Noritsugu Yabe

An ethanol-extract of Ampelopsis brevipedunculata (Vitaceae) berries decreases ferrous iron-stimulated hepatocyte injury in culture.

We characterized the effects of an ethanol-extract of the berries of Ampelopsis brevipedunculata (Maxim.) Trautv. (Vitaceae), a plant used in folk medicine to treat liver disease, on rat hepatocyte injury occurring spontaneously, stimulated with ferrous iron and with xanthine oxidase in combination with hypoxanthine or stimulated with ethanol in serum-free culture. Total intracellular and extracellular activities of lactate dehydrogenase (LDH) accumulating during incubation and the percentage of intracellular LDH activity released into culture medium were routinely measured, to evaluate the degree of the injury. The extract decreased a high level of LDH release spontaneously occurring and an elevated level of LDH release stimulated with ferrous iron to approximately the level caused by antioxidants, such as superoxide dismutase, pyruvate and dimethyl sulfoxide. Xanthine oxidase-stimulated LDH release was not decreased by the extract. Ethanol-stimulated LDH release was decreased by the extract when the spontaneous release level was comparatively high. These results indicate that the extract inhibits intact hepatocytes from degrading, by the toxic effect of iron released from primary injured hepatocytes through the generation of reactive oxygen species. The major antitoxic activity of the extract was found in an undialyzable fraction. Sugars were necessary to exert the activity as estimated by periodate oxidation of the extract.

Effects of Ampelopsis brevipedunculata (Vitaceae) extract on hepatic M cell culture: function in collagen biosynthesis

A spirits-extracted fraction of berries of Ampelopsis brevipedunculata (Maxim.) Trautv. (Vitaceae) is used in Japanese folk medicine to treat liver disease. Since such an extract has been shown to inhibit formation of collagen fibers by rat hepatic M cells, it was felt that the extract acts as an inhibitor of hepatic fibrosis. Amino acid analysis of fibrous substances developing on M cell layers and of a cell lysate fraction indicated that an A. brevipedunculata extract inhibited collagen formation. Biosynthesis of non-collagenous proteins and collagen was evaluated by measuring the extent of [3H]tryptophan incorporation into a protein fraction and the rate of [3H]proline incorporation into a collagenase-digestible fraction, respectively. In contrast to the results of the analysis of the fibrous substances, the A. brevipedunculata extract failed to decrease synthesis of non-collagenous proteins and collagen unless cell proliferation was inhibited. There was no detectable level of collagenolytic activity in the M cell culture with the A. brevipedunculata extract. The decrease in accumulation of collagen, therefore, appeared to be a consequence of the proliferation-inhibitory effect of the A. brevipedunculata extract. Such inhibitory activity was found in a macromolecular fraction that contained abundant sugars but lacked proteins.

Ampelopsis brevipedunculata (Vitaceae) extract stimulates collagen synthesis through superoxide generation in the serum-free cultures of rat dermal fibroblasts and Ito cells

We describe the effects of an ethanol-extracted fraction of berries of Ampelopsis brevipedunculata (Maxim.) Trautv. (Vitaceae), a plant used in folk medicine to treat liver disease, on the synthesis of non-collagenous proteins and collagen by rat collagen-producible cells such as dermal fibroblasts and liver non-parenchymal Ito cells. The generation of superoxide and hydroxyl radical was assessed by measuring the reduction of cytochrome c and the formation of thiobarbituric acid-reactive substances from deoxyribose, respectively. The synthesis of non-collagenous proteins and collagen as evaluated by measuring the extent of [3H]tryptophan incorporation into a total protein fraction of culture products and the [3H]proline-incorporating rate into a collagenase-digestible protein fraction, respectively. Both types of cells promptly synthesized only collagen in response to a dialyzable fraction of the extract. Major activity to generate oxygen free radicals accumulated in the dialyzable fraction whereas activity to decrease ferrous iron-mediated generation of the radicals accumulated in an undialyzable fraction of the extract. Stimulation of collagen synthesis was caused by superoxide because addition of superoxide dismutase but not pyruvate, an antioxidant of hydrogen peroxide, or dimethyl sulfoxide, an antioxidant of the hydroxyl radical, abrogated the stimulatory effect. The extract may arrest the progress of liver injury mediated by oxygen free radicals generated in the presence of ferrous iron.

A hidden mitogenic activity ofVitis (grape) extract for human lymphocytes in the presence of monocytes or serum

Dear Editor: Plant lectins are widely distributed in commonly consumed foods (7). It has been shown that a lectin is not always mitogenic (5). The discovery of mitogenic activity in lectins, however, seems to depend on culture conditions. In the conventional culture supplemented with serum it is probable that serum affects or masks activities of effector molecules. Serum-free culture systems may permit qualitative and quantitative studies to precisely identify those nature. An agricultural grape species Vitis vinifera has a long history of utilization for foods and beverages with the advance of civilization. Although such grape species was found to have a lectin activity, whether it is mitogenic has not been defined. We had already observed that extracts derived from berries of nonagricultural wild edible grape species V. coignetiae and V. ficifolia, but not of wild inedible species Ampelopsis brvipedunculata (4), exquisitely agglutinate human erythrocytes (unpublished results). Our attention was focused on other biological activities, in particular mitogenic activity, of such grape species. As shown in Fig. 1, under serum-free culture conditions an apparent proliferative response was observed when human peripheral blood lymphocytes (PBL) depleted of monocytes were exposed to undialyzable 40% ethanol-extracted fractions (4) obtained from ripe berries of Vitis species including I/. vinifera (not shown) but not of A. brvipedunculata. Unfractionated peripheral blood mononuclear ceils (PBMC) had merely a weak capacity of [3H]thymidine uptake or were inhibited to proliferate (Table 1). This is clear contrast with the response induced by the mitogenic lectin such as concanavalin A (Con A) because PBMC were more stimulated to proliferate with Con A than PBL were. Monocytes-macrophages are known to play an important role as accessory ceils in the T cell activation induced with antigens or some mitogens (2,12), especially under serum-free culture conditions (8). We prepared PBL fraction depleted of the majority of monocytes from PBMC fraction using cell adherence on a culture substratum in combination with cell phagocytosing of silica gel. Silica was proven to inhibit the lymphocyte proliferative response if it was added to culture before the Con A stimulation (11). Rabbit T lymphocytes at a high density (2 × 105 lymphocytes/weil) were found to proliferate in response to phytohemagglutinin in the absence of macrophages, but those at a low density (2 X 104 lymphocytes/well) failed to proliferate (10). Our employing cell density (1 × 105 cells/well) exhibiting the proliferative response of PBL induced with the extract in the absence of monocytes seems to be the former case. Alternatively, activated B cells were suspected to function as accessory ceils (3,12). Ethanol extract obtained from berries of V. vinifera also possessed a similar effect (not shown). Disappearance of [3H]thymidine uptake in the presence of relatively large amounts of Vitis extracts (higher than 10/.tg/ml) was due to ceil death caused by toxic effect because such ceils cultured overnight did not exclude erythrosine B dye. Extract obtained from Ampelopsis berries scarcely had a capacity of inducing [ZH]thymidine uptake by lymphocytes nor influenced deleterious effect on ceil survival even at a concentration of as high as 40 #g/ml. On the other hand, cytotoxicity was not seen when PBL were exposed to Vitis extracts at concentrations up to 200 #g/ml in 10% human serum-containing RPMI 1640 medium. The stimulatory effect of V. ficifolia extract on [3H]thymidine uptake in the culture supplemented with serum was roughly estimated 20 to 40 times less of that in serum-free culture using GG-404 medium (Fig. 2). Precipitate formed when the extract was mixed with serum. It is probable that lymphocyte proliferation-stimulating factor (LPSF) in the extract preparation reacts with some serum constituents, resulting in loss of the activity. In the presence of 0.5 to 1 #g/ml of indomethacin, PBMC significantly incorporated [3H]thymidine with a relatively larger extent in

Lysozyme as a regulator of interleukin-2-activated lymphocyte proliferation

SummaryLysozyme at 1 to 100µg/ml of exposure levels augmented or inhibited proliferative response of human peripheral blood lymphocytes stimulated with interleukin-2 (IL-2). This contradictory effect of lysozyme depended on IL-2 concentration, activating state of lymphocytes, addition time of lysozyme, and serum existence. Lymphocytes increased their IL-2-mediated proliferating ability in response to lysozyme when stimulated with less than suboptimal concentration of IL-2. Lymphocyte activation with anti-CD3 antibody changed the augmented proliferative response into the inhibited response by lysozyme addition whereas elimination of MHC class II molecule-expressing cells augmented the response. Addition of lysozyme within 1 h after IL-2 exposure was most effective in promoting the proliferation whereas additions after 16 to 24 h were ineffective or inhibitory. Addition after longer than 24 h inversely restored the proliferative response. Serum seemed to retard lysozyme action because either sequential serum addition 1 h after exposure of IL-2 and lysozyme to cells or exposure of IL-2 and serum after pretreatment of cells with lysozyme changed the proliferative responsiveness from inhibition into augmentation. Thus lysozyme may regulate lymphocyte proliferation responding to a magnitude of antigenic stimuli and to the progression of cellular events that periodically occur.

MODULATORY EFFECT OF RAT SMALL INTESTINAL EPITHELIAL CELL-CONDITIONED MEDIUM ON LYMPHOCYTE PROLIFERATION

SummaryThe small intestinal epithelium plays an important role in the mucosal host defense. Intestinal epithelial cells have been known to release substances that suppress lymphocyte proliferation, suggesting an immunoregulatory function. We investigated how intestinal epithelial cells affect lymphocyte proliferation. Serum-free medium that was conditioned by incubating epithelial cells, particularly crypt cells, of the rat small intestine affected proliferation of allogeneic spleen lymphocytes stimulated with concanavalin A, as assessed by measuring cellular [3H]thymidine incorporation. Less than 1% and greater than 2% of the conditioned medium enhanced and suppressed, respectively, lymphocyte proliferation. The causative substances found in the conditioned medium were dialyzable and heat-stable. Suppression was not due to toxicity to splenocytes. Exposure of splenocytes to a suppressive concentration of the conditioned medium beginning at 30 min before an onset of lectin stimulation decreased the suppression of lymphocyte proliferation. Splenocyte exposure to the suppressive concentration of the conditioned medium beginning at 30 min to 4 h after the onset of the stimulation inversely strengthened the suppression. A brief exposure of splenocytes to the conditioned medium for the last 4 h during a total 72-h culture period still suppressed lymphocyte proliferation. Thus, intestinal epithelial cells produce low-molecular-weight lymphocyte proliferation-modulating substances that suppress the proliferation of lectin-activated lymphocytes, but not resting ones, by affecting earlier intracellular events and the following DNA synthesis when incubated in culture medium.

EFFECTS OF IRON CHELATES ON THE TRANSFERRIN-FREE CULTURE OF RAT DERMAL FIBROBLASTS THROUGH ACTIVE OXYGEN GENERATION

SummaryEffects of nonchelating and chelating agents at 10 mM on the serum-free culture of rat dermal fibroblasts were investigated. A strong iron-chelating agent, iminodiacetic acid (IDA), and a weak one, dihydroxyethylglycine (DHEG), decreased iron permeation into preconfluent fibroblasts. A weak iron-chelating agent, glycylglycine (GG), a nonchelating agent, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and human apotransferrin (10 µg/ml) increased the permeation with time. Iron may be essential for survival of fibroblasts because subconfluent fibroblasts exposed to 100 µM FeSO4 in combination with transferrin, HEPES, or GG significantly decreased to release lactate dehydrogenase into the medium. Superoxide dismutase and dimethyl sulfoxide blocked the enzyme release, suggesting that superoxide and hydroxyl radical induce cellular damage but hydrogen peroxide (H2O2) generated by superoxide dismutation does not. GG significantly reduced H2O2 cytotoxicity. DHEG acted as a potent promoter of the iron-stimulated cellular damage if ascorbate or H2O2 was added to the medium. FeSO4 and FeCl3 (50 to 100 µM) individually combined with IDA maximally promoted fibroblast proliferation. Ascorbate increased formation of thiobarbituric acid-reactive substances from deoxyribose in the medium supplemented with FeSO4 and either IDA or DHEG. Conversely, ascorbate decreased the formation in the medium with FeSO4 and with or without other agents. Fibroblast proliferation may thus be stimulated through the active oxygen generation mediated by a redox-cycling between Fe3+ and Fe2+, which are dissolved in the medium at a high concentration, rather than through delivery of iron into the cells.

Synergistic effect of amino acids on production of lymphocyte proliferation-suppressing substances by rat intestinal epithelial cells

Conditioned culture medium of rat small intestinal epithelial cells suppressed proliferation of spleen lymphocytes stimulated with concanavalin A (approx. 10% of its control [3H]thymidine incorporation) whereas conditioned phosphate-buffered saline of the epithelial cells did not. On the other hand, conditioned saline of the epithelial cells exposed to a mixture of total 22 amino acids at their concentrations in the culture medium suppressed the proliferation (approx. 45% of its control [3H]thymidine incorporation). Neither conditioned saline of the epithelial cells exposed to other medium components nor lysates of freshly harvested epithelial cells suppressed the proliferation. Thus, amino acids synergistically stimulated intestinal epithelial cells to produce substances with the ability to suppress lymphocyte proliferation.