Comparative study of intracellular glutathione content in rat lymphocyte cultures treated with 2-mercaptoethanol and interleukin-2

The level of intracellular glutathione (GSH) in mitogen-stimulated mouse lymphocytes is increased in the presence of 2-mercaptoethanol (2-ME), an enhancer of lymphocyte activation and proliferation. Since proliferation of lymphocytes in response to mitogens involves direct activation by a mitogen followed by continued proliferation in response to interleukin-2 (IL-2), we have investigated the effect of 2-ME and exogenous IL-2 on the GSH content and cell proliferation of rat lymphocytes stimulated with phytohemagglutinin (PHA). PHA stimulation increased both GSH content and the magnitude of the proliferative response, as measured by thymidine incorporation into cellular DNA. However, incubation of stimulated lymphocytes with 2-ME or IL-2 for 72 hr produced a significant further elevation of GSH levels and thymidine incorporation. 2-ME also increased the GSH content in unstimulated cultures, but it had little effect on thymidine incorporation. IL-2 increased GSH content and decreased thymidine incorporation in unstimulated lymphocytes. Exposure of cells to DL-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH biosynthesis, significantly depleted GSH and lowered the proliferative response, suggesting a crucial role of de novo GSH synthesis for lymphocyte activation. The data suggest that both 2-ME and IL-2 promote lymphocyte proliferation, although the mechanisms by which intracellular GSH levels are increased by the agents are apparently different.Copies of articles are available through ISI Document Delivery Services c/o The Genuine Article, 3501 Market Street, Philadelphia, PA 19104.     

Phylogenetic studies on T cells. I. Lymphocytes of the shark with differential response to phytohemagglutinin and concanavalin A

Peripheral blood lymphocytes of the nurse shark (Ginglymostoma cirratum) respond to stimulation by concanavalin A (Con A) as evidenced by increased incorporation of tritiated thymidine. Separation by means of Ficoll-Isopaque yields two or more bands and a sediment, all of which contain lymphocytes responsive to Con A. Only the bottom cells react to phytohemagglutinin (PHA). This reaction cannot be detected in the unseparated lymphocyte population. Thus, only a unique subset of lymphocytes appears to be responsive to PHA and is probably blocked in its response by other cells. The findings suggest that differentiation toward Con A responsiveness may have preceded phylogenetically the responsiveness to PHA. Judging by the requirement for high concentrations of both mitogens the receptor sites on shark lymphocytes appear to be present in lower densities than on lymphocytes of higher vertebrates.     

Structure and functional alterations in lymphocytes induced by cryopreservation

Surface markers, Con A-induced capping, blastogenic transformation stimulated by PHA and allogeneic mononuclear cells, and natural killer activity of Ficoll — Hypaque-separated lymphocytes were studied before and after varying periods of cryopreservation. An increase was observed in the relative number of E rosetteforming cells and in the incorporation of [³H]thymidine into DNA, by the unstimulated cryopreserved cells after thawing. On the other hand, a substantial drop occurred in the Con A-induced capping and the natural killer activity of cryopreserved cells. The possible causes for the variation in the effects of cryopreservation on lymphocyte functions as reported by different investigators were discussed. It was concluded that until universally accepted, standardized procedures for the assessment of lymphocyte functions in vitro become available, each laboratory should establish the changes induced by cryopreservation in lymphocyte function with the methods employed locally to allow the observations made on cryopreserved lymphocytes to be meaningful.     

Inhibition of lymphycyte reactivity in vitro by autologous polymorphonuclear cells (PMN).

The influence of autologous polymorphonuclear cells (PMN) on lymphocyte reactivity was investigated by monitoring the uptake of tritiated thymidine by unstimulated, phytohemagglutinin (PHA)-stimulated, and fetuin-stimulated lymphocytes in vitro. Addition of PMN at PMN-to-lymphocyte ratios (P:L) of 0.5 to 2.0 progressively inhibited lymphocyte reactivity. Soluble extracts, obtained by sonication and ultracentrifugation (100,000g for 90 min) of PMN, also inhibited lymphocytes. The PMN-derived inhibitor(s) is noncytotoxic, heat labile (56 °C for 60 min), resistant to freeze-thawing (20 cycles), and appears to be nondialyzable. Inhibition was more marked when the factor was added at the initiation of lymphocyte cultures than when added with the tritiated thymidine 24 hr prior to cell harvest. Thus thymidine released by PMN which diluted the radiolabeled nucleotide and degradation of the tritiated thymidine did not explain these results. Lymphocytes incubated for 3 days in the medium containing the inhibitor reacted normally to PHA following washing, indicating that inhibition was reversible. These results suggest that a PMN-derived lymphocyte inhibitor(s) may modulate lymphocyte-mediated immune reactivity.     

The role of the thymus in maturational development of phytohemagglutinin and pokeweed mitogen responsiveness

A sensitive culture system for measuring lymphocyte transformation under physiological conditions by thymidine incorporation into DNA has been developed to study mouse and chick cell responses to mitogens. Both phytohemagglutinin (PHA) and pokeweed mitogen (PWM) stimulated thymus and spleen lymphocytes. Reduced but definite responses were obtained with lymph nodes, but negligible response with bone marrow cells.Thymocytes of newborn mice did not respond to PHA, but responded well to PWM. PHA responsiveness of thymocytes increased with aging until 12 weeks of postnatal life and then decreased in older animals. The level of background thymidine incorporation increased with advancing age. Spleen cells of 2-week-old mice were transformed by PHA and PWM, but in contrast to mouse thymus there was no decrease in older animals.Neonatal thymectomy of mice reduced the response of spleen cells to both PHA and PWM, especially in younger animals. The reduction was almost complete in the case of the PHA response, but only partial with the PWM response. Spleen cells from bursectomised chickens, checked for absence of B cell function, still responded well to both PWM and PHA.The results suggest PHA is a marker for T-lymphocytes in a certain “mature” stage of differentiation. PWM appears to stimulate a wider spectrum of cells.     

Enhanced uptake of calcium by transforming lymphocytes

Phytohemagglutinin caused a rapid increase in calcium accumulation by lymphocytes. The enhanced uptake was observed within 1 hr of initiation of transformation in both human lymphocyte and mouse spleen cell cultures. Increased uptake was also found in mixed lymphocyte cultures although not until late in the response. The rate of calcium uptake increased with time after stimulation and depended upon the PHA concentration. The lowtemperature coefficient (Q₁₀) for calcium permeability in unstimulated cells was indicative of a passive diffusion process, but the Q₁₀ was slightly greater for PHA-stimulated cells. Various chemical agents which alter membrane properties and/or cellular metabolism inhibited uptake to a greater extent in stimulated cultures than in control cultures. Ouabain did not affect the calcium permeability of controls or stimulated cells within 1 hr after PHA addition, but it partially inhibited calcium uptake 12 hr after PHA treatment. Cyclic AMP, dibutyryl cyclic AMP, and theophylline also altered calcium transport providing evidence for an effect of cyclic AMP on an early event in the transformation process.     

Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes

Summary The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of ³H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase a and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h.10^–6 M ara-C inhibited the incorporation of ³H-thymidine by 90–95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of ³H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 °C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of ³H-thymidine.Ara-C (10^–6 M) abolished also the PHA induced elevation of DNA polymerase a and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.     

Demonstration of significant differences in the proliferative capacity of lymphocytes from normal human subjects

Lymphocytes were obtained from six normal human subjects on three to five occasions. Lymphocyte response to pokeweed mitogen (PWM), phytohemagglutinin (PHA), and a pool of allogeneic lymphocytes was measured by incorporation of [³H]thymidine in cultures established in flat- and round-bottom multiwell plates. Unstimulated and mitogen-stimulated thymidine incorporation were not correlated. Therefore, incremental counts rather than stimulation indices were used to express data. Individual lymphocyte responses were more reproducible in round-bottom well plates. Using these data, correlation was found between the responses to PWM and PHA. Neither of these responses was correlated with the mixed lymphocyte reaction. There were no significant differences in the responses of these subjects to allogeneic lymphocytes. This response may, therefore, allow clear distinction between normal and abnormal lymphocyte reactivity. Among these normal subjects significant differences were found in their responses to both PWM and PHA. The biological import of these differences is not known.     

Lymphocyte transformation in casein-induced murine amyloidosis.

Spleen lymphocytes from casein-induced amyloidotic mice demonstrate diminished transformation in vitro to PHA-P, concanavalin A, and pokeweed mitogen but a normal response to lipopolysaccharide. Thymic and peripheral blood lymphocytes respond normally to these mitogens. The amyloidogen casein acted as a mitogen in both normal and casein injected mice. The diminished PHA responsiveness of spleen lymphocytes in vitro could have resulted from an inhibitory effect of amyloid fibrils on lymphocyte proliferation and did not indicate a generalized diminished cellular immunologic responsiveness of amyloidotic mice.     

Enhancing effect of lithium and potassium ions on lectin-induced lymphocyte proliferation in aging and down's syndrome subjects

The effect of different concentrations of LiCl or KCl (0.6–20 meq/liter) on PHA-stimulated lymphocytes from young, old, and Down's syndrome subjects was studied. LiCl showed a dramatic enhancing effect on [³H]thymidine incorporation induced by a suboptimal dose of PHA in old subjects and Down's syndrome patients. An increase of [³H]thymidine incorporation in human lymphocytes stimulated by a suboptimal dose of PHA was also observed with KCl. This effect was higher in old subjects than that observed in young and Down's subjects. LiCl and KCl can modulate and partially restore the derangement in early events of mitogen stimulation which seems to be present in lymphocytes from both old and Down's syndrome subjects.     

Effects of removing PHA from cultures of PHA-stimulated lymphocytes

The proliferative capacity of PHA-stimulated lymphocytes following removal of PHA from the cultures was investigated. Lymphocytes were incubated with different PHA concentrations for 3 or 24 h and were then cultured in fresh medium with or without PHA in the original concentration. Cell proliferation was measured by incorporation of ³H-TdR. The effect of removing PHA was found to vary with the PHA concentration used for stimulation. Thus removal of PHA at 3 and 24 h from cells stimulated with half the optimal and at 3 h from cells stimulated with optimal PHA concentrations inhibited thymidine incorporation almost completely. Removal at 24 h from the latter cells resulted in a moderately decreased thymidine incorporation, whereas no decrease was seen after the removal of PHA from cells stimulated with twice the optimal concentration. When the cells were stimulated with very high PHA concentrations (20 × optimal), removal of PHA even resulted in an increased thymidine incorporation, a phenomenon that most probably has to do with the utilization of exogenous thymidine being inhibited by high PHA concentrations.The decreased thymidine incorporation after removal of low PHA concentrations was due to a reduction in the number of cells entering the proliferation cycle as well as to a decreased multiplication of cells already in DNA synthesis. This shows that PHA stimulates the cells even after they have initiated DNA synthesis. Various explanations for the results are discussed.     

Differential production of interferon and lymphotoxin by human tonsil lymphocytes.

Lymphotoxin (LT) production, interferon (IF) production, and DNA synthesis were investigated after mitogen stimulation and in the mixed lymphocyte culture (MLC) reaction using human tonsil lymphocytes. Both LT and IF were assayed in parallel and from the same lymphocyte supernatants. An analysis of the PHA, PWM, one-way and two-way MLC reactions showed that the amounts of LT and IF produced could not be correlated. Polyriboinosinic: polyribocytidylic acid (poly(I: C)) failed to induce either LT production or [³H]TdR incorporation but did induce IF production. Removal of glass-adherent cells (GAC) had no effect on mitogen induced LT production but their removal reduced LT production in MLC reactions. GAC were necessary for IF production and optimal [³H]TdR incorporation in both mitogen stimulated cultures and in MLC reactions. IF and LT activities were shown to be the result of different molecules by using a Sephadex G-75 column. These results indicate that mitogen stimulation differs from MLC reactions in the cell type or control mechanisms involved for LT production, and that in mitogen stimulated cultures all three of these in vitro phenomena are probably the results of either different cell types or of different cell to cell interactions.     

The inhibitory effects of K+ channel-blocking agents on T lymphocyte proliferation and lymphokine production are "nonspecific"

The effect of K+ channel-blocking agents, tetraethylammonium (TEA) and 4-aminopyridine (4AP), on the responses of cloned murine helper and cytolytic T lymphocytes stimulated with mitogen, anti-T cell receptor monoclonal antibody, or interleukin 2 was examined. The addition of TEA and 4AP reduced [3H]thymidine incorporation and lymphokine production to levels observed in unstimulated cells. However, thymidine incorporation by the tumor cell lines P-815 and SP2/0, which replicate autonomously, also was inhibited by these drugs. Treatment of cloned murine helper T lymphocyte, L2, with TEA appeared to inhibit uptake of [3H]thymidine and [3H]phenylalanine after stimulation with interleukin 2. These results suggest that the inhibitory effects of the K+ channel-blocking agents TEA and 4AP may not be specific for the sequence of events that are initiated by activation of T lymphocytes through the antigen receptor. Instead, the observed inhibitory effects by these agents may result from inhibition of transport of thymidine, amino acids, and other essential metabolites across the cell membrane.     

Stimulators and inhibitors of lymphocyte DNA synthesis in supernatants from human lymphoid cell lines.

Some T and B lymphoid cell lines (LCL) were found to secrete into their supernatants a substance able to stimulate lymphocyte proliferation. This substance produced an increase in [3H]thymidine uptake by mononuclear cells when added to unstimulated cultures (mitogenic effect) or when added to cultures stimulated with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) (potentiating effect). When complete supernatants were used, the potentiating effect was sometimes masked by an inhibitor of DNA synthesis. Fractionation on Sephadex G-100 separated these two activities. The stimulatory substance eluted at a m.w. range of 15,000 to 30,000, and the inhibitor eluted with the albumin peak. B cells with or without monocytes were the most sensitive to the mitogenic effect, whereas T cells were unaffected. Responses to PHA and PWM were potentiated when T cells were present, but the maximum effect was observed when the proportion of T cells was less than 50%. The stimulatory material may be similar to lymphocyte mitogenic factor and may function as a T cell-replacing factor in B cell stimulation.     

Selenium status and phytohemagglutinin-stimulated T lymphocytes from beef calves

The effect of selenium (Se), supplemented by subcutaneous injections, upon in vitro phytohemagglutinin (PHA)-stimulated T lymphocyte blastogenesis was evaluated in crossbred calves at 45, 124, and 270 d of age. Peripheral blood lymphocytes (5×10⁵) obtained from control (n=4) and Se-treated (n=4) calves were cultured with 0, 0.4, and 0.8 μg PHA and 0.5 μCi [³H]-thymidine in a complete culture medium. At 60h, cultures were harvested for the quantitation of thymidine incorporation. The degree of lymphocyte blastogenesis or stimulation to PHA, expressed as the stimulation index (SI), was assessed for treatment and age effects. At the onset and end of the experimental period, plasma Se and blood glutathione peroxidase concentrations revealed that control calves [0.045 ppm and 48.4 units/g hemoglobin (Hb), respectively] were marginally deficient and treated calves (0.075 ppm and 143.0 units/g Hb, respectively) were adequate in Se status. The SI values were not influenced (P>0.01) by Se treatment, but were affected (P<0.003) by age. For combined treatments and PHA dosages, mean SI values were greater (P<0.05), when lymphocytes were tested at 45 (SI=25.9) than at 124 and 270 (SI=13.7 and 15.5, respectively) d of age. Lymphocyte viability and number were not affected by Se treatment, but lymphocyte number tended (P<0.1) to increase with age. In conclusion, T lymphocyte responsiveness to mitogen stimulation was normal in beef calves residing in a geographical area considered deficient in Se.     

Opposite modulation of lymph node cell and spleen cell responses to mitogens by muramyl dipeptide and its desmethyl analog

N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), the minimal structure necessary for adjuvant activity of mycobacterial cell wall preparations, was evaluated as an immunostimulant in mice. MDP treatment, which increased carbon clearance and nonspecific resistance to lethal Klebsiella challenge, induced lymph node cellular hyperplasia (4-fold). In contrast, spleen and resident peritoneal cell recovery was comparable to controls. Lymph node cells (LNC) from MDP-treated mice had enhanced [³H]thymidine uptake in unstimulated (4-fold) and lipopolysaccharide (LPS) (5-fold)-, concanavalin A (Con A) (2-fold)-, and phytohemagglutinin (PHA) (1.5-fold)-stimulated cultures. In contrast, spleen cells exhibited depressed responses when stimulated with LPS (2-fold), Con A (2- to 5-fold), and PHA (3-fold). Depressed responses of spleen cells to mitogens were demonstrated over a range of mitogen concentrations. The desmethyl analog produced similar effects, although spleen cells were not as hyporeactive. Opposing modulations of the immune system by MDP resembles that reported after BCG infection, and correlates with increased nonspecific host resistance to microbial challenge.     

Response of baboon peripheral blood lymphocytes to phytohemagglutinin: time-course and dose-response relationships.

Optimal conditions for studying phytohemagglutinin(PHA)-induced transformation of baboon peripheral blood lymphocytes were determined. Maximal stimulation, as determined by uptake and incorporation of tritiated thymidine (total cell and trichloroacetic-acid-precipitable radioactivity), occurred at a PHA level of 12.5 microgram in a culture volume of 0.25 ml containing 2 x 10(5) lymphocytes. Optimal stimulation occurred after a total incubation period of 114 h, the last 18 h of which was in the presence of 1muCi of the labeled DNA precursor per culture. While there was considerable variation in the extent of responsiveness of lymphocytes from individual animals, the shape of the dose-response and time-course curves for most mitogen concentrations was generally similar.     

Dose-dependent hyporreactivity to phytohemagglutinin in systemic lupus erythematosus

The response of peripheral blood lymphocytes to stimulation with optimal and suboptimal doses of PHA was measured in patients with active SLE before initiation of therapy. The [³H]thymidine uptake of SLE patient's lymphocytes was significantly lower than that of their matched controls when cells were stimulated with suboptimal PHA doses in the presence of autologous plasma. A moderate improvement in the PHA response was observed by culturing washed patient's lymphocytes in medium supplemented with pooled normal human plasma, but only in one case the response reverted to normal values. A significant inhibitory effect of SLE plasma on the response of normal donor's lymphocytes to stimulation with low PHA doses, which was independent from the presence of lymphocytotoxic antibodies and persisted after complement inactivation was observed in further experiments.The results indicate that depression of lymphocyte transformation could be demonstrated in patients with active SLE using suboptimal doses of PHA and suggest that this depression may be caused by both a defect in the responding lymphocyte populalation and the presence of inhibitory factor(s) in SLE plasma.     

KINETICS OF HUMAN LYMPHOCYTE RESPONSES IN VITRO: DETERMINATION OF CLONE SIZE AND INITIAL RATE OF ENTRY INTO DNA SYNTHESIS

In this third paper on the kinetics of lymphocyte stimulation we present a simple stochastic model for the entry of mitogen stimulated human lymphocytes into the proliferative cycle. The model is based on the assumption that responder ‘recruitment’ is a process of simple exponential decay. The model can be applied to the initial rapid rise in thymidine uptake after stimulation and successfully predicts the behavior of colchicine inhibited mitogen responses. Application of the model allows the estimation of the following constants; the size of the responding clone, the rate of entry of committed cells into the initial cell cycle, the duration of the lag period before uptake of thymidine increases above background and the average duration of thymidine uptake in responding lymphocytes (T_s). If we analyze the experimental results of mitogen stimulation experiments in these terms we can show that the first three constants are sensitive functions of both the dose of mitogen and the source of the responding lymphocytes. The most interesting finding may be the fact that low doses of mitogen seem to decrease the rate of entry of committed lymphocytes into cell cycle. This would imply that the rate determining step in this process is not of an all or none type.     

Stimulation of chicken lymphocytes by T- and B-cell mitogens.

Cultures of chicken spleen, peripheral blood, thymus, and bursal lymphocytes were tested for mitogenic stimulation by phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), bacterial lipopolysaccharide (LPS), trypsin, and insulin. Spleen and blood leukocytes were stimulated by both the lectins and LPS, and also to some degree by trypsin and insulin as judged by increased incorporation of [³H]thymidine into acid-insoluble material. This was observed in cultures incubated in serum-free medium as well as in the presence of foetal bovine serum or autologous plasma. Thymus cells were reproducibly stimulated by high concentrations of PHA. No significant responses were obtained in bursal cell cultures with any of the compounds tested. Removal of cotton wool-adherent cells from the spleen cell suspensions resulted in a subpopulation of cells which were stimulated by PHA but showed little response to ConA, PWM, or LPS. This procedure did not remove surface immunoglobulin-bearing cells from the original suspension. Both these enriched spleen lymphocytes and the unfractionated spleen, blood and thymus leukocyte cultures were effectively stimulated by a partially purified PHA but with a highly purified PHA preparation only at very high concentrations. These and other results suggest that the mitogenic components in crude PHA preparations are different for chicken and human or mouse cells.