Effect of cytochalasin B on lymphocyte stimulation induced by concanavalin A or periodate

The effects of cytochalasin B on lymphocyte stimulation induced by concanavalin A (Con A) and by periodate were investigated. At low concentrations (0.1 – 1 μg/ml) cytochalosin B greatly potentiated the responses to these two mitogens. Cytochalasin B was most effective when added with the mitogens at the beginning of incubation. The action of cytochalasin B at low concentration was suggested to be on an early process of DNA synthesis induced by these mitogens.     

Effects of thromboxane A2 on lymphocyte proliferation

The main cyclooxygenase-dependent arachidonic acid derivatives produced by monocytes and macrophages have been shown to be thromboxane A2 and prostaglandin E2. The immunomodulatory effects of thromboxane A2 were examined using a specific thromboxane synthase inhibitor (dazoxiben), a thromboxane A2 analog (U46619), and a thromboxane A2 receptor blocker (BM13.177). Dazoxiben inhibited lymphocyte proliferation in response to mitogens (PHA and OKT3), but also reoriented cyclic endoperoxide metabolism towards the production of prostaglandin E2. Prostaglandin E2 has been shown previously to inhibit mitogen-induced lymphocyte proliferation. U46619, a stable thromboxane A2 analog, slightly enhanced lymphocyte responses to mitogens in the presence of dazoxiben and in the presence of a cyclooxygenase inhibitor (indomethacin). This occurred at concentrations of U46619 which are probably supraphysiological in view of the short half-life of natural thromboxane A2. Finally, the thromboxane A2 receptor blocker BM13.177 did not have any effect on mitogen-induced lymphocyte proliferation. It is concluded that thromboxane A2 has no or minimal modulatory effects on lymphocyte proliferative responses to mitogens and that the effect of thromboxane A2 synthase inhibition is rather due to reorientation of cyclic endoperoxide metabolism, resulting in increased prostaglandin E2 production.     

Lysozyme-induced inhibition of the lymphocyte response to mitogenic lectins

Both human lysozyme (HL) and hen egg white lysozyme (HEWL) inhibited the proliferative response of peripheral blood lymphocytes to T cell mitogens such as the lectins phytohemagglutinin and concanavalin A. This inhibition was observed both when HL or HEWL was added to the lymphocyte cultures in combination with phytohemagglutinin or concanavalin A and when lymphocytes were pretreated with either lysozyme and extensively washed prior to culture with mitogens. Under both conditions, the effects were strictly dose dependent; the lysozyme concentrations yielding maximal inhibitory effect were 5 micrograms/ml for HL and 1 microgram/ml for HEWL, while both lower and higher concentrations were less effective. Specific antilysozyme rabbit sera completely prevented the inhibitory effects of both HL and HEWL on the proliferative response of lymphocytes to phytohemagglutin or concanavalin A. Chitotriose (a lysozyme inhibitor) caused a strong reduction in the inhibitory effects of the two lysozymes on the lymphocyte response to either lectin. HL and HEWL also were found to markedly inhibit the polyclonal B cell proliferation and differentiation induced by pokeweed mitogen and T cells. A less marked inhibition was also obtained when T cells, but not B cells, were pretreated with HL or HEWL. Again, as in the experiments with T cell mitogens, the effects were dose dependent and 5 micrograms/ml HL and 1 microgram/ml HEWL proved to be the most effective concentrations. The possible mechanisms by which lysozyme inhibits the lymphocyte response to mitogenic lectins are considered and discussed. The enzymatic activity seemed to perform an essential function, as shown by the loss of effect when the heat- or trypsin-inactivated lysozymes were used and by the fact that only the enzymatically active compound, among certain semisynthetic derivatives of HEWL, inhibited the lymphocyte response to the mitogens. However, the cationic properties of the lysozyme molecule appeared to be essential too, since enzymes with a similar specificity of action showed effects similar to those observed with HL or HEWL only when they carried a strong positive charge. It is suggested that lysozyme, which is naturally secreted by monocytes and macrophages, might interact with lymphocyte surface receptor sites and participate in the complex mononuclear phagocyte-lymphocyte interactions and in the modulation of lymphocyte activation.     

Inhibition of phytohemagglutinin-, periodate- and A23187-induced lymphocyte transformation by Vinca alkaloids

The effect of the Vinca alkaloids, vincristine and vinblastine, on mitogen-induced transformation of isolated human peripheral blood lymphocytes has been investigated. Cells were subjected to a variety of mitogens (PHA, ionophore A23187 and sodium periodate) whose mechanism and site of action differ. Addition of vincristine or vinblastine to lymphocyte cultures prior to mitogen produced a concentration-dependent inhibition of cell transformation as determined by measurement of DNA synthesis and blast formation. The inhibitory effects were not due to decreased cell viability, since the drugs had little or no effect on cell viability. Vincristine and vinblastine were also found to impair [³H]thymidine incorporation by prestimulated blast cells at the higher drug concentrations tested. The results presented in this communication show that the Vinca alkaloids block lymphocyte transformation induced by either lectin or non-lectin mitogens. This suggests that the inhibitory step(s) may occur after mitogen stimulation.     

Antigenic markers on rat lymphocytes. I. Characterization of A.R.T., an alloantigenic marker on rat thymus and thymus-derived cells.

Drugs with pharmacologic activities on cell membrane components were tested for their effects on responses of murine lymphocytes to mitogens. The effects of the drugs were found to be partly selective and related to the type and dose of the mitogen. Most striking were the effects of cytochalasin B, which inhibited the responses to low doses of concanavalin A or phytohemagglutinin, but potentiated the reactions to the high doses of the lectins. Chloroquine and chlorpromazine, on the other hand, were more inhibitory to cultures containing supraoptimal doses of the lectins. Colchicine inhibited the responses against lipopolysaccharide more than those against the two lectins. The inhibitory effects of colchicine were almost unchanged when the drug was added at intervals up to 24 hr after the onset of culture, whereas the effects of the other tested drugs diminished markedly when added to cultures 6 hr or more after the mitogens. The results are discussed in view of their relationship to the poorly understood mechanisms which regulate the lymphocyte responses in the presence of different doses of mitogens.     

Kinetics of lymphocyte stimulation in vitro by non-specific mitogens

The role of mitogens during lymphocyte activation was studied with kidney bean leucoagglutinin, concanavalin A and kidney bean phytohemagglutinin. The mitogens were removed by treatment with appropriate antisera, which was demonstrated to remove also mitogens already attached to the cells. The process of lymphocyte activation in vitro can be divided into four distinct steps, three of which are mitogen-dependent and the fourth is mitogen-independent. The first step consists of attachment of the stimulatory molecules to the cell membrane. The second step consists of reaction between mitogen and an activating system. During these two phases the cells become preactivated. The establishment of a preactivated state involves at least some synthesis of cytoplasmic RNA. The preactivated state is reversible and during the third step of lymphocyte activation the final result of preactivation is determined. Depending on the presence or absence of mitogen the cells may remain preactivated for over 60 h, they may return to the resting state or they may proceed through the final stages of the proliferation cycle and eventually divide. This fourth step is independent of the presence or absence of mitogen. A prolonged contact between cells and mitogen is required during the mitogen-dependent steps. The process of lymphocyte activation by mitogen is thus continuously being regulated by the stimulatory molecules on the lymphocyte membrane, which may be of considerable significance also for in vivo immunologicai reactions at the cellular level.     

Studies on the interaction between mitogens and human lymphocytes in vitro

The initial events in interaction between mitogens and lymphocytes were studied with kidney bean phytohemagglutinin (PHA-W), concanavalin A (Con A), kidney bean leucoagglutinin (LA) and antilymphocyte globulin (ALG). The lectins were characterized by disc electrophoresis and immunoelectrophoresis. LA was found to be homogeneous while PHA-W was separated in three bands and showed two antigenic components. When lymphocytes were incubated with mitogen for a short time (1 h) and in experiments according to the described technique for transfer of mitotic stimulation between lymphocytes it was found that the binding of PHA-W to the cell differed from that of LA and ConA. In binding experiments with labelled mitogens PHA-W was found to have twice as many binding sites per cell as LA and ConA, although similar affinity constants were found. The relationship between mitogens and lymphocyte receptors was studied in lymphocytes incubated with two mitogens simultaneously for a short period. Both inhibitory and synergistic effects were found. The results indicate that (a) mitogens with different receptor specificities give a synergistic response; (b) mitogens reacting with the same or closely related receptors are inhibitory to each other. The interpretation of the binding of PHA-W to lymphocytes and of the inhibitory and synergistic effects of mitogens are discussed.     

Effects of N-acyl-2-hydroxymethyl aziridines on in vitro proliferative responses of human lymphocytes stimulated by mitogens

Aziridines have been shown to possess marked immunotropic activity. The aim of this work was to study the in vitro effects of different concentrations of three novel aziridines, 2-hydroxy-methyl-1-(N-phtaloylglycyl) aziridine (aziridine 1), 2-hydroxy-methyl-1-(N-phtaloylalanyl) aziridine (aziridine 2) and 2-hydroxy-methyl-1-(N-phtaloylphenylalanyl) aziridine (aziridine 3), on the proliferative responses of human lymphocytes stimulated by mitogens (concanavalin A (Con A) and lipopolysaccharide (LPS)), and interleukin-2 (IL-2), interleukin-6 (IL-6) secretion. The results showed that aziridines 1 and 3 significantly stimulated the resting and Con A or LPS lymphocyte proliferation at concentrations between 1 micromol/l and 1 mmol/l, in a dose-dependent manner, the action of aziridine 3 being the highest. They also increased IL-2 and IL-6 secretion. However, aziridine 2 had no effect on the resting lymphocyte proliferation in the absence of mitogens, at any concentration used, reduced Con A-stimulated T lymphocyte proliferation and LPS- stimulated B lymphocyte proliferation in a dose dependent manner and diminished IL-2 and IL-6 production. None of the three aziridines affected cell viability. In conclusion, the three aziridines used in this study displayed immunomodulatory properties. Aziridines 1 and 3 are potentially immunostimulant while aziridine 2 is immunosuppressive and could be used to provide nonspecific cell-mediated immune responses.     

Lymphocyte reaction in schizophrenia patients to the phytomitogens, concanavalin and phytohemagglutinin]

The capability of the schizophrenic patients' lymphocytes and lymphocytes of healthy persons to respond to the stimulating action of T-mutagens -- concanavalin A and PHA -- was studied. The T-cell count was determined by the method of rosette formation; the influence of adhesive cells on the lymphocyte response to mitogens was ascertained. The response to both the mitogens in the patients' lymphocyte cultures was reduced as compared to control, and the T-cell count failed to differ from the normal. The removal of adhesive lymphocytes results in the disappearance of differences between the response of the patients' lymphocytes and normal lymphocytes to both the mitogens.     

Stimulation of human peripheral blood lymphocytes by periodate, galactose oxidase, soybean agglutinin, and peanut agglutinin: differential effects of adherent cells.

Blastogenic responses of normal human peripheral lymphocytes to three distinct groups of mitogens were studied: Group I--phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM); Group II--soybean agglutinin (SBA) and peanut agglutinin (PNA); and Group III--galactose oxidase (GO) and sodium periodate (IO4-). SBA was mitogenic for human cells, and this effect was enhanced by treating the cells with neuraminidase (NA). PNA was mitogenic only after cells had been treated with NA. GO was effective before and activity was increased after lymphocytes were treated with NA. Responses to Group II and III mitogens were more variable than were those to Group I mitogens. Studies with purified T and B cells indicated that SBA and PNA were T cell mitogens, whereas IO4- and GO failed to stimulate either T or B cells. Adding macrophages back to this system indicated that they were both T cell mitogens with strict macrophage requirements. T cell responses to SBA and PNA were enhanced over responses to unfractionated cells to a degree that could not be explained simply by enrichment of the cultures with T cells. Removal of adherent cells from unfractionated cell suspensions again revealed a marked enhancement of responses to SBA and PNA, a consistent decrease in responses to IO4-, and a variable decrease in responses to GO. Similar results were found with 14C-leucine and 3H-uridine incorporation, as well as 3H-thymidine for the assessment of bastogenic response. Mechanisms responsible for these differential effects of macrophage depletion on lymphocyte responses to different groups of mitogens are yet to be determined. Either different mitogens require different lymphocyte to macrophage ratios for optimal stimulation, or some mitogens (i.e., SBA and PNA) form inhibitory complexees in the lymphocyte-macrophage mixture. In any case, variability in response to mitogenic agents in normal as well as pathologic states may be dependent on adherent cell populations, rather than on the lymphocytes themselves.     

Human lymphocyte subpopulations: giant SRBC rosettes.

Human thymus-derived lymphocytes have the ability to form rosettes with sheep red blood cells (SRBC) in vitro. In the investigation of rosettes of peripheral blood lymphocytes of 10 normal subjects, the number of SRBC adhering to the lymphocyte in each of 100 rosettes was assessed. The percentage of rosettes with SRBC greater than or equal to 36 per rosette was only 1.2 +/- 0.5. These were defined as giant SRBC rosettes. Peripheral blood lymphocytes were stimulated in vitro by four mitogens: sodium periodate, neuraminidase plus galactose oxidase, pokeweed mitogen, and concanavalin A. The lymphocytes were then cultured at 37 degrees C. The giant rosette-forming lymphocytes became significantly increased 4 to 24 hr after stimulation, prior to the appearance of lymphoblasts or increased incorporation of tritiated thymidine. The giant rosettes were not caused by the hemagglutinating properties of pokeweed mitogen and concanavalin A that were adsorbed on the lymphocyte surfaces. This was shown by the fact that, on removal of the receptors by trypsinization, they were regenerated on culture in vitro in the absence of the mitogens. It was concluded that giant SRBC rosettes constituted a marker for some of the activated lymphocytes. Their appearance was independent of the increase in size of the cells or of DNA synthesis. These receptors were intrinsic to lymphocytes and not caused by mitogens adsorbed on their surfaces.     

Regulation of lymphocyte responses by human gangliosides. I. Characteristics of inhibitory effects and the induction of impaired activation

Gangliosides obtained from normal human brain were found to inhibit the in vitro activation of human lymphocytes by nonspecific mitogens and allogeneic cells at concentrations between 3 to 50 microgram/1.5 to 1.7 X 10(5) lymphocytes/0.2 ml culture. Ganglioside inhibition did not represent cytotoxic effects or altered lectin binding and was independent of the mitogen concentration. In addition to concentration, the degree of inhibition was dependent on the mode of presentation to lymphocytes, since gangliosides incorporated within liposomal membranes displayed a synergistic inhibitory effect greater than predicted from the cultures receiving either gangliosides or liposomes alone. In binding experiments, radiolabeled ganglioside GM1 became associated with human lymphocytes within 10 min. However, approximately 72 hr pre-exposure of human lymphocytes to gangliosides was required to induce impaired lymphocyte responses to mitogens and allogeneic cells. Thus, concentrations of human gangliosides equivalent to the levels occurring in the sera of patients with certain malignancies are capable of actively inhibiting lymphocyte stimulation in addition to inducing impaired lymphocyte responses.     

Regulatory effects of macrophage-secreted factors on T-lymphocyte colony growth.

Human fibroblast and leukocyte interferons were found to suppress lymphocyte mitogenesis induced by optimal doses of phytohemagglutinin and concanavalin A. In certain situations (low doses of mitogen and/or low doses of interferon), however, interferon significantly enhanced mitogenesis. In experiments using varying concentrations of interferon, dose-response curves with different slopes were obtained for fibroblast and leukocyte interferons. The effect of interferon was apparently exerted during early stages of the lymphocyte cell cycle. There was no inhibitory effect of interferon if the lymphocytes were washed with medium before being exposed to mitogen. Interferon increased the binding of radiolabeled mitogens to cells. The results suggest that the immunological effects of interferon are consequences of actions on lymphoid cells. Fibroblast and leukocyte interferons seem to have different modes of action, or to bind differently to target cells. Possible mechanisms for the suppressive and enhancing effects of interferons on lymphoid cells are discussed.     

Role of calcium and cyclic AMP on the activation of lymphocyte glycogen phosphorylase by mitogens

1. Various mitogens such as concanavalin A, phytohaemagglutinin, the pokeweed mitogen and trypsin were found to produce a rapid and transient activation of glycogen phosphorylase activity of lymphocytes incubated in a Krebs-Ringer-bicarbonate-glucose buffer. 2. Activation of the enzyme by these mitogens was always accompanied by an increase in the intracellular cyclic AMP concentration. 3. The presence of calcium ions in the incubation buffer was essential for obtaining the mitogen effects. Addition of ionophore A-23187 also produced an activation of glycogen phosphorylase, similar to that found in mitogen activation but without increase in intracellular cyclic AMP concentration. Dibutyril cyclic AMP also produced lymphocyte phosphorylase activation, even in the absence of extracellular calcium ions. 4. It is proposed that phosphorylase activation by mitogens occurs through a mechanism that involves the participation of both calcium ions and cyclic AMP.     

Decrease in mitogen responsiveness of mononuclear cells from peripheral blood after epinephrine administration in humans

A single subcutaneous injection of 0.2 mg epinephrine into healthy human subjects caused a transient lymphocytosis in peripheral blood. Mononuclear cells (MNC), isolated at various times after epinephrine administration, were cultured in the presence of mitogens. The blastogenic responses to pokeweed mitogen (PWM) and phytohemagglutinin (PHA) were significantly reduced for up to 60 min post-epinephrine (p less than 0.05); the response to concanavalin A (Con A) was reduced in the 15-min samples only. All responses returned to pre-injection levels by 120 min post-injection. Removal of adherent monocytes from MNC isolates before culture did not restore normal mitogen responsiveness. When MNC were cultured in the absence of mitogens, there was no difference in survival between pre- and post-epinephrine samples. Incubation of untreated MNC for 2 hr or 18 hr in vitro with various concentrations of epinephrine (10(-5) to 10(-1) mg/ml) had no effect upon the subsequent blastogenic response to mitogens. Other workers have reported that epinephrine administration causes alterations in the composition of the circulating lymphocyte pool. Taken together, these data suggest that the reduction in mitogen responsiveness after epinephrine is the result of changes in the distribution of lymphocyte subclasses in peripheral blood.     

In vitro glucocorticoid inhibition of steroid-treated residual circulatory human lymphocytes

Various doses of glucocorticoids given in vivo caused a similar degree of maximal lymphopenia. The sensitivities of mitogen-induced proliferation to the suppressive effects of glucocorticoid added in vitro were studied in residual lymphocytes obtained after steroid injection. Methylprednisolone (MP) administered intravenously depleted circulatory lymphocytes and reduced markedly the proliferative responses of residual cells to mitogens (phytohemagglutinin and concanavalin A) 4 to 8 hr after the injection. The addition of MP in vitro to the residual cells further inhibited the cell proliferation. The degrees of proliferation inhibition induced by in vitro MP were compared in cells obtained at various intervals after MP injection. At each specific mitogen concentration, lymphocytes obtained at various intervals were inhibited to a similar degree by MP in the cultures. There was no evidence that cells obtained at the period of maximal lymphopenia, 4 to 8 hr after MP injection, were more resistant to the inhibition of glucocorticoid added in vitro. Hence, the residual lymphocytes were not “steroid-resistant” in the sense of proliferative responses to T-cell mitogens. These results indicate the mechanism of lymphocyte sequestration is unrelated to the suppressive effects of glucocorticoid on cell proliferation.     

In vitro effects of retinoids on murine thymus-dependent and thymus-independent mitogenesis

The effects of three retinoids: all-trans-retinoic acid (RA), 13-cis-retinoic acid (13-cis-RA), and N-(4-hydroxyphenyl)retinamide (4-HPR) on murine splenic lymphocyte proliferative responses to mitogens were evaluated. The responses to T-cell mitogens, PHA and Con A, and a T-cell-dependent B-cell mitogen, PWM were significantly potentiated by these retinoids. However proliferative responses to a B-cell mitogen, Escherichia coli LPS were unaffected or inhibited. All three retinoids at concentrations ranging from 10(-6) to 10(-15) M significantly potentiated Con A-induced proliferative responses. In response to PWM, 10(-13) M RA, 10(-12) M 13-cis RA, and 10(-11) M 4-HPR were the lowest concentrations producing significant potentiation. Endpoint concentrations of retinoids significantly potentiating responses to PHA were; 10(-9) M RA, 10(-8) M 13-cis RA, and 10(-6) M 4-HPR. These responses were independent of retinol contained in fetal calf serum supplemented medium since responses were reproduced in serum-free medium devoid of retinol. Optimal potentiation by retinoids of responses to these T-cell-dependent mitogens were found at superoptimal concentrations of mitogen suggesting a selective inhibition of T-suppressor cells. Thus, potentiation of T-cell-dependent mitogen responses provides the most sensitive biological assay yet described for detection of retinoid activity and is a reproducible system to explore the cellular and molecular mechanisms of retinoid-mediated immunopotentiation.     

The effects of corticosteroid hormones and thyroid hormones on lymphocyte viability and proliferation during development and metamorphosis of Xenopus laevis

Abstract. Metamorphosis in the South African clawed frog, Xenopus laevis , is characterized by a striking loss of lymphocytes in the thymus, liver, and spleen. Changes in the proliferative responses of splenocytes and thymocytes to T cell mitogens and semi-allogeneic cells are also observed at metamorphosis. Because the levels of circulating thyroid hormones (TH) and corticosteroid hormones (CH) increase dramatically during the climax of metamorphosis, we have investigated the possible role of TH and CH as mediators of the changes in lymphocyte numbers or lymphocyte function. Here we report on the in vitro effects of CH and TH on lymphocyte viability and on phytohemagglutinin-P (PHA)-stimulated lymphocyte proliferation at prometamorphosis and climax of metamorphosis. We have observed consistently significant inhibition of proliferation by corticosterone. In contrast, we have observed inconsistent inhibition of proliferation by both thyroxine (T₄) and triiodothyronine (T₃). In short-term studies, the viability of thymocytes and splenocytes was reduced in the presence of CH but not TH.
These observations are consistent with a hypothesis that loss of larval lymphocytes and changes of lymphocyte function at metamorphosis may be due to elevated concentrations of CH rather than TH.
Because CH have been shown to enhance TH-induced effects during metamorphosis, we looked at the combined effects of these agents on PHA-stimulated lymphocyte proliferation. While each agent was inhibitory in several experiments, there was no significantly greater inhibition when splenic lymphocytes were cultured with both.     

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.     

Magnetic field inhibits isolated lymphocytes' proliferative response to mitogen stimulation

We aimed to find out how the exposure of isolated lymphocytes to a pulsed magnetic field (MF) affected their in vitro proliferative response to mitogenic stimulation. Cells were exposed to MF of various intensities (0.3, 0.6, and 1.2 T) at a constant frequency of 30 Hz, for a period of 60, 180, and 330 s. Then, the proliferative response of splenocytes was induced by optimal concentrations of concanavalin A (Con A; mitogenic toward T cells), bacterial lipopolysaccharide (LPS; mitogenic toward B cells), or pokeweed mitogen (PWM; mitogenic toward both populations). We found that the exposure of lymphocytes to the MF profoundly inhibited their proliferative response to mitogens. The suppressive action of the MF on B and T cell proliferation was intensified when a cooperative response of those two lymphocyte populations was simultaneously induced by PWM. The inhibitory effect of MF depended on the exposure time and MF intensity. Prolonged exposure and/or a stronger intensity of the MF weakened its inhibitory influence on the response of lymphocyte to mitogenic stimulation. The data show that an exposure to MF may influence the activity of lymphocytes in their response to mitogenic stimuli.