferation and melanogenesis and the mechanism(s) activated by hrbFGF.     

A method for quantifying melanosome transfer efficacy from melanocytes to keratinocytes in vitro

Several different in vivo and in vitro bioassays are used to evaluate melanosome transfer efficacy from melanocytes to keratinocytes. However, these methods are complicated and time consuming. Here, we report on a simple, rapid, direct, and reliable in vitro method for observing the process of melanosome transfer from melanocytes to keratinocytes. First, we selected and tested a melanoma cell line RPMI-7951 that can normally synthesize melanin and transfer from mature melanosomes to keratinocytes in vitro. We cocultured these cells with a human ovarian teratoma transformed epidermal carcinoma cell line, which is also capable of accepting melanosomes transferred from melanocytes, as in normal keratinocytes. The cells were cocultured for 24-72 h and double labeled with FITC-conjugated antibody against the melanosome-associated protein TRP-1, and with Cy5-conjugated antibody against the keratinocyte-specific marker keratin 14. The cells were examined by fluorescence microscope and flow cytometry. Melanosome transfer from melanocytes to keratinocytes increased in a time-dependent manner. To verify the accessibility of this method, the melanosome transfer inhibitor, a serine protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and a melanosome transfer stimulator, alpha-melanocyte-stimulating hormone, were added. The serine protease inhibitor decreased melanosome transfer, and alpha-melanocyte-stimulating hormone increased melanosome transfer, in a dose-dependent manner. In conclusion, this is a simple, rapid, and effective model system to quantify the melanosome transfer efficacy from melanocytes to keratinocytes in vitro.     

The exocyst is required for melanin exocytosis from melanocytes and transfer to keratinocytes

Skin pigmentation involves the production of the pigment melanin by melanocytes, in melanosomes and subsequent transfer to keratinocytes. Within keratinocytes, melanin polarizes to the apical perinuclear region to form a protective cap, shielding the DNA from ultraviolet radiation‐induced damage. Previously, we found evidence to support the exocytosis by melanocytes of the melanin core, termed melanocore, followed by endo/phagocytosis by keratinocytes as a main form of transfer, with Rab11b playing a key role in the process. Here, we report the requirement for the exocyst tethering complex in melanocore exocytosis and transfer to keratinocytes. We observed that the silencing of the exocyst subunits Sec8 or Exo70 impairs melanocore exocytosis from melanocytes, without affecting melanin synthesis. Moreover, we confirmed by immunoprecipitation that Rab11b interacts with Sec8 in melanocytes. Furthermore, we found that the silencing of Sec8 or Exo70 in melanocytes impairs melanin transfer to keratinocytes. These results support our model as melanocore exocytosis from melanocytes is essential for melanin transfer to keratinocytes and skin pigmentation and suggest that the role of Rab11b in melanocore exocytosis is mediated by the exocyst.     

Melanosomes, melanocytes and keratinocytes in the human epidermis in incontinentia pigmenti

A functioning epidermal melanin unit implies a melanocyte capable of transferring melanosomes to keratinocytes; this requires not only melanocytes with adequate dendrites but also "receptive" keratinocytes. Skin with incontinentia pigmenti was examined by electron microscopy. Premelanosomes were occasionally found within keratinocytes and deposits of extracellular granular material that came from vacuolar degeneration of keratinocytes adjacent to melanocytes.     

Elutriation of human keratinocytes and melanocytes fromin vitro cultured epithelium

Summary Human epidermal keratinocytes grown in culture and at different stages of differentiation are shown to be viably separated by elutriation. A specific fraction enriched in melanocytes was obtained. Elutriation of cells obtained fromin vitro cultured epithlium could prove useful in studies concerning the biochemistry and molecular markers of cells isolated from normal epithelium and from different pathologies.     

Differential effects of selenite and selenate on human melanocytes, keratinocytes, and melanoma cells.

Among the substances that attracted the attention of oncologists in recent years are selenium-containing compounds, both inorganic and organic. Several epidemiological studies have shown an inverse correlation between selenium intake and cancer incidence. In the experiments reported here, we compared the effects of 2 inorganic selenium-containing salts that differed in the level of selenium oxidation, selenite IV and selenate VI. We tested the effects of these 2 compounds on cell survival and growth, cell cycle processing, cell morphology, cytoskeleton, and lipid peroxidation in 3 human skin cell types: normal keratinocytes, melanocytes, and human melanoma cell line HTB140. The different effects of selenite and selenate on the viability, growth, and morphology of normal cells and tumor cells are reported and provide a base for future research and treatment of some neoplastic diseases. The attention is paid to cell apoptosis induced by selenite and not by selenate, and the effects of tested substances on thioredoxin reductase system are postulated.     

Effects of commonly used mitogens on the cytotoxicity of 4-tertiary butylphenol to human melanocytes

Summary In the search for environmental compounds responsible for contact or occupational vitiligo, it was found that the most potent was 4-tertiary butylphenol (4-TBP). Exposure to 4-TBP is widespread both in industry and in consumer items including synthetic leather, plastic, glues, and germicidal phenolic detergents. How 4-TBP causes depigmentation and the death of melanocytes is currently unclear. Growth mitogens for human melanocytes include α-melanocyte stimulating hormone (α-MSH), basic fibroblast growth factor (bFGF) and 12-o-tetradecanoylphorbol-13-acetate (TPA). The former two mitogens are physiological growth factors for melanocytes. We have studied the effects of these mitogens on the cytotoxicity of 4-TBP in human melanocytes. Our results demonstrated that deprivation of α-MSH or bFGF from melanocyte cultures resulted in reduced cytotoxicity to 4-TBP. Similar results were obtained upon treatment of melanocytes with an inhibitor of cAMP-dependent protein kinase A (PKA), that is known to be activated by α-MSH, or with an inhibitor of the tyrosine kinase bFGF receptor. In contrast, removal of fetal bovine serum or TPA from the culture medium did not influence the susceptibility of melanocytes to 4-TBP. These results suggest that activation of the cAMP and tyrosine kinase signaling pathways, both of which are involved in the mitogenic response of melanocytes, increase the susceptibility of these cells to the cytotoxic effects of 4-TBP. This work was presented in part at the seventh meeting of Pan-American Society for Pigment Cell Research, June 15–18, 1997, Providence, Rhode Island, USA.     

Influenza viruses are T cell-independent B cell mitogens.

UV-inactivated influenza virus A strains of subtypes H1, H2, H3, and H6 were shown to be mitogenic for unprimed splenic lymphocytes from BALB/c mice. Representative viruses of these four subtypes all behaved as T cell-independent B cell mitogens. The magnitude of the proliferative response was determined by the subtype of the hemagglutinin molecule: H2 and H6 viruses were the most potent mitogens, and H3 viruses were moderately mitogenic, whereas H1 viruses induced only low, but significant, levels of proliferation. Mitogenesis was inhibited by antiviral sera and by monoclonal antibodies directed against hemagglutinin.     

Divergent regulation of proteoglycan and glycosaminoglycan free chain expression in human keratinocytes and melanocytes

Summary Keratinocytes and melanocytes, which together form units of structure and function within human epidermis, are known to differ in expression of autocrine growth factors, particularly those with heparin binding affinity. Because such cytokines could be regulated by the endogenous heparinlike glycosaminoglycan, heparan sulfate, proteoglycan synthesis was compared between human keratinocytes and melanocytes cultured from a common donor. Following steady-state isotopic labeling under conditions of active growth (low density cultures) and growth inhibition (high density cultures), the sulfated polymers were isolated from conditioned media and cell extracts. We found that keratinocytes produced substantially more sulfated glycosaminoglycans than did the melanocytes. There was no evidence for hyaluronic acid synthesis by the melanocytes. The majority of [³⁵S]-sulfate labeling was in the heparan sulfates of the keratinocytes and in the chondroitin sulfates of the melanocytes. During the transition from active growth to growth inhibition, there was increased heparan sulfate proteoglycan and free chain synthesis by keratinocytes but not by melanocytes, and chondroitin sulfate proteoglycan production declined in both cell lineages. The differences may reflect divergent evolution as each cell type came to exploit those complex polysaccharides in different ways to regulate molecular pathways of growth and differentiation. The coupling of growth inhibition with augmented synthesis of heparan sulfates observed for the keratinocytes suggests a regulatory role in growth factor signaling in that cell type.     

Non-specifically activated human peripheral blood mononuclear cells are cytotoxic for human keratinocytes in vitro

Cultured human keratinocytes were lysed by activated PBMC in a 4-h 51Cr release assay. PBMC were activated by incubation with 50 U/ml of rIL-2 for 4 days. The cytotoxic precursors were found to be NKH1+ and included both CD2+ and CD2- phenotypes. This cytotoxicity was not genetically restricted, as cells killed both allogeneic and autologous keratinocytes without priming. Cytotoxicity was blocked by pre-incubation of effector cells with mAb against LFA-1 alpha-(TS1/22) and beta-chains (TS1/18), but not by antibodies directed against CD4, CD8, or leukocyte common Ag (T200) suggesting that LFA-1 is an important interactive molecule in this cytotoxicity. IFN-gamma is reported to upregulate ICAM-1, the ligand for LFA-1. Pre-treatment of target keratinocytes with IFN-gamma was also found to greatly increase the sensitivity of keratinocytes to lysis. This increased sensitivity to lysis was blocked by anti-LFA-1 and anti-ICAM-1, but not by anti-DR (L243), and thus was not the result of increased DR expression. Such treated targets were lysed at low levels (15 to 18%) by an Ag-specific CD8+ cytotoxic clone as well as a T cell line derived from a skin lesion of allergic contact dermatitis. In contrast, control keratinocytes were only sensitive to IL-2-activated PBMC as described above. The above findings may be relevant to a variety of conditions in which epidermal damage is associated with lymphocytic infiltrate. These conditions include graft-vs-host disease, erythema multiforme, and lupus erythematosus. DR+ keratinocytes, which may be a marker for IFN-gamma are also found in the above conditions. It is suggested that epidermal pathology may be mediated by non-specific cytotoxicity induced in the course of an immune response.