Guinea Pig Keratinocytes Research Articles

The effect of orally-administered retinoid on lectin binding in guinea pig keratinocytes.

Retinoids influence proliferation and differentiation of epithelial cells. The present paper was designed to examine the change of lectin binding in isolated keratinocytes from retinoid-treated guinea pigs. Aromatic retinoid, etretinate, in peanut oil (5 mg/kg/day) was given orally for a period of 14 days. Skin samples were obtained from animals on days 1, 3, 7, and 14 after beginning the administration. Free keratinocytes were obtained by treatment with EDTA and trypsin. They were separated into 3 fractions by centrifugation on a continuous colloidal silica (Percoll) density gradient. The cells in each fraction were stained by biotinyl lectins and avidin-FITC, and the fluorescence intensity was measured by cytofluorometry. The lectins used were Concanavalin A (Con A), soybean agglutinin (SBA), peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA), and Ricinus communis agglutinin (RCA). The PNA-binding was diminished in both the intermediate and lower density fractions at 7 and 14 days. The binding of SBA was reduced in all fractions from 1 to 7 days. With Con A, RCA and DBA, no changes in intensity were found.

Surface Charge of Fractionated Guinea Pig Keratinocytes Measured by Free-flow Cell Electrophoresis.

Keratinocytes differentiate from basal cells to spinous, granular, and horny layer cells. It is known that alterations in the surface charge of cell membranes in most cases reflect the processes of differentiation. By using a continuous colloidal silica (Percoll) density gradient, keratinocytes may be separated into three fractions which correspond to their arrangement in vivo. Using a free-flow cell electrophoretic technique, we measured the electrophoretic mobility of guinea pig keratinocytes. Electrophoretic mobility histograms of basal and granular cells showed slow and fast monophasic patterns, respectively. In spinous cells, a biphasic pattern of slow and fast electrophoretic mobility was present. The electrophoretic mobility level of guinea pig keratinocytes was slightly reduced with neuraminidase digestion. Those of human red blood cells and lymphocytes, however, were markedly decreased. These results indicate that membrane charge density is lower in basal cells and higher in granular cells and that the membrane charge density of guinea pig keratinocytes involves not only neuraminic acid residues but also other substance(s). Our results illustrate the alterations of cell membrane charge properties during epidermal cell differentiation.

The inhibition of EGF-dependent proliferation of keratinocytes by tyrphostin tyrosine kinase blockers.

Protein tyrosine kinase blockers of the tyrphostin family inhibited the EGF-dependent proliferation of human and guinea pig keratinocytes grown in culture and induced their growth arrest. These blockers also significantly inhibited the growth of epidermal keratinocytes, but not of dermal cells, in whole skin organ culture from both guinea pig and human origin. The antiproliferative activity of these tyrphostins correlated quantitatively with their potency as inhibitors of EGF receptor autophosphorylation and the EGF-dependent protein phosphorylation of intracellular target proteins in the keratinocyte. Furthermore, no significant cell cytotoxicity or reduction in serine and threonine phosphorylation of many intracellular polypeptides were observed upon incubation of the cells with tyrphostins like AG213. The complete growth arrest induced by the tyrphostins is fully reversible and upon their removal the keratinocytes resumed their growth with the original growth rate. Because of the nontoxic nature of these compounds and their growth-arresting properties, we suggest their use as agents to treat hyperproliferative conditions of human skin.

Alterations in membrane fluidity during keratinocyte differentiation measured by fluorescence polarization

The epidermis shows a distinctive pattern of differentiation wherein keratinocytes proliferate in the basal cell layer and mature into spinous and granular cells. Using a discontinuous density-gradient centrifugation method, guinea-pig keratinocytes were separated into high (HDF), intermediate (IDF), and low (LDF) density fractions. Morphological and flow cytometrical observations demonstrated that HDF, IDF, and LDF were basal, spinous, and granular cell-rich fractions, respectively. Membrane fluidity of the fractionated keratinocytes was measured by diphenylhexatriene fluorescence polarization. Polarization (p)-value of keratinocytes was negatively correlated with temperature. At each temperature, HDF cells showed a lower p-value than IDF or HDF cells except at 40 degrees C. Since a low p-value indicates a high degree of Brownian motion, membrane fluidity is higher in basal cells and lower in spinous and granular cells. Our results indicate that membrane fluidity of guinea-pig keratinocytes decreases during their maturation.

Modulation of the Binding and Endocytosis of Concanavalin A by Guinea Pig Keratinocytes: Reversible Antagonistic Effects of Cholesterol and Phospholipid-Liposomes

Alteration of guinea pig keratinocyte membrane microviscosities (eta) by liposomes of varying composition was determined by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Measurements performed either with whole cell suspensions or Percoll-separated cell subpopulations, indicate a similar membrane microviscosity (eta = 3.37 poise +/- 10%) compared to those microviscosities reported for other cell types. Our findings show that treatment of guinea pig keratinocytes with liposomes composed of phospholipids results in a decreased membrane microviscosity (1.95 poise), whereas treatment of the cells with an emulsion of cholesterol hemisuccinate, or liposomes composed of cerebrosides, causes an increase in membrane microviscosity (3.85 poise and 5.55 poise +/- 10%, respectively). Changes in membrane fluidity had no significant effect on cell viability. A reduced membrane microviscosity resulted in a decrease in the binding of Concanavalin A to keratinocytes, whereas an increased microviscosity resulted in an increased binding of Concanavalin A. Furthermore, endocytosis of Concanavalin A bound to keratinocytes plasma membranes was not significantly affected by a reduced membrane microviscosity, whereas an increased membrane microviscosity completely blocked the endocytosis of Concanavalin A. Another novel observation was that membranes "fluidified" by phospholipid liposomes could be "rigidified" by treatment with cholesterol hemisuccinate and vice versa. Moreover, these alternate changes in membrane microviscosity resulted in simultaneous alternate changes in the binding of Concanavalin A to the keratinocyte surface.

Binding modes of IgG from Pemphigus autoimmune sera onto guinea pig keratinocytes and the fate of bound IgGs

Pemphigus is an intraepidermal autoimmune blistering disease of humans caused by circulating IgGs. We have investigated the binding mode and the fate of bound antibodies from Pemphigus sera (P-IgG) on guinea pig keratinocytes in suspension in order to find clues to the loss of cell adhesion in vivo (acantholysis). Flow cytometry, following indirect immunofluorescent labeling of the keratinocytes, and dead cells' staining with ethidium bromide, demonstrated the specific surface binding of P-IgG onto living keratinocytes only. This was shown with several Pemphigus sera or purified P-IgG. This technique, used with various Pemphigus sera, showed that the specific binding is increased when the serum titer is higher, and "Km" values for P-IgG were roughly and inversely correlated to the titers. Upon saturation the same average number of Pemphigus IgG sites per cell were found for the sera of different patients. Analysis of the specific binding of [125I]-P-IgG onto Percoll-separated (living) keratinocytes showed the existence of two classes of sites: 2 x 10(6) sites/cell high-affinity sites (Kd = 1.5 x 10(-6) M total IgG) and 25 x 10(6) sites/cell low-affinity sites (Kd = 6 x 10(-5) M total IgG). Cell sorting and flow cytometry of individual cells allowed us to correlate the light-scattering signal, the RNA content, the size and morphology, and the P-IgG binding to the cells. The results indicated that P-IgG binding is homogeneous within the living keratinocytes and increases with cell size (cell maturity). Cell-sorter analysis of cells with membrane-bound P-IgG, coupled to direct determination of P-IgG released in the medium, revealed the fate of bound P-IgG: 40-60% of the P-IgGs were released in the medium within 30 minutes at 37 degrees C. This was accompanied and followed by a much slower, metabolic energy-dependent, internalization process of the membrane-bound P-IgG. The internalization has been confirmed by electron microscopy of bound P-IgG labeled with protein A-gold. Internalized IgGs were seen in the cells in coated membranous vesicles and other endocytic compartments. Similar behavior was also observed with two other membrane ligands: i.e., concanavalin A and multispecific rabbit "antisurface" antibodies.

Cytofluorometric study on lectin binding of isolated guinea pig keratinocytes

Lectin binding was cytofluorometrically measured on fractionated keratinocytes of guinea pig. Free keratinocytes were obtained by treatment of EDTA and trypsin. After the treatment, they were separated into 3 fractions by centrifugation on a continuous colloidal silica (Percoll) density gradient. Cells in each fraction were stained by biotinyl lectins and avidin-FITC, and fluorescence intensity was measured by cytofluorometry. Results obtained indicate that little cell surface glycoconjugate is lost during the preparation of free keratinocytes.

Primary mitochondrial activity of gossypol in yeast and mammalian cells

Gossypol showed primary antimitochondrial activity in yeast cells in that the drug (1) inhibited growth of cells utilizing mitochondrial substrates as carbon and energy sources, and (2) selectively inhibited mitochondrial protein synthesis. Primary antimitochondrial activity was demonstrated in guinea-pig keratinocytes (GPK) by early arrest of growth and loss of viability in medium with glutamine (a mitochondrial substrate) as carbon and energy source compared with cells utilizing glucose. Gossypol depressed oxygen uptake directly in respiring cells. Gossypol interacted with the known antimitochondrial agents ethidium bromide and 5-fluorouracil (FU), potentiating the activity of FU but reversing that of ethidium bromide in yeast and GPK. Also, the activity of the mitochondrial inhibitor oligomycin was reversed by the presence of gossypol in yeast cells but not tested in GPK. The uptake and retention of the mitochondria-specific dye rhodamine 123 were much depressed by gossypol in GPK. Gossypol showed little or no inhibitory effects in yeast or GPK in the presence of ethanol (0.2–0.5%). The drug was not mutagenic with respect to the yeast mitochondrial system. It was tentatively suggested that mitochondrial perturbation could explain the antifertility effect of gossypol if it is assumed that mitochondria have a special role to play in spermatogenesis and sperm motility, making these tissues more sensitive to mitochondrial inhibitors than somatic cells.

Change of intracellular fluidity during keratinocyte differentiation measured by fluorescence polarization

The fluorescence polarization method was applied to measure the intracellular fluidity of fractionated guinea pig keratinocytes. Guinea pig epidermal cell suspension was obtained by treatment with EDTA and trypsin, and was separated into high, intermediate, and low density fractions using Percoll density gradient centrifugation. Morphological observation and cytofluorometric analysis of DNA content in the fractionated epidermal cells showed that the high, intermediate, and low density fractions were basal, spinous, and granular cell-rich fractions, respectively. Intracellular fluorescence polarization of each fraction was determined by a polarization spectrofluorometer (Hitachi MPF-4, prototype) with fluorescein diacetate. The P-values were calculated for high, intermediate, and low density fractions as 0.192 +/- 0.021, 0.172 +/- 0.019, and 0.147 +/- 0.012, respectively. Since low P-values indicate a high degree of fluidity, the results indicate that intracellular fluidity of keratinocytes is lower in basal cells and higher in granular cells. Dye-binding experiments showed that fluorescein-binding proteins were not detected in the soluble fraction of the epidermal cells. The present findings suggest that intracellular fluidity of the guinea pig keratinocyte increases during the process of its differentiation.

Cytofluorometric study of thiol and disulphide groups in guinea pig epidermis

Content of thiol and disulphide groups was cytofluorometrically measured on guinea pig keratinocytes, which were separated into three fractions. Free keratinocytes were obtained by the treatment of EDTA and trypsin. After the treatment keratinocytes were separated into three fractions by centrifugation on a continuous colloidal silica (Percoll) density gradient. Cells in each fraction were stained with N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide, and measured by cytofluorometry. Thiol concentration was low in basal cells, and higher in both spinous and granular cells. Disulphide group content was low and fairly constant in them. Human peripheral lymphocytes and guinea pig spleen lymphocytes were used to examine the influence of EDTA-trypsin treatment. No remarkable loss of either thiol or disulphide groups was found in them after the treatment.

Distribution patterns of cytoplasmic microtubules in epidermal keratinocytes.

The distribution of cytoplasmic microtubules in cultured guinea-pig keratinocytes was investigated using immunofluorescence (IF) microscopy with monospecific anti-tubulin antibodies and electron microscopy (EM). In culture, adherent cells displayed networks of thin fluorescent fibres, while a homogeneous and/or granular cytoplasmic IF was shown in the cells of upper layers as well as in trypsinized cells. By EM many microtubules were shown in adherent cells but there were fewer or none in the upper layers. An increase in calcium ion (Ca2+) concentration and the addition of an ionophore (X537A) to the culture medium caused disassembly of microtubules. This effect was cancelled by a calmodulin inhibitor. Cryostat sections of normal human and guinea-pig epidermis stained with anti-tubulin antibodies showed a homogeneous and/or granular cytoplasmic IF from basal to granular layers but no detectable IF was seen in the horny layer. These results suggest that keratinocytes contain a cellular pool of tubulin in various states of polymerization and that microtubule disassembly may occur during differentiation, probably being regulated by Ca2+-calmodulin complexes.

Retinoids increase the response of guinea pig but not human keratinocytes to agonists of adenylate cyclase in vitro.

Primary cultures of adult human and guinea-pig keratinocytes were treated with various retinoids for up to 7 days. The cells were exposed to 3H-adenine and then challenged with several agonists of adenylate cyclase. 3H-labeled adenine nucleotides were extracted and 3H-cAMP purified chromatographically. All retinoids increased the formation of 3H-ATP from 3H-adenine and the conversion of 3H-ATP to 3H-cAMP by guinea-pig cells, the extent being dependent on the particular retinoid and agonist used. Human cells were relatively sensitive to isoproterenol and not to prostaglandins or histamine, but their response was little affected by retinoids. Guinea-pig cells were most sensitive to prostaglandins. The proliferogenic effects of retinoids on the latter cells may be mediated by increased sensitivity to agonists leading to generation of cAMP.

Retinoic Acid Delays the Terminal Differentiation of Keratinocytes in Suspension Culture

The effect of retinoic acid (RA) on the terminal differentiation of guinea pig keratinocytes maintained in suspension culture was studied. Keratinocytes obtained from trypsinized guinea pig skin were suspended in medium containing 20% calf serum and 1.2% methyl cellulose. RA, which was added at the beginning of culture, delayed differentiation as judged by a decrease in the percent of cells that developed disulfide cross-linked keratin (sodium dodecyl sulfate insoluble cells) and cornified envelopes (sodium dodecyl sulfate and 2-mercaptoethanol insoluble cells). RA inhibited differentiation maximally at 5 microgram/ml on day 3 of 5 day culture; concentrations as low as .005 microgram/ml were also inhibitory. Because the disulfide cross-linking of keratin and the formation of cornified envelopes are thought to occur when the cell membrane becomes permeable, we determined whether RA inhibited these processes by stabilizing the cell membrane. Two agents (ionophore X537A and Triton X-100) which permeate cell membranes rapidly reversed the inhibitory effect of RA on cornified envelope formation. In addition, when cultured with RA, the percent of cells which became permeable to trypan blue was reduced, also suggesting that RA acts on the cell membrane. These studies show that RA can inhibit keratinocyte differentiation by stabilizing the cell membrane thereby delaying transition from a living epidermal cell to a dead cornified cell.

Dynamic redistribution of concanavalin A binding sites on isolated guinea pig keratinocytes.

Modifications in the distribution of the binding sites for concanavalin A (Con A) were studied on trypsin isolated living guinea pig keratinocytes. Fluorescein-labelled Con A was used and the in vitro procedure has included short-term cultures, experiments at 37 degrees C and 4 degrees C, the study of colchicine and vincaleucoblastine effects. It was possible to induce different patterns of staining corresponding to distinct redistribution of Con A binding sites; the distinct redistribution was correlated to the effects of Con A, colchicine and vincaleucoblastine. These findings demonstrated that the system used was appropriate to the study of some dynamic events on the keratinocytes membranes.

EPIDERMAL CELL MEMBRANE AS THE ORIGIN OF PEMPHIGUS ANTIGEN

ABSTRACTThe substance bound to the pemphigus antibody (P‐Ab), pemphigus antigen, was removed from the surface of the keratinocytes of both human and guinea pigs with high concentrations of trypsin. When keratinocytes were cultured for more than 24 hours, the antigen reappeared on the surface. Furthermore, P‐Ab was recovered from the P‐Ab treated cultured cells by acid treatment.Cell membranes were prepared from human keratinocytes in order to determine whether or not the antigen was situated on their surface. The antigen was also detected on the surface of the cell membrane. As the membrane was found to be able to absorb P‐Ab, the antigen was extracted from the membrane by papain digestion and P‐Ab coated Sepharose column chromatography. The eluate from the column was also able to block P‐Ab activity. At the same time, the culture supernatant of both human and guinea pig keratinocytes was examined for its ability to block P‐Ab activity. The culture supernatant showed a slight ability to block P‐Ab. The origin and nature of the antigen was discussed.

Epidermal Lysosomes and Ultraviolet Light

Secondary lysosomes of guinea-pig keratinocytes were labeled, in vivo, with an electron microscopic marker (Thorotrast) that can he identified both within stable lysosomes and, after lysosome lysis, in extralysosomal locations. Immediately after irradiation with 10 and 20 minimal erythemal doses delivered from an ultraviolet light source in vivo, the large majority of epidermal lysosomes was intact but occasional lysosomes exhibited ruptured membranes and a spilling at tracer into the cytoplasm. Morphologic signs of early UV damage to the cytoplasm were present immediately alter irradiation but these occurred also in the absence of lysosome Lysis. Unirradiated controls showed no cytoplasmic damage but the same amount of lysosome lysis as the irradiated animals. With regard to lysosomes and lysosome lysis there was no difference between nonirradiated and irradiated animals biopsied immediately after UV-exposure. Two, 6, and 12 hr after irradiation the irradiated animals exhibited a greater degree of lysosome lysis than the controls. It is concluded that lysosome lysis accompanies the UV reaction of the epidermis but the present study provides no evidence that it represents an initial pathogenic event.

Keratinization in dispersed cell cultures of adult guinea-pig ear skin.

SUMMARY A study of the behaviour of dispersed cell cultures of adult guinea-pig keratinocytes has been made using time-lapse cinephotomicrography and electron microscopy. Attention has been directed mainly to the formation of aggregates and the ultrastructure of the cells involved. At the periphery of the clumps and at the edges of spaces within the clumps the cells were organized in a layered system and fully keratinized cells occurred only within a three dimensional system. Some partial keratinization and some autolysis was also noted.

Experimental pigment donation in vivo

The uptake of mouse melanosomes by guinea pig keratinocytes was induced experimentally in vivo without the action of melanocytes. The uptake mechanisms were discriminatory as small melanosomes, isolated from B16 mouse melanomas, were taken up in groups, maintaining membrane-bound aggregates within the cytoplasm of the recipient epidermal cells. By contrast, large melanosomes, prepared from the hair bulbs of C57 BI mice, were incorporated as singles into keratinocytes and were dispersed singly within their cytoplasm. The intracytoplasmic distribution patterns of melanosomes obtained were identical with the distribution patterns which occur normally in keratinocytes of different species and races; the appearance of the single melanosomes and their envelopes as well as that of the aggregated melanosomes and their surrounding membranes did not differ from the single and aggregated melanosomes occurring under normal, physiologic conditions. Since these results were obtained in a system which bypasses the melanocyte, they suggest that the distribution patterns of melanosomes within keratinocytes, i.e., aggregation or dispersion, are independent of the actions of the pigment cell and do not represent different stages of a single, uniform process. They appear to be determined by the mechanisms by which melanosomes are taken up into keratinocytes, and these in turn are influenced by the sizes of the individual melanosomes.

Control of Keratinocyte Division In Vitro

Aqueous extracts of newborn rat epidermis inhibit mitosis of cultivated keratinocytes obtained from guinea pigs and humans. The mitotic activity of cultivated human keratinocytes and fibroblasts obtained from the uninvolved skin of subjects with psoriasis is similar to the mitotic activity of these cells obtained from involved psoriatic skin. This observation is consistent with the model of repression of mitotic activity in normal skin and a derepression of mitotic activity in psoriatic epidermis. Extracts of liver also inhibit mitosis of guinea pig keratinocytes. Cultivated keratinocytes do not exhibit the same specificity of inhibition by epidermal extracts that has been reported for surviving fragments of mouse ear.