Guinea Pig Epidermal Cells Research Articles

Anti-proliferative and cytotoxic activity of surface-associated material from periodontopathogenic bacteria

The easily solubilized surface-associated material from three bacterial species associated with periodontal diseases, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Eikenella corrodens, produced dose-dependent inhibition of thymidine incorporation by human fibroblasts, the human monocytic cell line U937 and guinea pig epidermal cells. In contrast, lipopolysaccharides from A. actinomycetemcomitans and P. gingivalis were either inactive or substantially less active over the dose range tested. One of the constituents of surface-associated material from a ‘non-leucotoxic’ strain of A. actinomycetemcomitans was highly cytotoxic to human peripheral blood polymorphonuclear cells, with 50% killing from less than 1 ng/ml. A constituent of the surface-associated material from P. gingivalis was approximately one log order less active. The lipopolysaccharides from these bacteria were at least three log orders less active in neutrophil killing. These findings add weight to the hypothesis that easily solubilized exopolymers from periodontopathogens play a major part in the pathology of periodontal diseases.

Changes of Electron Spin Resonance Membrane Fluidity in Hexadecane-Induced Hyperproliferative Epidermis

To study some of the biochemical and physical states of membranes associated with hyperproliferation, the effect of topical hexadecane on membrane fluidity in guinea pig epidermis was investigated by electron spin resonance using a 5-doxylstearic acid spin labeling agent. Guinea pig epidermal cells were separated into three regions of keratinocytes by Percoll density gradient centrifugation. Membrane fluidity and Na+, K+-ATPase activity were higher in hyperproliferating epidermal cells than in control. The free cholesterol content and the molar ratio of free cholesterol to phospholipid were found to decrease significantly. Also elevated levels of palmitic acid, stearic acid and omega-3 unsaturated fatty acid derived from phospholipid were observed. Normal differentiation of epidermis was found to be accompanied by a decrease in membrane fluidity, whereas a relatively high membrane fluidity was maintained in the hexadecane-induced hyperproliferation.

Changes of membrane fluidity and Na+,K+-ATPase activity during cellular differentiation in the guinea pig epidermis.

The dynamic properties of plasma membrane in epidermal cells were determined by means of electron spin resonance using two kinds of doxyl stearic acid spin labeling agents: 5-DSA and 12-DSA. 5-DSA and 12-DSA are stearic acid analogues with a nitroxide radical ring at the 5th and 12th carbon positions, and these motions reflect molecular motion of lipid bilayer surrounding the hydrophilic region and the hydrophobic region, respectively. Guinea pig epidermal cells were separated into three regions of keratinocytes by Percoll density gradient centrifugation; the upper, middle, and lower epidermal cells. The order parameter S values for 5-DSA and 12-DSA incorporated into the isolated keratinocytes increased, suggesting a decrease in the plasma membrane fluidity, as cells approached the upper epidermal cell layer. The Na+,K+-ATPase activity as a plasma membrane-bound enzyme was determined in each epidermal cell region, and was found to decrease gradually as the cells approached the upper layer. Accordingly, the differentiation of epidermal cells in the keratinization process was found to be associated with a decrease in plasma membrane fluidity and with a decline of Na+,K+-ATPase activity.

Epidermal cell growth-dependent arylhydrocarbon-hydroxylase (AHH) activity in vitro.

Cytochrome P-450-dependent arylhydrocarbon-hydroxylase (AHH) activity and inducibility by benzanthracene (BA) was measured in cultured guinea pig and human epidermal cells. Basal AHH-activity (AHHb) in guinea pig epidermal cells was much higher than in human epidermal cells. AHHb in guinea pig epidermal cells was directly related to the labeling index and decreased to the original level between the 5th and 7th day of cell culturing. On the other hand, the induction-ratio of AHH reached its maximum level when the number of cells began to rise (proliferation phase) and remained high at day 7 of the cell culture. These results suggest a cell growth dependent activity and inducibility of carcinogen-metabolizing enzymes, such as AHH, in isolated epidermal cells.

Effects of colchicine and cytochalasin B on distribution of concanavalin A receptors in isolated and cultured guinea pig epidermal cells.

Regulation of the distribution of concanavalin A (Con A)/receptor complexes by the cytoskeletal contracture system was studied in guinea pig epidermal cells in suspension and culture using the fluorescence double staining method. After treatment with 100 micrograms/ml of Con A at 37 degrees C for 30 min lectin/receptor complexes were endocytosed by the less-differentiated cells in suspension and by the adherent cells in 1- and 3-day cultures that represent a growing cell fraction. The same treatment resulted in diffuse surface distribution of the complexes in the well-differentiated cells in suspension. Colchicine (10(-5) and 10(-6) M) inhibited internalization of the complexes with resultant diffuse distribution in 60% of the adherent cells in culture. Cytochalasin B (5 and 10 micrograms/ml) not only inhibited endocytosis but promoted formation of surface patchy clumps of the complexes in suspended, less-differentiated cells and cultured adherent cells. The distribution profile was not influenced by these drug treatments in the well-differentiated cells. SDS polyacrylamide gel electrophoresis and autoradiography of 125I-labelled epidermal membranes revealed several Con A-reactive polypeptides common to the cells at various differentiation steps. The progressive decrease in endocytosis and mobility of Con A/receptor complexes was suggested to occur with differentiation. In the germinative cells the distribution of lectin/receptor complexes seemed to be regulated by microfilaments and microtubules.

Phorbol Ester Serves As a Coepibolin in the Spreading of Primary Guinea Pig Epidermal Cells

In earlier studies, it was shown that the human plasma-spreading glycoprotein, epibolin (the 65 kD species of serum-spreading factor or vitronectin), requires a second plasma component, termed coepibolin, in order to support maximal dissociated epidermal cell spreading in tissue culture. Whereas epibolin alone in defined medium supports some cell spreading, the purified plasma coepibolin preparations do not effect spreading in the absence of epibolin. Although not yet entirely purified, coepibolin associates with some plasma fractions but not with others; it is certainly not a property of all proteins, e.g., while bovine serum albumin (BSA) has coepibolin activity, ovalbumin does not. The data presented here show that the phorbol ester, 12-tetra-decanoyl-1-phorbol-13-acetate (TPA) can act as a potent coepibolin and support maximal spreading over a concentration range of 10-100 ng/ml. In the absence of epibolin TPA does not stimulate the spreading of epidermal cells when given alone or in the presence of BSA or ovalbumin. Coepibolin activity appears to associate with tumor-promoting activity in that the phorbol derivative, phorbol-12,13-didecanoate, shows coepibolin activity, while its inactive non-tumor-promoting isomer, phorbol-4 alpha-phorbol-12,13-didecanoate, does not. These data suggest that the proteinaceous plasma-derived cofactor acts in a fashion similar to TPA and that this as yet unexplained mechanism of TPA action is important to the full expression of epibolin and to the early phase of epidermal cell spreading.

Ultraviolet-evoked prostaglandin biosynthesis in varying stages of keratinocyte differentiation in guinea pig skin

Prostaglandin (PG) production by guinea pig epidermal cells was evaluated at various incubation intervals in normal and UV-exposed cultures. Prostaglandins have been implicated as mediators of the early phase of erythema in skin exposed to sunlight or UV-radiation. Using a density gradient centrifugation procedure, the epidermal cells were fractionated according to the various maturation sta s of epidermal keratinocytes: high-density epidermal cells (HDEC) consisting of round, less mature cells; low-density epidermal cells (LDEC) consisting of polygonal keratinized cells; and intermediate-density epidermal cells (IDEC) consisting of both HDEC and LDEC. When cultures of 1 × 10 6 cells were incubated at 37°C in 5% C0 2 the highest concentrations of five PG moieties measured were present in supernatants from the LDEC cultures as compared to those of IDEC or HDEC. Levels of PGF 2α were much higher than the rest, which were found in the order PGF 2α > PGE 2 > PGE 1 > 6-keto-PGF 1α > thromboxane (TX)B 2. UV-irradiation induced increases in all but TXA 2 production. These results identify and quantitate five compounds produced as a result of exaggerated activity of the cyclooxygenase induced by UV-irradiation.

Distribution and mobility of concanavalin A receptors on isolated guinea pig epidermal cells at various stages of differentiation.

Trypsinized guinea pig epidermal cells were separated by velocity sedimentation at unit gravity. Based on the relationship between cell size and both morphological and functional aspects of differentiation, the cells were classified as lower (a diameter less than 12.5 micron), middle (a diameter between 12.5 and 15 micron), and upper (a diameter greater than 15 micron) epidermal cells. Fresh cells exposed to rhodaminated concanavalin A (Con A) were sedimented and reacted with fluoresceinated anti-Con A serum to distinguish cell surface Con A from intracellular lectin. Labeling at 4 degrees C resulted in a uniform surface distribution of Con A irrespective of cell size. After a 1-hr incubation of Con A-labeled cells in lectin-free medium at 37 degrees C, lower epidermal cells and approximately half of middle epidermal cells internalized Con A/receptor complexes by endocytosis while lectin remained diffusely on the remainder of middle epidermal cells and upper epidermal cells. By electron microscopy, ferritin-Con A was clustered on surface areas and invaginations of the plasma membrane before being endocytosed. We concluded that the differentiation of epidermal cells was accompanied by progressive decrease in endocytosis and, most probably, mobility of Con A receptors.

Distribution of anionic surface sites on human melanocytes and human melanoma cells in culture.

With cationized ferritin (CF) as an ultrastructural marker for anionic cell surface sites, cultured guinea pig melanocytes display a uniquely homogeneous labelling pattern and a striking absence of redistribution of marker material. In the present study, we applied the same technique to normal human melanocytes and melanoma cells. Unfixed primary human mixed epidermal cell cultures displayed CF labelling patterns identical to those in guinea pig epidermal cells: on keratinocytes, CF was found in distinct aggregations which, upon prolonged incubation, clustered and were shed. Melanocytes, in contrast, bound CF to the cell surface as a uniform diffuse monolayer. There were no signs of cluster formation or shedding. Melanoma cell cultures were derived from 10 primary (2 lentigo maligna, 1 lentigo maligna melanoma, 4 superficial spreading melanomas, 2 nodular portions of superficial spreading melanomas, 1 nodular melanoma) and from 7 metastatic melanomas (4 cutaneous, 3 lymph node metastases). The CF labelling patterns encountered were heterogeneous. Three out of 10 primary tumors and 5 out of 7 metastases showed alterations of the normal melanocyte labelling pattern: regions of typical CF distribution were irregularly interrupted by stretches of membrane free of marker. In some areas, CF occurred in small globular aggregates. There was considerable heterogeneity of CF labelling patterns in different clones of a given culture. Altered CF binding patterns in melanoma cells appear to be associated with high metastasizing protential of the cell clones and may thus represent an unfavourable prognostic sign.

Subsets of Epidermal Langerhans Cells as Defined by Lectin Binding Profiles

In this study we characterize the cell surface glycoconjugate moieties of strain 2 guinea pig epidermal Langerhans cells (LC) in single cell suspension by using a battery of 17 fluorescent lectins. All LC displayed binding sites for concanavalin A, succinylated concanavalin A, Lens culinaris agglutinin, Pisum sativum agglutinin, wheat germ agglutinin, succinylated wheat germ agglutinin, Griffonia simplicifolia agglutinin I, Ricinus communis agglutinin I, Phaseolus vulgaris E agglutinin, and Phaseolus vulgaris L agglutinin, but failed to bind Sophora japonica agglutinin (SJA), Dolichos biflorus agglutinin (DBA), and Ulex europaeus agglutinin I (UEA I). Neuraminidase pretreatment rendered LC reactive for SJA, but not for DBA and UEA I. The binding profiles of certain lectins point to the existence of LC subpopulations in that Griffonia simplicifolia I-B4 isolectin, peanut agglutinin (PNA), Helix pomatia agglutinin, and soybean agglutinin bound to only 80% (range 70-90%) of Ia-positive epidermal cells; binding sites for these lectins on primarily unreactive Ia-positive cells were unmasked when epidermal cells were treated with neuraminidase prior to lectin labeling. Ultrastructural PNA labeling studies revealed that the vast majority of Birbeck granule-containing LC displayed PNA binding sites, whereas indeterminate cells were consistently PNA-negative. Identification of carbohydrate configurations expressed on LC surfaces by lectin binding may provide a clue for the elucidation of the mechanisms of established LC functions and possibly the discovery of as yet unknown properties of this cell type.

Deacylation of ceramide, triacylglycerol and phospholipids in guinea PIG epidermal cells

The epidermal cells obtained by mild trypsinization of guinea pig skin were first labeled with [ 3H]palmitate. Interestingly, free ceramide was one of the major radioactive products. The radioactivity of ceramide was located exclusively in the N-acyl moiety and palmitate was incorporated as such, without detectable chain modification. Sphingomyelin and glucosylceramide were not significantly labeled in spite of the active synthesis of ceramide. In order to investigate the action of lipolytic enzymes, the labeled cells were chased in a tracer-free medium. Bovine serum albumin (or fetal calf serum) and ionophore A23187 markedly stimulated the deacylation of lipids. The effects of the two stimulators were, however, distinctly different from each other. Bovine serum albumin (or fetal calf serum) caused principally the deacylation of triacylglycerol and ceramide, with little effect on that of phospholipids. In contrast, the effect of the ionophore was confined to phospholipid deacylation. In the presence of the ionophore, phosphatidylcholine and phosphatidylethanolamine were degraded to a similar extent. Palmitate was cleaved from both C-l and C-2 positions of phosphatidylcholine. The results suggest that the lipolytic systems of the epidermal cells can respond to extracellular stimuli and that the action of triacylglycerol lipase and ceramidase is controlled differently from that of phospholipases.

Effect of vitamin A acid on homeostasis and differentiation of epidermal cells cultured on collagen.

Cultured guinea pig epidermal cells were treated with vitamin A acid (VA;2, 5, 10 micrograms/ml) and controlled by autoradiography and histochemical method. Cells attached to the bottom of the plates were counted and their number correlated with the number of cells sloughed off into the medium (A/D ratio). VA reduced the size of multilayered cell colonies, favoured cell spreading and accelerated confluence. It altered the mode of shedding since the cells sloughed off into the medium as isolated cells rather than cell complexes, as in untreated controls. It increased the A/D ratio in early cell cultures and decreased the A/D ratio in late cell culture. The higher the concentration of VA, the earlier and more prominent was the decrease of the A/D ratio. Low concentration of VA (2micrograms/ml) given once at seeding favoured the formation of keratohyalin and cornified envelopes whereas high concentration (5, 10micrograms/ml) given continuously prevented their formation and caused mucoid vacuolar degeneration of single cells. VA inhibited complete keratinization in all cases, but appeared to allow pre-keratinization, particularly at low concentrations. The results indicate that VA controls epidermal homeostasis in vitro by its influence on both proliferation and shedding. Its effect on the markers of differentiation is heterogeneous since it prevented keratinization in every case, but induced keratohyalin formation under certain conditions.

Multiple mechanisms of dissociated epidermal cell spreading.

To test the possibility that epidermal cells use a common basement membrane protein whenever they spread, in vitro experiments were conducted using trypsin-dissociated guinea pig epidermal cells and the following proteins: human serum, bovine serum albumin, serum fibronectin, Type IV collagen, laminin, and epibolin (a recently described serum glycoprotein which supports epidermal cell spreading; Stenn, K.S., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:6907.). When the cells were added to media containing the specific proteins, all the tested proteins, except for serum albumin, supported cell spreading. Added to protein-coated substrates in defined media, the cells spread on fibronectin, epibolin, and laminin-Type IV collagen, but not on albumin or whole serum. In none of these experiments were the results qualitatively affected by the presence of cycloheximide. Antibodies to a specific protein blocked cell spreading on that protein but not on the other active proteins, e.g. whereas antibodies to epibolin blocked cell spreading on epibolin, they did not affect spreading on fibronectin, collagen, or laminin. In a second assay in which the cells were allowed to adhere to tissue culture plastic before the protein-containing medium was added, the cells spread only if the medium contained epibolin. Moreover, under these conditions the spreading activity of whole serum and plasma was neutralized by antiepibolin antibodies. These results support the conclusion that dissociated epidermal cells possess multiple spreading modes which depend, in part, on the proteins of the substrate, proteins of the medium, and the sequence of cell adhesion and protein exposure.

Receptors for Complement on Langerhans Cells

A population of guinea pig epidermal cells, identified as Langerhans cells, formed rosettes with sheep erythrocytes coated with IgM immunoglobulin and C3b (EAC1-3b) but not with intermediates carrying only C1, C14b, or C14b2a. These findings were unusual since most other cells expressing C3b receptors also express C4b receptors. To investigate whether these results were the consequence of a limited number of C4b molecules on the cells, EAC14b cells were prepared with increased amounts of C4b (15 to 250 micrograms on 10(9) cells). EAC14b rosette formation was found with a maximal number when there were 3,000 molecules per cell, a 6-fold excess over that found in previous experiments. One could inhibit this rosette formation by fluid phase C3b as well as by fluid phase C4b, suggesting proximity or possible identity of these 2 receptors.

Establishment of a Cell Line Retaining Langerhans Cell Characteristics

A discontinuous gradient of polyethylene glycol (PEG) with dimethyl sulfoxide (DMSO) was used to induced fusion between guinea pig epidermal cells (GPEC) enriched in Langerhans cells (LC) and cells of a human fibrosarcoma line (T-1080) deficient in hypoxanthine phosphoribosyl transferase (HPRT). The rapidly proliferating carrier T-1080 cells lack plasma membrane ATPase activity, Fc receptors, C3b receptors, guinea pig Ia-antigens, and nonspecific esterase activity, all characteristic of LC, and absent on other guinea pig epidermal cells. GPE/T-1080 hybrid cells were selected in hypoxanthine/aminopterin/thymidine (HAT) medium and by C3b and Fc rosette formation. A resultant continuous cell line was characterized karyotypically, histochemically, ultrastructurally and immunologically.

Complement receptors on guinea pig epidermal Langerhans cells.

Abstract A population of guinea pig epidermal cells possessed the ability to form rosettes with IgM antibody-sensitized sheep erythrocytes coated with purified human C3b (SEAIgMC1-), but not with intermediates carrying C1, C4, or C2. This population of rosette-forming epidermal cells was identified as Langerhans cells (LC) ultrastructurally and by LC depletion and enrichment studies. In addition, these cells were able to bind fluid phase C3b, which inhibited any subsequent SEAIgMC1-3b rosette formation. Rosette formation was also inhibited after SEAIgMC1-3b pretreatment with beta 1H and C3b-inactivator. LC were shown to possess trypsin-resistant C3b and Fc receptors, and unlike the temperature-independent nature of LC Fc-rosette formation, the ability of LC to form C3 rosettes was a temperature-dependent process. The finding of C3b receptors on the surface of guinea pig epidermal LC supports the concept that they are of the phagocytic monocyte-macrophage family.

CYCLIC AMP AND EPIDERMAL CELL DIVISION

ABSTRACTThe relationship of cyclic AMP to epidermal cell division has been studied in cultured guinea pig epidermal cells. Tritiated thymidine uptake by cultured cells reached a peak the second day after seeding. A significant drop in the concentration of cyclic AMP occurred simultaneously with the increased uptake of tritiated thymidine into DNA. Those cells in which the concentration of cyclic AMP decreased did not respond to external prostaglandin E1 (PGE1) stimulation. The response was restored in the cultured cells on the fourth and fifth when intracellular concentration of cyclic AMP started to rise. The results indicate that there is a cyclic alteration of the surface membrane of epidermal cells during active DNA synthesis which may be related to fluctuation in the intracellular concentration of cyclic AMP.

Binding specificity of rabbit anti-guinea pig epidermal cell sera: comparison of their receptors with those of concanavalin A and pemphigus sera

Rabbit anti-guinea pig epidermal cell sera (AES) which were prepared by immunizing rabbits with enzymatically dispersed viable guinea pig epidermal cells were shown to react to the intercellular substances of stratified squamous epithelia of guinea pigs and monkeys in a pattern similar to that seen with concanavalin A (ConA) or pemphigus sera (PS) by immunofluorescence. Reciprocal blocking tests were performed on guinea pig lip mucosa after removal of the non-specific binding substances between preincubated substances and subsequently incubated ones. No definite inhibition was noted in the subsequent reactions upon preincubation with AES or PS. However, preincubation with ConA weakened the subsequent reaction with PS, but did not block the reaction with AES. Effects of solvents on the receptors for AES, ConA and PS were also examined. ConA receptor was resistant to both saline solution and ethanol (95% and 99%). PS receptor was labile to both saline solution and ethanol, while AES receptor was labile to ethanol, but resistant to saline solution. These observations suggest that the receptor for AES differs from that for ConA or PS.

Distribution and mobility of specific antigens on isolated guinea pig epidermal cells

Surface distribution and mobility of epidermis-specific antigens were studied under various conditions in trypsinized, isolated guinea pig epidermal cells by means of immunoferritin electron microscopy. Fresh or paraformaldehyde-fixed cells were treated with rabbit antiserum specific for epidermal cell surface antigens of the guinea pig at 4°C and then labeled with ferritin-conjugated sheep anti-rabbit IgG at 4° or 37°C for 15 min, respectively. In fresh cells labeled at 4°C ferritin was bound uniformly over the entire cell surface in all keratinocytes with some tendency to form small aggregates. In fresh cells labeled at 37°C there were large aggregates of ferritin that were separated by long unlabeled regions (patch formation) and pinocytosis of ferritin in all basal and lower spinous cells. Some upper spinous cells also showed formation of small clusters and pinocytosis of ferritin. Other upper spinous and granular cells were uniformly labeled on the entire cell surface and showed no evidence of pinocytosis. The surface antigens of isolated epidermal cells were demonstrated to be mobile in the membrane plane of basal and lower spinous cells but immobile within the membrane of granular cells. Immobilization of the antigens seemed to occur first in upper spinous cells. It is possible that decrease in fluidity is one of the qualitative changes of the epidermal cell membrane associated with keratinization. Fixed basal and spinous cells showed many small aggregates of ferritin while fixed granular cells displayed uniform labeling. It was suggested that modification of the antigenicity due to paraformaldehyde fixation resulted in the patchy appearance of labeling in lower keratinocytes.

Surface Distribution of Pemphigus Antibody-Binding Substance(s) on Isolated Guinea Pig Epidermal Cells. An Immunoferritin Electron Microscopic Study

The surface distribution of pemphigus antibody-binding substance(s) on trypsinized, isolated guinea pig epidermal cells was studied by means of immunoferritin electron microscopy. Both fresh and 4% paraformaldehyde-fixed cells were incubated with pemphigus serum at 4°C for 15min and then labeled with ferritin-conjugated goat antihuman IgG at 4°C or 37°C for 15 min, respectively. In these cells clusters of ferritin particles were distributed randomly on the surface of nondesmosomal membranes and hemidesmosome. Fresh basal and lower spinous cells showed clusters of 10 to 20 ferritin particles at 4°C while they displayed various sizes of clusters of 10 to more than 50 particles, some of which were interpreted as patches, at 37°C. Fresh upper spinous and granular cells showed clusters of 10 to 20 particles at both 4°C and 37°C. Fixed cells displayed clusters of no more than 20 ferritin particles irrespective of the layers from which they derived and of labeling conditions. Occurrence of patches implied that aggregation of binding substance(s) was induced by pemphigus antibodies. It was suggested that pemphigus antibody-binding substance(s) were movable in the membranes of basal and lower spinous cells while they were less mobile or immobile in upper spinous and granular cells.