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.     

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 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.     

Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts

We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria.     

Modulatory effect of zinc on the proliferative response of murine spleen cells to polyclonal T cell mitogens

To study the effect of zinc on the proliferative response to polyclonal T cell mitogens, spleen cells from C57BL/6 mice were cultured with or without ZnCl2 and stimulated with graded doses of concanavalin A or phytohemagglutinin. Addition of 10(-4) M ZnCl2 inhibited proliferation whereas 10(-5) to 10(-6) M ZnCl2 did not modify the response to suboptimal doses of mitogen but increased DNA synthesis in cultures stimulated with high doses of mitogen (10 or 20 micrograms/ml of concanavalin A and 10 or 25 microliters/ml of phytohemagglutinin) which are supraoptimal for C57BL/6 mice, and inhibited proliferation in cultures of spleen cells from animals of this strain, low responder to T cell mitogens. In contrast, supplementation with ZnCl2 did not enhance the response to mitogen of spleen cells from high responder BALB/c mice. The enhancing effects of ZnCl2 on the proliferative response of C57BL/6 cells were not observed following depletion of adherent cells or in cultures supplemented with 5 X 10(-5) M 2-mercaptoethanol, both conditions capable of abrogating the inhibitory effect of high mitogen doses on the response of C57BL/6 cells.     

Activation of murine lymphocytes by cyclic guanosine 3′,5′-monophosphate: Specificity and role in mitogen action

Cyclic guanosine 3′,5′-monophosphate (cyclic GMP) stimulates nucleic acid synthesis in lymphocytes, and has been implicated as the intracellular effector of the actions of mitogenic agents on these cells. In the present study, we examined the specificity of the mitogenic activity of cyclic GMP and of its 8-bromo (Br) derivatives, and the effects of the T cell mitogens, concanavalin A, phytohemagglutinin, and staphylococcal entertoxin B (SEB) on the cyclic GMP content and guanylate cyclase activity of mouse splenic lymphocytes. Cyclic GMP and guanosine modestly increased the incorporation of [³H]thymidine into DNA by cultured lymphocytes, but were far less effective than their 8-Br-derivatives. However, on a molar basis the mitogenic activity of both 8-Br-guanosine and 8-Br-5′-GMP exceeded that of 8-Br-cyclic GMP, when tested in the presence and absence of serum in the culture media. Combined addition of maximal doses of these nucleotides did not give additive stimulatory effects, suggesting an action on a common subpopulation of cells, and possibly a common mechanism. By contrast, cyclic AMP, 8-Br-cyclic AMP, 8-Br-adenosine, cholera toxin and prostaglandin E₁ suppressed both basal [³]thymidine incorporation and stimulation of this parameter by T-cell line mitogens and the guanosine nucleotides. Rapid effects of concanavalin A, phytohemagglutinin, SEB, guanosine, 5′-GMP, 8-Br-guanosine, and 8-Br-5′-GMP on the cyclic GMP content of murine lymphocytes could not be demonstrated. Similarly, concanalin A, phytohemagglutinin and SEB failed to alter guanylate cyclase activity when added directly to cellular homogenates or pre-incubated with intact cels. Conversely, carbamylcholine rapidly increased lymphocyte cyclic GMP but was not mitogenic.These results are consistent with the hypothesis that cyclic GMP and cyclic AMP are antagonistic in their influence on lymphocyte mitogenesis. However, they also demonstrate that related nucleotides are more potent mitogens than cyclic GMP and suggest that activation of murine lymphocytes by concanavalin A, phytohemagglutinin and SEB may not be mediated by rapid increases in cellular cyclic GMP content. Since high concentrations of exogenous cyclic GMP and related nucleotides must be used to influence DNA synthesis, the biologic significance of this effect remains uncertain.     

Effects of alveolar surfactant aggregates on T-lymphocyte proliferation

The effects of alveolar large aggregate (LA) and small aggregate (SA) surfactant subfractions isolated from healthy adult rats on mitogen-stimulated proliferative responses of human peripheral blood mononuclear cells (PBMC) was examined. Various concentrations of total surfactant suppressed proliferation of stimulated lymphocytes by up to 95% of mitogen-stimulated cells alone. LA subfractions of total surfactant had no effect on proliferation, whereas SA significantly enhanced the lymphocyte proliferation at lower concentrations (7.8 microg/ml) compared to mitogen-stimulated cells alone. Higher concentrations of SA (62.5 microg/ml) inhibited lymphocyte proliferation. This concentration-dependent effect of SA on proliferation of PBMC was also present when cells were stimulated with various lectins including anti-CD3, concanavalin A and phytohemagglutinin. Analysis of the supernatant of mitogen-stimulated cell cultures treated with inhibitory concentrations of SA showed decreased amounts of interleukin (IL)-2, compared to cells alone, which could be reversed by adding exogenous IL-2 to the cell cultures with the SA. These results suggest that alveolar surfactant subfractions have distinct functions within the alveoli, both biophysically and with respect to their effects on the host's immunomodulatory responses.     

The regulatory effect of histamine on the immune response: characterization of the cells involved

Any immunological response is the end result of the equilibrium between many positive and negative regulatory factors. It has been recently demonstrated that histamine receptor-bearing T lymphocytes could play a role in this regulation. This work aims to study the effects of different cell populations after incubation with histamine on the proliferative response of normal lymphocytes. The histamine-incubated peripheral blood lymphocytes (PBL) lower the proliferative response of normal cells toward mitogens (phytohemagglutinin, concanavalin A) and antigens (mixed lymphocyte culture). In order to precise the cell subpopulations involved in this suppression, PBL have been depleted of adherent cells and B and T lymphocytes have been purified by a standard rosette technique. The enriched B cells do not suppress the normal response but the suppressor activity of T cells, as well as adherent cell-depleted PBL, are significantly reduced compared to the one of PBL. The initial suppressor activity is restored by addition of 1% adherent cells (and not 5 or 10%) to adherent cell-depleted lymphocytes and 10% adherent cells (not 1 or 5%) to T-enriched population. These observations suggest a role for adherent cells in this regulation.     

Interleukin 2-induced lymphocyte proliferation is independent of increases in cytosolic-free calcium concentrations

Activation of lymphocytes by mitogenic lectins initiates a sequence of events that culminates in DNA synthesis and cell proliferation. The mitogenic effects of lectins on T lymphocytes leads to the production of a group of lymphokines including the interleukins. The binding of interleukin 2 (IL 2) to its receptor results in activation of the cell leading to DNA synthesis. An increase in cytosolic-free Ca++ ([Ca++]i) is associated with activation of lymphocytes by mitogenic lectins and also appears to be a prerequisite for induction of DNA synthesis and cell proliferation. We have determined whether the proliferative response triggered by IL 2 binding to its receptor is associated with or requires an increase in [Ca++]i. Using human and murine IL 2-sensitive cell lines, we have demonstrated that the IL 2-induced proliferative response, in contrast to that induced by mitogens such as phytohemagglutinin or concanavalin A, is not accompanied by an increase in [Ca++]i as monitored by the fluorescent indicator quin-2. Furthermore, IL 2-dependent triggering of lymphoblasts occurs in the presence of extremely low extracellular calcium concentrations that prevent transmembrane calcium flux. Activation of IL 2 receptor-bearing T cells, therefore, does not appear to be associated with or to require an increase in [Ca++]i as part of the activation and signaling process. The critical step requiring calcium flux in cell signaling by mitogenic lectins must therefore occur elsewhere in the activation cascade.     

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.     

Separation of human lymphocyte subclasses by rosettes with latex-lectin particles

The specificity and affinity of eight lectins (concanavalin A, L. culinaris, P. sativum, phytohemagglutinin P, D. biflorus, soybean agglutinin, T. purpureus, and T. vulgaris) to B, T, T gamma, and T mu lymphocytes from the blood of normal subjects were determined. Lectins attached to latex particles were used to evaluate the binding of each lectin to individual cells. The rosette percentage found in each lymphocyte population expresses the specificity index and the specific sugar concentrations needed to decrease the rosette percentage by 50% is taken as the affinity index. B Lymphocytes showed a major subclass, with respect to T lymphocytes, with receptors for WGA, SBA, D. biflorus, L. culinaris, and P. sativum lectins. In contrast, T lymphocytes exhibit a greater number of cells with specific receptors for Con A, T. purpureus, and PHA lectins than B lymphocytes, the T gamma subpopulation being responsible for the specificity of the first two lectins and the T mu subpopulation for the PHA lectin.     

Optimal conditions for blastogenic response of peripheral blood lymphocytes to T and B cell mitogens in cynomolgus monkeys

Optimal conditions for the mitogen-induced proliferation of T and B lymphocytes of cynomolgus monkeys were determined. The T cell mitogens concanavalin A and phytohemagglutinin, at concentrations of 1.25–10 μg/ml and 1.25–10 μg/ml, respectively, and the T and B cell mitogen pokeweed mitogen, at concentrations of 0.2–10 μg/ml, induced high lymphoproliferative responses, the average stimulation index (SI) being 34–93. Since suitable mitogens have not been reported for monkey B cells, we tested three types of lipopolysaccharide (LPS): two derived from Escherichia coli—one extracted with phenol and one extracted with trichloroacetic acid (TCA); and one derived from Salmonella typhimurium, extracted with phenol. All three LPS had a high mitogenic effect for monkey lymphocytes, with SI of 2.3–6.4. The highest response was observed for 25 μg/ml of Salmonella LPS, and the addition of trypsin to the culture augmented the proliferative response of low or non-responder lymphocytes. © 1994 Wiley-Liss, Inc.     

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.     

Inhibition of mitogen-stimulated T lymphocyte proliferation by calcitonin gene-related peptide

The effect of calcitonin gene-related peptide (CGRP) on mouse lymphocyte proliferation stimulated by mitogens was studied. CGRP (10(-10)-10(-7) M) dose-dependently inhibited the proliferative response of mouse lymph node cells and spleen cells stimulated by T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA), whereas a B cell mitogen lipopolysaccharide (LPS) did not inhibit this response. The maximal inhibition by this peptide was 50% to 80% at 10(-8) and 10(-7) M. The addition of 10(-8) and 10(-7) M CGRP to lymph node cell cultures 24 hr after stimulation with Con A or PHA also had a significant inhibitory effect on the proliferative response. Furthermore, in the same concentration range (10(-10)-10(-7) M) CGRP increased intracellular cyclic AMP concentration in nylon wool nonadherent cells, but not in nylon wool adherent cells. CGRP had no significant effect on intracellular cyclic GMP concentration. In addition, specific binding of CGRP was observed in mouse spleen cells. Our present study suggests that CGRP inhibits the proliferative response of T lymphocytes to the mitogens by interacting with cell receptors coupled with adenylate cyclase. CGRP may be implicated in the regulation of T cell function.     

Co-mitogenic tumor promoters suppress the phosphatidylinositol response in lymphocytes during early mitogenesis

The tumor-promoting agents 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phorbol-12,13-dibenzoate inhibited the increased accumulation of [32P]phosphatidylinositol (PI) induced in mouse spleen lymphocytes by mitogenic lectins in the presence of [32P]orthophosphate. Similar inhibition of [32P]PI levels by TPA was seen in human tonsil T-lymphocytes stimulated with phytohemagglutinin. Only co-mitogenic phorbol esters prevented the [32P]PI accumulation during early mitogenesis. No increased 32P-labelling due to mitogen or decreases due to TPA was observed when cells were equilibrated with [32P]orthophosphate for 24 h prior to stimulation with mitogen, from which it is concluded that the total concentrations of phosphatidylcholine (PC) and PI are unaffected by mitogen or co-mitogen. The [32P]PI elevation but not the [32P]PC elevation was proportional to T-cell mitogenic potency for the lectins concanavalin A, divalent succinyl concanavalin A and phytohemagglutinin, and was prevented in each case by 5 X 10(-8) M TPA. Escherichia coli lipopolysaccharide did not give increased 32P incorporation into PI or PC, and TPA had no effect on 32P labelled phospholipid levels in the presence of this B-cell mitogen. The results indicate that the phosphatidylinositol response is not an invariable correlate of T-cell mitogenesis by polyclonal mitogens.     

Effect of cell-free murine liver extract on lymphocyte blastogenesis in vitro.

The effect of cell-free liver extract (LE) on the proliferation of spleen cells in vitro was examined using [³H]thymidine incorporation. LE inhibited the blastogenic response of murine lymphocytes stimulated with plant mitogens, phytohemagglutinin, and concanavalin A and in the mixed lymphocyte reaction (MLR). Suppression of cell proliferation occurred whether the LE was syngeneic or allogeneic to the responding cells. This effect was observed only when LE was present in cultures, as preincubation of cells with LE did not impair their capacity to respond to stimulation. Profound suppression of proliferation was achieved with the addition of LE to the culture up to 48 hr after the onset of stimulation. However, the inhibitory effect was readily reversible upon removal of LE from the culture. Furthermore, although LE was capable of suppressing the generation of cytotoxic lymphocytes, LE did not interfere with their capacity for cytolysis. These findings indicate the presence of a potent inhibitor of lymphocyte proliferation in a cell-free extract of murine liver.     

Increased mitogenic reactivity of normal spleen cells to T lectins induced by thymus humoral factor (THF)

When normal spleen cells were incubated for 24 hr in medium containing thymic humoral factor (THF) and then stimulated by phytohemagglutinin (PHA) or concanavalin A (Con A), a significant increase in the mitogenic reactivity of these cells was observed. When stimulation to T lectins was performed simultaneously with THF, a strong inhibition in cell reactivity was found. It seems that these opposite effects of THF on cell reactivity to T lectins are determined by the sequence of events which lead to maturation of lymphoid cells. Thymic humoral factor does not modify the response of cells to B mitogen lipopolysaccharide (LPS), thus suggesting that this maturative effect on lymphoid cells is exerted on T lymphocytes only.     

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.     

Potentiation of the T lymphocyte response to mitogens. V. Inhibition of the response to concanavalin A by supraoptimal doses or by other lectins and its aversion by LAF.

The enhanced thymidine incorporation in murine lymphocytes induced by Concanavalin A (Con A) was markedly inhibited in the presence of other lectins, which are poorly mitogenic (phytohemagglutinin {PHA} or pokeweed mitogen), or non-mitogenic (soybean agglutinin {SBA}). The level of inhibition was found to be inversely proportional to the mitogenic effect of the lectins. Our results did not support the notions that the lectins inhibit the lymphocyte responses by competing with Con A, or by activating suppressor cells. Rather, the data suggest that the lectins cause cytotoxic or cytostatic effects. The effects of the inhibitory lectins were found to resemble those of supraoptimal doses of Con A. In particular, both effects were partly averted by the lymphocyte activating factor (LAF). The mitogenic effect of LAF was not inhibited by the non-mitogenic lectin, SBA, whereas the poor responses to PHA or to moderately supraoptimal doses of Con A were markedly potentiated by this factor. It is thus suggested that LAF activity counteracts the inhibitory processes provoked by the lectins.