Cell-density-regulated chemotactic responsiveness of keratinocytes in vitro.

Keratinocytes represent the main constituents of the epidermis and have been found to play a regulatory role in a variety of inflammatory skin diseases. The functional activity of keratinocytes is highly heterogeneous, and depends on the cell localization in the epidermal architecture, and the maturation or differentiation state of the cells. Spontaneously proliferating HaCaT cells, showing several similarities to basal epidermal keratinocytes, were found to respond to external chemoattractants, including the chemokines RANTES (regulated on activation normal T cell expressed and secreted) and interleukin-8 and the mu-opioid agonist DAMGO ([d-ala2, N-Me-Phe4, Gly-ol5]enkephalin) in migration assays. The chemotactic responsiveness was highly dependent on the cell density of the monolayer, with greatest chemotactic activity at the highest cell density. Whereas RANTES was found to be the most potent chemoattractant, constitutive RANTES production was also detected in the HaCaT cultures. We found an inverse correlation between constitutive RANTES production and chemotactic responsiveness toward external RANTES, suggesting a possible functional down-modulation of the RANTES receptors, CC chemokine receptor 1 and CC chemokine receptor 5, during culture. Results from confocal laser scanning microscopy showed reduced CC chemokine receptor 1, but not CC chemokine receptor 5, expression by HaCaT cells at low cell densities, which was abolished in the presence of neutralizing antibodies against RANTES. The total CC chemokine receptor 1 pool (surface and intracellular receptors), however, showed no significant change during in vitro culture. Chemotactic responsiveness toward RANTES was directly correlated with the level of CC chemokine receptor 1 surface expression. Taken together these results show that with keratinocyte proliferation and the progressive increase in cell density there are dramatic alterations in keratinocyte function.     

Enkephalins modulate differentiation of normal human keratinocytes in vitro

Abstract Opioid peptides are a group of neuropeptides which include enkephalins, endorphins and dynorphins. In addition to their central and peripheral antinociceptive function, opioids can modulate immune activity and cell proliferation. Previously, we have shown that enkephalins are present in macrophages infiltrating the dermal papillae in involved psoriatic skin and that the amount of enkephalin is significantly increased in involved psoriatic skin. Because enkephalins were detected close to the epidermis, we examined the effects of opioid peptides on the differentiation (transglutaminase type 1 activity and cytokeratin 10 expression) and proliferation (MTT assay) of cultured human keratinocytes. Enkephalins (methionine-enkephalin, leucine-cnkephalin and the synthetic DADL) inhibited cell differentiation dose-dependently, while β-endorphin had no effect. The opioid receptor antagonist naltrexone completely antagonized the inhibitory effect of methionine-enkephalin and leucine-enkephalin. but not that of DADL. Furthermore, methionine-enkephalin had a slight inhibitory effect on the proliferation of keralinocytcs. Enkephalin was detected in unstimulated keratinocyte cultures, and naltrexone alone stimulated keratinocyte differentiation. These results indicate that enkephalins may play a role in the differentiation of epidermal keratinocytes. It remains to be determined whether the enkephalin detected in psoriatic skin are sufficient to affect epidermal differentiation in vivo.     

Dicarboxylic acids inhibit the growth of keratinocytes in vitro

The antiproliferative effect of three straight-chained saturated dicarboxylic acids was examined with neonatal mouse keratinocyte cultures. Adipic acid (C6), azelaic acid (C9), and sebacic acid (C10) were added to the cultures in concentrations ranging from 1 to 50 mmol/l. Proliferation was assayed by liquid-scintillation counting of 3H-thymidine incorporation into DNA and by autoradiography. Fifty percent inhibition of 3H-thymidine incorporation was observed with 50 mmol/l adipic acid, 20 mmol/l azelaic acid, and 10 mmol/l sebacic acid, respectively. The antiproliferative effect was completely reversible after cessation of treatment. Moreover, treated cultures then showed a rebound effect with increased DNA synthesis. These results show that dicarboxylic acids exert reversible antiproliferative effects on keratinocytes.     

Minoxidil: inhibition of proliferation of keratinocytes in vitro

Previous studies have suggested that minoxidil stimulates growth of keratinocytes, possibly in a manner similar to the action of epidermal growth factor. Using both a short-term assay, thymidine incorporation, and a longer term assay, cell counting, to assess proliferative growth, we tested the activity of minoxidil in human keratinocyte cultures grown in 0.1 mM Ca(++). Minoxidil failed to stimulate growth in these assays. At concentrations of 5-10 micrograms per ml, minoxidil showed half-maximal inhibition of both EGF- and placental extract-stimulated thymidine incorporation. Minoxidil also inhibited proliferative growth in the presence or absence of placental extract. Direct measurement of the ability of minoxidil to compete for binding to the EGF receptor indicated that minoxidil probably does not bind to the EGF receptor. Minoxidil was not toxic, as keratinocytes continued to survive and grow, although at a slower rate, in the presence of minoxidil.     

Localization of ABCA12 from Golgi apparatus to lamellar granules in human upper epidermal keratinocytes

ABCA12 is an ATP-binding cassette transporter and is thought to act as a transmembrane lipid transporter. We reported that deleterious ABCA12 mutations cause a disturbance in lamellar granule (LG) lipid transport in the epidermal granular layer keratinocytes, resulting in harlequin ichthyosis, a severe genodermatosis. Detailed localization of ABCA12 in comparison with glucosylceramide and Golgi apparatus markers were studied in order to obtain clues to clarify the function(s) of ABCA12 in human skin. We performed double-labelling immunofluorescent staining using antibodies against ABCA12, glucosylceramide and two Golgi apparatus markers (TGN46 and GM130) in normal human skin and cultured keratinocytes. Immunogold electron microscopy for ABCA12 and glucosylceramide was studied on postembedding and cryoultrathin sections of normal human skin. Confocal laser scanning microscopy demonstrated that ABCA12 and glucosylceramide co-localized in the granular layer keratinocytes as well as in keratinocytes cultured in high Ca2+ conditions through the Golgi apparatus to the cell periphery. Postembedding immunogold electron microscopy revealed that both ABCA12 and glucosylceramide labellings were associated with the LG of the uppermost granular layer keratinocytes. Using cryoultramicrotomy, lamellar structures in the LG were more clearly observed, and ultrastructural localization of ABCA12 and glucosylceramide was better demonstrated to LG in the uppermost granular layer cells. These results indicate that ABCA12 plays an important role in lipid transport from the Golgi apparatus to LG in human granular layer keratinocytes.     

Aminoglycosides suppress tRNA processing in human epidermal keratinocytes in vitro

The ever-growing resistance of pathogens to antibiotics and the lack of potent antibacterial drugs constitute major problems in the treatment of infectious diseases. Thus, the better understanding of the mode of action of antibiotics at the molecular level is of essential importance. Accumulating evidence points towards RNA as being a crucial target of antibacterial and antiviral drugs. Interestingly, aminoglycosides, one of the most important families of antibiotics, apart from their inhibitory effect on ribosome function, reportedly interfere with various RNA molecules and in vitro suppress the proliferation of human keratinocytes. In this study we investigated the effect of the aminoglycosides neomycin B, paromomycin, tobramycin and gentamycin on ribonuclease P activity from normal human epidermal keratinocytes. All aminoglycosides tested revealed a dose-dependent inhibition of tRNA maturation, which was reduced by increasing Mg(2+) ion concentrations, indicating competition of the cationic aminoglycosides with magnesium ions required for catalysis. Our in vitro findings suggest that the inhibitory effects of aminoglycosides on tRNA processing may be implicated in the mechanisms of their antiproliferative action on human epidermal keratinocytes.     

Minocycline modulation of alpha-MSH production by keratinocytes in vitro.

The anti-inflammatory mechanisms of minocycline, an antibiotic used in the treatment of the inflammatory component of acne, are only partially understood. In addition to inflammation due to cytokines (IL-1, IL-6, TNF-alpha, etc.), recent studies have shown that neuropeptide-mediated neurogenic inflammation may play an important role in cutaneous inflammation. The purpose of this study was to investigate minocycline-induced modulation of cutaneous production of alpha-melanocyte-stimulating hormone (alpha-MSH), a neuropeptide with known anti-inflammatory activity. Two different skin models were used: explants of inflammatory skin and reconstituted skin, both incubated with minocycline at different concentrations and for different time periods. Epidermal production of alpha-MSH, as evaluated by immunofluorescence and immunoperoxidase techniques, showed increased expression in both models. This neuropeptide, which has an anti-inflammatory activity (notably through production of IL-10, antagonism of IL-1 and inhibition of the chemotaxis of polymorphonuclear leukocytes), thus plays a role in the anti-inflammatory action of minocycline.     

Cytotoxic effects of antimicrobial photodynamic therapy on keratinocytes in vitro

BACKGROUND: Previous work has shown that cutaneous microbial species associated with skin conditions of microbial aetiology are susceptible to killing by photodynamic therapy (PDT) using visible light and methylene blue. Antimicrobial PDT (APDT) in vivo would require a therapeutic regimen where bacteria could be killed without damaging adjacent tissue. OBJECTIVES: To study keratinocyte killing in vitro using APDT. METHODS: We used a combination of methylene blue (100 microg mL(-1)) and visible light (42 mW cm(-2)), previously used for microbial killing, to study cytotoxic effects on keratinocytes. Kill rates and subsequent D-values were determined against a human keratinocyte cell line (H103) using trypan blue and neutral red dye viability tests. RESULTS: The kill rates for keratinocytes were exponential over the 90- and 180-min period of the experiment for neutral red and trypan blue, respectively. The corresponding D-values were shown to be 198 and 205 min using trypan blue exclusion and neutral red uptake viability tests, respectively. CONCLUSIONS: The kill rates for keratinocytes were 18-200-fold slower than those previously determined for cutaneous microbial species, suggesting that in vivo, APDT sufficient to reduce microbes by seven log cycles would have little cytotoxic effect on keratinocytes. This approach may offer a safe alternative to conventional antimicrobial treatment.     

An in vitro outgrowth culture system for normal human keratinocytes

BACKGROUND: Normal human epidermal keratinocytes usually proliferate in low-calcium and differentiate in high-calcium without a feeder layer, but they stop proliferating and differentiate at confluency even in low-calcium, serum-free medium. OBJECTIVE: We speculated that this contact inhibition would be mediated in part by mechanical tension. To prove this, we created a new assay system. METHODS: A 10 mm diameter cloning ring was put on the center of a 60 mm dish coated with type I collagen. Keratinocytes were plated in the ring and incubated for 4h, then we had a circular epidermal monolayer sheet. We changed the mechanical tension by removing the ring and measured the diameter of the sheet under various conditions. RESULTS: When we used keratinocyte-serum free medium (SFM) whose calcium concentration is below 0.1 mM as a medium, the keratinocytes in the perimeter migrated individually, and the keratinocytes in the center portion started differentiation. However, when we added calcium chloride to SFM (final concentration more than 0.5 mM), keratinocytes at the periphery showed marked lamellipodia without losing contact with the surrounding cells. These keratinocytes showed coordinate sheet-like outgrowth as a whole even in high concentrations of calcium. CONCLUSION: These results suggest that other than calcium concentration, change of the mechanical tension would be one of the factors that mediate proliferation or differentiation of keratinocytes and that this new assay can be useful in analyzing proliferation, differentiation, and migration of keratinocytes.     

Human keratinocytes in vitro have receptors for leukotriene B4

We have demonstrated a high-affinity binding site for leukotriene B4 (LTB4) on human epidermal keratinocytes in vitro. In substrate saturation studies, one population of binding sites with a dissociation constant (Kd) of 1.03 +/- 0.3 nM and a maximal binding capacity (Bmax) of 148.2 +/- 45.3 fmol/mg protein could be demonstrated. On average 5,500 binding sites were found on individual keratinocytes in culture. The affinity constant of this binding site correlates well with previous reports on the proliferative effect of LTB4 on keratinocytes in vitro. These findings confirm that LTB4 may in part be responsible for epidermal hyperproliferation in inflammatory skin diseases.     

The effect of cyclosporin on human epidermal keratinocytes in vitro

Cyclosporin A has been shown to be effective in the treatment of severe, recalcitrant psoriasis, but it is uncertain whether the mode of action is primarily by immune suppression or by other mechanisms. Cyclosporin-dependent growth-inhibition has recently been demonstrated in vitro using several non-human and transformed epithelial cell lines. In this study the effect of cyclosporin on human epidermal keratinocytes and skin fibroblasts was investigated. Secondary cultures of human epidermal keratinocytes were grown on collagen-coated dishes in the presence of increasing concentrations of cyclosporin. Inhibition of growth was observed at 6-8 microM. An almost identical dose-response curve was obtained for the cytotoxic drug, cis-platin. Short-term exposure (I h) to cyclosporin did not have any effect on epidermal cell growth, suggesting that direct membrane-related effects were not involved. Analysis of cellular proteins by SDS-PAGE indicated no effect of continuous cyclosporin exposure on in vitro differentiation. The observation that human epidermal keratinocyte growth is inhibited by cyclosporin suggests that a topical form of therapy for psoriasis may be an effective alternative to oral treatment.     

Thymidine salvage changes with differentiation in human keratinocytes in vitro.

We compared the capacity of proliferating and differentiating keratinocytes to salvage and catabolize extracellular thymidine. Both populations of cells catabolized thymidine to thymine and possessed thymidine phosphorylase activity. As keratinocytes differentiate, thymidine phosphorylase activity ultimately increased twofold. In contrast, proliferating and differentiating keratinocytes differed markedly in their capacity to salvage extracellular thymidine. Proliferating keratinocytes readily salvaged extracellular thymidine to form nucleotides, whereas differentiating cells rapidly lost this capacity. The inability of differentiating cells to form nucleotides from thymidine was not attributed to reduced availability of thymidine due to catabolism but rather was the result of the rapid loss of thymidine kinase activity. As keratinocytes differentiate in suspension culture, they lose 41% of thymidine kinase activity in 8 h and over 90% of activity in 12 h. Our data indicate that loss of capacity to salvage extracellular thymidine for synthesis of nucleotides closely parallels the onset of differentiation in keratinocytes.     

E‐FABP induces differentiation in normal human keratinocytes and modulates the differentiation process in psoriatic keratinocytes in vitro

Epidermal fatty acid‐binding protein (E‐FABP) is a lipid carrier, originally discovered in human epidermis. We show that E‐FABP is almost exclusively expressed in postmitotic (PM) keratinocytes, corresponding to its localization in the highest suprabasal layers, while it is barely expressed in keratinocyte stem cells (KSC) and transit amplifying (TA) keratinocytes. Transfection of normal human keratinocytes with recombinant (r) E‐FABP induces overexpression of K10 and involucrin. On the other hand, E‐FABP inhibition by siRNA downregulates K10 and involucrin expression in normal keratinocytes through NF‐κB and JNK signalling pathways. E‐FABP is highly expressed in psoriatic epidermis, and it is mainly localized in stratum spinosum. Psoriatic PM keratinocytes overexpress E‐FABP as compared to the same population in normal epidermis. E‐FABP inhibition in psoriatic keratinocytes markedly reduces differentiation, while it upregulates psoriatic markers such as survivin and K16. However, under high‐calcium conditions, E‐FABP silencing downregulates K10 and involucrin, while survivin and K16 expression is completely abolished. These data strongly indicate that E‐FABP plays an important role in keratinocyte differentiation. Moreover, E‐FABP modulates differentiation in psoriatic keratinocytes.     

Adherence of dermatophyte microconidia and arthroconidia to human keratinocytes in vitro

The early interaction, adherence, between dermatophyte conidia and human keratinocytes has been studied in vitro. Two spore forms were used: microconidia and arthroconidia produced in vitro. The adherence of spores from three dermatophyte species, Trichophyton rubrum, T interdigitale, and T quinckeanum, was investigated using keratinocyte suspensions from different skin sites. Time-dependent adherence was demonstrated for all fungi studied with maximum adherence occurring between 3 and 4 h. There were no significant differences in adherence rates between the organisms studied. An order of affinity was established between keratinocytes from different sites and significant differences were demonstrated in adherence of microconidia to skin cells derived from sole versus knee. No differences in adherence rates were demonstrated in atopics versus patients with chronic dermatophytosis and normals. Adherence was inhibited, but not abolished, by subinhibitory concentrations of ketoconazole, itraconazole, and griseofulvin. The interaction between microconidia, arthroconidia, and keratinocytes was verified with scanning and transmission electron microscopy.     

In vitro cytotoxicity of antimicrobial agents to human keratinocytes

Backround The degree of bacterial contamination in the wound bed plays a key role in determining the level of “take” of keratinoeyte grafts in burn patients. This study was done to evaluate the best suited antimicrobial agent for topical application in relation to antimicrobial activity and cytotoxic effect on graft cells. Objective The cytotoxicity of 7 antibacterial agents (gentamicin sulphate, ciprofloxacin hydrochioride, bacitracin, polymyxin B sulphate, diethanolamin salt of fusidic acid, teicoplanin, vancomycin hydrochtoride), one anlifungal substance (amphotcricin B sodium deoxycholate) and one antiseptic agent (povidone iodine) on cultured human keratinocytes, HaCaT keratinocytes and fibroblasts was investigated. Material and methods The effects of these agents were tested using two separate assays - the neutral red assay and the Bradford protein assay. In order to assess toxicity of the agent, the obtained midpoint cytotoxicity (MC50) was compared to the respective minimal inhibitory concentration (MIC) for receiving the cytotoxic potential in microbiologically effective doses. Results Antibiotics effective against Staphylococcus aureus with a small potential of toxicity are Bacitracin, Fusidic acid, Vancomycin and Tcicoplanin. Gentamicin and Ciprofloxacin are effective against both Staphylococcus aureus and Pseudomonas aeruginosa with a low potential of toxicity. Conclusion The use of antimicrobial agents with a midpoint cytotoxicity (MC50) higher than the minimal inhibitory concentration (MIC) by at least a factor of 100 might reduce the risk of toxic injury to graft cells.