Modulation of thymocyte membrane potential by concanavalin A.

Binding of convanavalin A, a potent mitogenic lectin, to thymocyte surface membrane causes depolarization of membrane potential. The effect is suppressed by alpha-methyl-D-glucoside, a haptenic inhibitor of this lectin, or by low temperature. Colchicine and cytochalasin B aslso suppress the change. These data indicate that perturbation of thymocyte surface membrane receptors induced by concanavalin A might be linked to change in the functional state of cellular cytoskeletal systems in turn causing depolarization of thymocyte surface membrane. The initial event generated by receptor-ligand interaction on the outer surface could be translated into cellular interior action only under highly fluid conditions of membrane lipid. Depolarization of thymocyte plasma membrane may be involved in the triggering mechanism of metabolic burst associated with blastoid transformation.     

Enhancement of DNA synthesis and cAMP content of mouse thymocytes by mediator(s) derived from adherent cells.

Supernatants of adherent mouse peritoneal exudate cells or human mononuclear cells were used as the source of lymphocyte activation factor (LAF). LAF was found to potentiate the effect of mitogens such as PHA and Con A on DNA synthesis by mouse thymocytes. However, LAF also was capable of reducing vigorous thymosyte reactions to Con A. Thus, LAF usually enhanced the effect of PHA on DNA synthesis by BALB/c thymocytes to a relatively greater degree than that of Con A. This change in the ratio of Con A to PHA response of thymocytes suggests that LAF can serve as a regulator of thymocyte DNA synthesis. Moreover, in the presence of LAF, allogeneic thymocytes developed the ability to have bidirectional mixed thymocyte reactions. Exposure to LAF not only improved the ability of parental thymocytes to act as responder cells, but, in addition, led to increased stimulatory activity of F1 thymocytes, presumably by promoting the differentiation of stimulator cells. These indications that LAF affected differentiation were investigated further by studying its effect on the cAMP content of thymocytes. LAF stimulated significant immediate but transient elevations of intracellular cAMP and adenylate cyclase activity in thymocyte membranes. In contrast, the mitogens themselves failed to elevate or to influence the effect of LAF on the content of intracellular cAMP of thymocytes. Furthermore, the potentiating effect of LAF on mitogen-induced thymocyte DNA synthesis at times was enhanced by exogenous cGMP, carbachol, or imidazole. These findings suggest that LAF, through its stimulation of cAMP levels in thymocytes may in turn promote thymocytes to differentiate sufficiently to become competent to proliferative in response to mitogens.     

Effect of concanavalin A on the level of sulfhydryl groups in thymocytes

Fluorescein mercury acetate (FMA), a fluorescent probe, is used for the investigation of SH-groups of thymocytes' plasma membrane. It is found that mitogenic lectin Con A decreases the amount of membrane SH-groups and increases the fluorescence polarization degree of FMA (PFMA). The value of PFMA increases also during the incubation of cells with potassium ferricyanide and H2O2 but it decreases in the presence of NADH. The analysis of the data permits a conclusion that the thymocyte activation by Con A results in the selective oxidation of certain SH-groups with the formation of disulphide cross-linking between the plasma membrane receptors bound with the lectin molecules.     

Preparation and fractionation of membrane vesicles of thymocytes after osmotic cell disruption

The isolation of plasma membranes is often accompanied with a loss of sensitivity to stimulatory agents (e.g. mitogens). Changes in the structure of the cell membranes after cell breakage have also been reported. Concerning this problem, "mild" cell disruption conditions were tested by using osmotic shock. Different membrane fractions were isolated, resulting in an enrichment of plasma membranes in one fraction. All fractions were characterized by plasma membrane marker enzymes (alkaline phosphatase, gamma-glutamyltransferases), the amount of cholesterol, the molar ratio of cholesterol and phospholipid, by dodecyl sulfate-gelectrophoresis and by electronmicroscopy. Total balance sheets of the fraction were made for each of the different biochemical parameters. The enriched plasma membranes resulting by using the described method had also lost the sensitivity to the stimulatory effect caused by mitogens such as Concanavalin A.     

Effects of retinoic acid on the human lymphocyte response to mitogens

Nontoxic concentrations of retinoic acid enhance DNA synthesis of human peripheral blood lymphocytes in response to phytohemagglutinin or rabbit-antihuman thymocyte globulin, whereas the response to concanavalin A or pokeweed mitogen remained unaffected. Retinoic acid-induced stimulation of lymphocyte reactivity to phytohemagglutinin or antithymocyte globulin was most evident in T cell-enriched subpopulations and required the near-concurrent addition of retinoic acid and mitogens. Retinoic acid-mediated enhancement of lymphocyte proliferation in response to phytohemagglutinin or antithymocyte globulin was paralleled by a concomitant suppression of immune interferon production of lymphocytes stimulated with these mitogens. These findings allow further studies on the immunoregulatory action of retinoids in vitro.     

Pitfalls in measuring fluorescence polarization in mitogen-stimulated T-lymphocytes

Fluorescence polarization measurements with the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed to detect changes in the fluidity of plasma membranes from T-lymphocytes stimulated with mitogens. When the cells were incubated with succinyl-concanavalin A an increase in fluorescence polarization was observed. This, however, could be shown to be due to the interaction of the mitogen with the label DPH and did not reflect changes in the plasma membrane. In purified plasma membranes a decrease rather than an increase of fluorescence polarization was observed.     

The production of regulatory cytokines for thymocyte proliferation by murine thymic epithelium in vitro

Two separate cultures of pure, morphologically distinct thymic epithelial cells have been generated and maintained in culture for one year (A.C. Nieburgs et al., Cell. Immunol. 90, 439-450, 1985). Supernatants from one of these cell lines, TECs, were examined for functional activity on thymocytes in vitro. These supernatants contained three distinct intercellular mediators, each capable of modulating thymocyte responses to T-cell mitogens. Enhancement of thymocyte proliferation to suboptimal doses of mitogen was associated with a factor that eluted in the 97,000-Da region on molecular sieve chromatography and was functionally and physicochemically distinct from interleukin-1 and interleukin-2 (IL-1 and IL-2). Suppression of the thymocyte response to optimal doses of mitogen was mediated by a 1000- to 5000-Da factor. These two intercellular components have different susceptibilities to heat treatments and are trypsin insensitive. In addition, thymic epithelial cells produced significantly high levels of prostaglandin E2 (PGE2) which also suppressed thymocyte responses to mitogen, but only at high doses of supernatant. These epithelial cell-derived enhancing and inhibitory effects on thymocytes could play a role in regulating intrathymic events.     

Spin-labeled stearates as probes for microenvironment of murine thymocyte adenylate cyclase-cyclic adenosine 3':5'-monophosphate system.

The interaction of various spin-labeled compounds with the murine thymocyte adenylate cyclase-cyclic AMP system was investigated. Electron paramagnetic resonance spectra from spin-labeled compounds were used to calculate the order parameter, S, and indicated that the thymocyte plasma membrane is a relatively rigid structure. Increasing concentrations of spin-labeled stearates, but not their corresponding methyl esters, resulted in increased membrane fluidity, partial lysis, and concomitant complete inhibition of cholera toxin-mediated increases in cyclic AMP content. Upon subsequent isolation of plasma membranes from these cells, cholera toxin-stimulated adenylate cyclase activity was also completely inhibited. Direct addition of spin-labeled stearates, but not spin-labeled methyl stearates, to thymocyte homogenates caused a dramatic reduction of basal, cholera toxin-, isoproterenol-, NaF-, and prostaglandin E1-stimulated adenylate cyclase activity. Inhibition was complete within the first minute of addition to homogenates and required approximately 0.2 mM spin-labeled stearate I(12,3) for half-maximal inhibition. This inhibition occurred in the presence or absence of an ATP-regenerating system and was not readily reversible. Furthermore, since the membrane cyclic phosphodiesterase activity was not altered by spin-labeled stearates, their inhibition was attributed to a direct action of stearate spin labels on adenylate cyclase. Neither stearate, methyl stearate, spin-labeled methyl stearates nor 2,2,6,6,-tetramethylpiperidine-1-oxyl (Tempo) altered cell viability or enzyme activities at the concentrations studied. Spin-labeled stearates seemed to intercalate into different areas of the plasma membrane than their corresponding methyl esters. Furthermore, the action of spin-labeled stearates appeared to be on the exterior of the plasma membrane rather than the interior. These results illustrate the presence of multilipid domains and the importance of selected lipids and lipid-protein interactions in the adenylate cyclase-cyclic AMP system. Thymocyte adenylate cyclase is described in terms of a current model for membrane proteins.     

Alterations in the electrical properties of T and B lymphocyte membranes induced by mitogenic stimulation. Activation monitored by electro-rotation of single cells

Stimulation of either B or T lymphocytes using specific mitogens results in changes in the passive electrical properties of the cell surface. These effects can be related to growth and secretion. This was possible because the high resolution of the contra-field electro-rotation method, combined with the use of very low conductivity media, allowed accurate and analytically-derived values for the cell surface properties. Increases in effective CM (membrane capacity) and changes in apparent membrane conductivity (reflecting the additive effects of true membrane conductivity GM and surface conductance KS) were measured. After 72 h treatment with concanavalin A, thymocyte CM had increased from 0.76 muF/cm2 to 1.24-1.46 muF/cm2 (7.6 to 12.4-14.6 mF/m2). Allowing for the stimulation-induced size increase (cell radius increased from 2.8 to 4.4 micron) these data imply that the plasma membrane area per cell increases 5-fold during stimulation. Stimulation of B cells (by 3 days incubation with bacterial lipopolysaccharide) increased CM from 0.93 to 1.6-1.7 muF/cm2 (9.3 to 16-17 mF/m2). Incubation without mitogen gave no significant increase in CM or in radius. Control cells of different sizes showed no difference in membrane properties. The increases in effective CM are argued to reflect an increase in membrane ramification (microvilli, folding, etc.). The apparent membrane conductivity of T cells also increased during stimulation, from 5 to 21 mS/cm2 (50 to 210 S/m2). This increase is proportionately much greater than that in CM or in membrane area. It seems to be due to a real increase in GM, but a small increase in KS may also occur. The earliest changes in apparent membrane conductivity were evident between 3 and 5 h after stimulation, before the cells increased in size. This response parallels increases in transmembrane transport reported to follow mitogenic stimulation.     

Periodate induced cross-linking of concanavalin A-reactive membrane proteins of rabbit thymocytes.

Purified plasma membranes of rabbit thymocytes are exposed to sodium periodate and galactose oxidase at conditions similar to those used to induce mitogenic transformation of lymphocytes. The membrane proteins are then fractionated by dodecyl sulfate poly-acrylamide gel electrophoresis. At concentrations of 0.005 M, Na IO4 cross-links 55,000 D and 110,000 D glycoproteins which are known to specifically bind concanavalin A. Galactose oxidase has a similar cross-linking effect, but, at the same time causes proteolytic degradation of membrane proteins. Our data indicate that oxidizing agents, like NaIO4 and galactose oxidase, can indeed cross-link receptors of the thymocyte plasma membrane as has often been proposed as a possible mechanism of their action.     

Comparative study, using fluorescent methods, of cell membranes and their reconstituted liposomes

Cell plasma membranes and proteoliposomes reconstituted from solubilized plasma membranes of thymocytes and Ehrlich ascites carcinoma cells have been studied by fluorescent methods. It has been shown that proteoliposomes are characterized by greater polarization and rigidity of microsurroundings in membrane proteins and greater microviscosity of membrane lipids. Proteoliposomes from thymocyte membranes contain less membrane proteins and express lower polarity of the lipid bilayer than proteoliposomes from Ehrlich ascites cells.     

An NMR investigation of the changes in plasma membrane triglyceride and phospholipid precursors during the activation of T-lymphocytes.

Two-dimensional 1H-NMR spectroscopy was used to quantify the level of isotropically tumbling plasma membrane triglyceride and the intracellular concentrations of water-soluble phospholipid precursors during the activation of thymic T-lymphocytes. The concentration of "mobile" triglyceride in the plasma membrane was seen to increase 25-fold during 72 h of activation of murine thymic T-lymphocytes with ionomycin and phorbol myristate acetate. This is the first unequivocal demonstration of such a dramatic increase in mobile plasma membrane triglyceride during T-lymphocyte activation and leads to the suggestion that immune cell activation is associated with increased plasma membrane fluidity. The intracellular concentrations of choline- and ethanolamine-based phospholipid precursors were shown to increase during the early stages of T-lymphocyte activation and then remain at levels above those in resting cells. This may facilitate de novo phospholipid biosynthesis, which is presumably necessary since cell volume, and hence the plasma membrane surface area, was demonstrated to increase significantly during thymocyte activation.     

Stimulation of calcium-ATPase activity by 3,5,3''-tri-iodothyronine in rat thymocyte plasma membranes. A possible role in the modulation of cellular calcium concentration.

We have previously demonstrated that 3,5,3'-tri-iodo-L-thyronine (T3) produces a very rapid and transient increase in calcium uptake and cytoplasmic free calcium concentration in the rat thymocyte, and have postulated that Ca2+-ATPase may contribute to the overall effect of T3 on cellular calcium metabolism. In the present study, we show that in the rat thymocyte, T3 increased plasma membrane Ca2+-ATPase activity. This effect of T3 was very rapid, seen at 30 s after the addition of the hormone, and was concentration-related, evident at a physiological concentration as low as 1 pM. Evaluation of the effect of several thyronine analogues on Ca2+-ATPase activity revealed the following order of potency: D-T3 greater than or equal to 3'-isopropyl-L-T2 = L-T3 = L-T4 = D-T4 greater than L-rT3 greater than 3,5-L-T2 greater than DL-thyronine. Studies with the calmodulin antagonist trifluoperazine demonstrated that thymocyte Ca2+-ATPase activity and its stimulation by T3 are influenced by calmodulin. Other studies showed that several adrenergic agents, agonists and antagonists, had no effect on thymocyte Ca2+-ATPase activity and its stimulation by T3. From these and previous observations, we would suggest that in the rat thymocyte, the T3-induced increase in Ca2+-ATPase activity, which enhances the expulsion of calcium from the cell, plays a role in the diminution and transiency of the stimulatory effect of T3 on thymocyte calcium metabolism.     

Plasma membrane potential in thymocyte apoptosis.

Apoptosis is accompanied by major changes in ion compartmentalization and transmembrane potentials. Thymocyte apoptosis is characterized by an early dissipation of the mitochondrial transmembrane potential, with transient mitochondrial swelling and a subsequent loss of plasma membrane potential (DeltaP sip) related to the loss of cytosolic K+, cellular shrinkage, and DNA fragmentation. Thus, a gross perturbation of DeltaPsip occurs at the postmitochondrial stage of apoptosis. Unexpectedly, we found that blockade of plasma membrane K+ channels by tetrapentylammonium (TPA), which leads to a DeltaP sip collapse, can prevent the thymocyte apoptosis induced by exposure to the glucocorticoid receptor agonist dexamethasone, the topoisomerase inhibitor etoposide, gamma-irradiation, or ceramide. The TPA-mediated protective effect extends to all features of apoptosis, including dissipation of the mitochondrial transmembrane potential, loss of cytosolic K+, phosphatidylserine exposure on the cell surface, chromatin condensation, as well as caspase and endonuclease activation. In strict contrast, TPA is an ineffective inhibitor when cell death is induced by the potassium ionophore valinomycin, the specific mitochondrial benzodiazepine ligand PK11195, or by primary caspase activation by Fas/CD95 cross-linking. These results underline the importance of K+ channels for the regulation of some but not all pathways leading to thymocyte apoptosis.     

Lipid composition and microviscosity of subcellular fractions from rabbit thymocytes. Differences in the microviscosity of plasma membranes from subclasses of thymocytes

There are indications from freeze-fracture experiments that subclasses of rabbit thymocytes show different mobilities of plasma membrane components. Consequently, one would expect differences in the fluidity of the plasma membrane. For this reason, rabbit thymocytes were separated on a Ficoll/Metrizoate gradient yielding three subclasses representing various levels of cell differentiation. These thymocyte subclasses did not show any significant differences in the degree of fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. The fluorescence polarization of the plasma membrane may be overshadowed by the contribution of all cellular lipids due to penetration of the fluorescent probe into the cell. Therefore, plasma membranes were isolated from rabbit thymocytes using a cell-disrupting pump, differential centrifugation, and sucrose density gradient centrifugation. As shown by biochemical and electron microscopical analyses, plasma membranes with a high degree of purity were obtained. As expected the plasma membrane fractions showed a higher microviscosity than the other subcellular fractions. This was attributed to a higher cholesterol to phospholipid molar ratio and a higher degree of saturation of phospholipid fatty acid chains.Subsequently, the microviscosity was measured of plasma membrane preparations obtained from two main subclasses of thymocytes representing mature and immature lymphocytes. The immature thymocytes yielded two plasma membrane fractions with higher microviscosity than the mature cells.     

Characterization of Cell Growth-Inhibitory Factor in Inflammatory Peritoneal Exudate Cells of Rats

We characterized the nature and reaction mode of the cell growth-inhibitory factor (here designated CGIF) from rat peritoneal exudate cells (PEC). The soluble fraction separated from the lysate of Enterococcus faecalis-induced 24 hr PEC completely inhibited Con A-induced thymocyte mitogenesis. Gel filtration chromatography showed that CGIF has a molecular weight of approximately 23–25 kDa. Isoelectric focusing with Rotofor indicates that the factor has an isoelectronic point of 5.8–6.4. CGIF was inactivated by treatment at 70 C, for 30 min or by tryptic digestion, but the activity was not destroyed by the reduction with dithiothreitol. As well as thymocyte proliferation, CGIF completely suppressed ³H-thymidine incorporation of splenocytes which were stimulated by either Con A or LPS, suggesting the factor is effective on both T and B cells. The acting point of the inhibitor appeared to be a later stage of the lymphocyte activation sequence, since it was still effective when added 28.5 hr after the addition of Con A. CGIF also reduced the viability of these cells when added with mitogens such as Con A or LPS. CGIF thus appears to be distinct from interleukin-1 receptor antagonist or transforming growth factor-β.     

The effect of ionizing radiation on the Ca2+, Mg2(+)-ATPase activity of plasma membranes]

A study was made of a change in Ca2+, Mg(2+)-ATPase activity induced by the effect of ionizing radiation (5-10(4) Gy) on a thymocyte plasma membrane suspension. The Michaelis' constant and maximum rate of enzymic reactions were determined. With a dose of 10(3) Gy the structural changes in Ca2+, Mg(2+)-ATPase were shown to reduce the affinity of the substrate to an active enzyme center and to decrease the rate of the enzyme/substrate complex degradation.     

Induction of the non-selective mitochondrial pore in lymphoid cells. 1. Permeabilized rat thymocytes.

The opening of the cyclosporin-sensitive pore in the inner membrane of mitochondria in rat thymocytes was studied. In thymocytes with digitonin-permeabilized plasma membrane, the mitochondrial pore was induced by Ca2+ overload, by uncoupling, by oxidation or cross-linking of membrane dithiols, and by atractyloside, a specific inhibitor of the adenine nucleotide transporter. Pore opening was prevented by cyclosporin A (CsA) and by its non-immunosuppressive analog MeVal-CsA. The sensitivity of the pore to CsA was decreased by atractyloside and practically disappeared when it was added in combination with uncoupler. The main properties of the pore in mitochondria from thymocytes and from hepatocytes are the same. Release of Ca2+ from thymocyte mitochondria induced by uncoupling is mediated by a specific uniporter and by the pore with similar rates.     

Lipid composition and microviscosity of subcellular fractions from rabbit thymocytes. Differences in the microviscosity of plasma membranes from subclasses of thymocytes.

There are indications from freeze-fracture experiments that subclasses of rabbit thymocytes show different mobilities of plasma membrane components. Consequently, one would expect differences in the fluidity of the plasma membrane. For this reason, rabbit thymocytes were separated on a Ficoll/Metrizoate gradient yielding three subclasses representing various levels of cell differentiation. These thymocyte subclasses did not show any significant differences in the degree of fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. The fluorescence polarization of the plasma membrane may be overshadowed by the contribution of all cellular lipids due to penetration of the fluorescent probe into the cell. Therefore, plasma membranes were isolated from rabbit thymocytes using a cell-disrupting pump, differential centrifugation, and sucrose density gradient centrifugation. As shown by biochemical and electron microscopical analyses, plasma membranes with a high degree of purity were obtained. As expected the plasma membrane fractions showed a higher microviscosity than the other subcellular fractions. This was attributed to a higher cholesterol to phospholipid molar ratio and a higher degree of saturation of phospholipid fatty acid chains. Subsequently, the microviscosity was measured of plasma membrane preparations obtained from two main subclasses of thymocytes representing mature and immature lymphocytes. The immature thymocytes yielded two plasma membrane fractions with higher microviscosity than the mature cells. These finding is in line with earlier observed differences in the glycerol-induced clustering of intramembranous particles. Furthermore, the results of this study support the view that the fluorescence polarization technique applied to whole cells does not exclusively monitor the plasma membrane.     

Mechanism of changes in irradiated thymocytes detected by anilinonaphthalenesulfonate

In experiments on rat thymocytes obtained 15 min, 2 h and 4 h after irradiation of animals with the dose of 10 Gy it was found that 15 min and 4 h following irradiation changes occurred in their membranes increasing the lifetime of the excited state of anilinonaphthalene sulfonate and hydrofobicity and viscosity of its microenvironment. The indicated parameters did not vary from the controls 2 h following irradiation. The analysis of the data obtained prompted a suggestion that the observed changes were associated with the structural rearrangements in the plasma membrane rather than with its destruction. Model experiments on irradiated thymocyte suspensions showed that there was a correlation between changes in the cell membrane and meabolic processes.