Keratinocytes as depository of ammonium-inducible glutamine synthetase: age- and anatomy-dependent distribution in human and rat skin.

In inner organs, glutamine contributes to proliferation, detoxification and establishment of a mechanical barrier, i.e., functions essential for skin, as well. However, the age-dependent and regional peculiarities of distribution of glutamine synthetase (GS), an enzyme responsible for generation of glutamine, and factors regulating its enzymatic activity in mammalian skin remain undisclosed. To explore this, GS localization was investigated using immunohistochemistry and double-labeling of young and adult human and rat skin sections as well as skin cells in culture. In human and rat skin GS was almost completely co-localized with astrocyte-specific proteins (e.g. GFAP). While GS staining was pronounced in all layers of the epidermis of young human skin, staining was reduced and more differentiated among different layers with age. In stratum basale and in stratum spinosum GS was co-localized with the adherens junction component beta-catenin. Inhibition of, glycogen synthase kinase 3beta in cultured keratinocytes and HaCaT cells, however, did not support a direct role of beta-catenin in regulation of GS. Enzymatic and reverse transcriptase polymerase chain reaction studies revealed an unusual mode of regulation of this enzyme in keratinocytes, i.e., GS activity, but not expression, was enhanced about 8-10 fold when the cells were exposed to ammonium ions. Prominent posttranscriptional up-regulation of GS activity in keratinocytes by ammonium ions in conjunction with widespread distribution of GS immunoreactivity throughout the epidermis allows considering the skin as a large reservoir of latent GS. Such a depository of glutamine-generating enzyme seems essential for continuous renewal of epidermal permeability barrier and during pathological processes accompanied by hyperammonemia.

Enrichment of human beta 1 bri/alpha 6 bri/CD71 dim keratinocytes after culture in defined media.

Stem cell-like keratinocytes are responsible for the high regenerative potential of the skin. For clinical applications using keratinocytes in artificial skin constructs, it is suitable to work with serum-free medium under defined conditions. This is also true for the preceding expansion of the stem cell-like keratinocyte population. Therefore, we analyzed the effect of a serum-free medium on the population distribution in comparison to an established serum-containing standard medium for keratinocyte culture. We quantified the freshly isolated as well as cultured primary human keratinocytes by their expression of the beta(1) integrin (CD29) in combination with the expression of the alpha(6) integrin (CD49f) and the transferrin receptor (CD71) by flow cytometric methods. We were able to show that cultivation with serum-free medium induces a switch of the cell population to higher expression of the beta(1) integrin. In addition, the proportion of the alpha(6)(bri)/ CD71(dim)-expressing keratinocyte cell population was enhanced about 35.4 +/- 6.56% after cultivation with serum-free medium. Culture in serum-containing medium increased this proportion of the keratinocyte cell population only about 17.3 +/- 8.06%, when compared to the alpha(6)(bri)/ CD71(dim)-expressing keratinocyte cell population measured directly after isolation. Our data show that the applied culture conditions already have an enormous impact on the development of a stem cell-like phenotype of keratinocytes. This work demonstrates that the serum-free medium significantly increases the proportion of beta(1)(bri)/alpha(6)(bri)/CD71(dim)-expressing keratinocytes. In conclusion, these findings implicate new applications in keratinocyte stem cell research and regenerative medicine.

Effect of fatty acids on expression of endothelial leukocyte adhesion molecules.

BACKGROUND: With respect to linoleic acid both beneficial and proatherogenic effects have been described. However, the effect on expression of cell adhesion molecules on human coronary artery endothelial cells (HCAEC) is not yet established. The aim of the experiments was to evaluate the influence of linoleic acid in comparison with palmitic acid regarding the cytokine-induced expression of endothelial leukocyte adhesion molecules (intercellular cell adhesion molecule-1 ICAM-1, vascular cell adhesion molecule-1 VCAM-1, E-selectin). METHODS: HCAEC were cultured in microvascular endothelial cell growth medium. In the experiments, the cells were preincubated with linoleic acid and palmitic acid, respectively (10 micro mol/l, 2 days) or under control conditions, after which interleukin- 1alpha (IL-1alpha, 10 ng/ml in the test medium) was added for 1 day. The monoclonal antibodies used were fluorescein isothiocyanate (FITC)- labeled anti-ICAM-1, FITC-labeled anti-VCAM-1, and FITC-labeled anti-E-selectin. Expression was analyzed by flow cytofluorimetry. Next, to examine the effects of fatty acids on adhesion of monocytes to endothelial cells, adhesion experiments with the monocytic U 937 cell line were performed. RESULTS AND CONCLUSIONS: IL-1alpha increased ICAM-1,VCAM-1, and E-selectin expression compared to controls. Incubation with IL-1alpha together with linoleic acid reduced the expression of ICAM-1 and VCAM-1 in contrast to palmitic acid. Furthermore, in the presence of linoleic acid a tendency of diminished adhesion of monocytes is seen. The results indicate that a reduced expression of cell adhesion molecules may be relevant to the antiatherogenic effects of linoleic acid. This is in contrast to the properties of palmitic acid.