Interrelations of natural cellular cytotoxicity and interferon systems in immobilization stress

The suppressed cytotoxic natural killer activity was observed in the mouse spleen during 2 days after 6 h of immobilization stress. Both serum interferon level and interferon production by spleen cells under PHA, ConA and enterotoxin stimulation were also significantly decreased. The period of suppression was followed by the recovery of the activity of the two systems by day 7-9. The experimental data form the basis for the suggestion that endogenous interferon has major significance for the mechanism of disturbed natural killer activity after stress influences.     

Studies on rabbit lymphocytes in vitro : XX. Demonstration of cells reactive to more than one mitogen by mixed sequential stimulation

Rabbit peripheral blood lymphocyte (PBL) cultures stimulated by ConA and then blocked by the addition of competing sugar or antiserum after 6–15 h of ConA prestimulation respond to restimulation by PHA or PWM to a much greater extent than to continuous stimulation or delayed stimulation with PHA or PWM. This effect of mixed lectin sequential stimulation indicates that many of the same PBLs will respond to more than one mitogen, but that some cells require preactivation by one mitogen in order to respond fully to another mitogen. Thus, the number of PBLs which respond to PHA or PWM alone is much less than the number which respond following pretreatment with ConA when the pretreatment effect of ConA alone is blocked. Rabbit PBLs do not respond to LPS and preactivation by ConA does not prepare rabbit PBLs to respond to LPS.     

Mitogenic responses of peripheral blood mononuclear cells of vervet monkeys (Cercopithecus aethiops): Apparent role of adherent cells

Peripheral blood mononuclear cells (PBM) from normal vervet monkeys (Cercopithecus aethiops) were examined for blastogenic responses to concanavalin A (ConA), phytohemagglutinin (PHA), and pokeweed mitogen (PWM). The mitogen stimulated PBM in a dose-dependent manner. Response to ConA was apparently higher than for the other two mitogens. Cell density and mitogen concentration were critical parameters for optimal lymphocyte proliferation, an observation in line with that reported for other mammalian species. Depletion of an adherent cell population probably of monocyte/macrophage lineage from vervet PBM gave higher proliferative responses to both ConA and PHA, but the response without adherent cells to ConA was greater than the response without adherent cells to PHA. This latter finding is in contrast to what has been reported in many other species.     

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.     

Selenium promotes T-cell response to TCR-stimulation and ConA, but not PHA in primary porcine splenocytes

There is controversy in the literature over whether the selenium (Se) influences cellular immune responses, and the mechanisms possibly underlying these effects are unclear. In this study, the effects of Se on T-cell proliferation and IL-2 production were studied in primary porcine splenocytes. Splenocytes were treated with different mitogens in the presence of 0.5-4 μmol/L sodium selenite. Se significantly promoted T-cell receptor (TCR) or concanavalin A (ConA)-induced T-cell proliferation and IL-2 production but failed to regulate T-cell response to phytohemagglutinin (PHA). In addition, Se significantly increased the levels of cytosolic glutathione peroxidase (GPx1) and thioredoxin reductase 1 (TR1) mRNA, the activity of GPx1 and the concentration of reduced glutathione (GSH) in the unstimulated, or activated splenocytes. These results indicated that Se improved the redox status in all splenocytes, including unstimulated, TCR, ConA and PHA -stimulated, but only TCR and ConA-induced T-cell activation was affected by the redox status. N-acetylcysteine (NAC), a pharmacological antioxidant, increased T-cell proliferation and IL-2 production by TCR and ConA stimulated splenocytes but had no effect on the response to PHA in primary porcine splenocytes confirming that PHA-induced T-cell activation is insensitive to the redox status. We conclude that Se promotes GPx1 and TR1 expression and increases antioxidative capacity in porcine splenocytes, which enhances TCR or ConA -induced T-cell activation but not PHA-induced T-cell activation. The different susceptibilities to Se between the TCR, ConA and PHA -induced T-cell activation may help to explain the controversy in the literature over whether or not Se boosts immune responses.     

Synergistic effect of TPA and T-cell mitogens in nonmammalian vertebrates

Summary The phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) was used as a comitogen with the plant lectins phytohemagglutinin (PHA) and concanavalin A (ConA) in short-term cultures of whole blood from nonmammalian vertebrates. Stimulation with TPA in addition to standard mitogens resulted in a synergistic effect, consistently yielding more metaphases than cultures stimulated with either PHA, ConA, or TPA alone and is successful with blood samples as small as 0.1 ml. The increased mitotic index makes it possible to use different banding procedures for systematic studies. Also, because the amount of blood needed is so small, this procedure, unlike other published techniques, does not require the destruction of smaller animals to do chromosome studies. This work was supported in part by National Institute of Environmental Health Science Grant 5-T32-ES07015-08 to the Environmental Toxicology Center at the University of Wisconsin-Madison.     

Effect of orally administered plant lectins on intestinal liquor accumulation and amylase activity in rats

Short-term effects of orally administered plant lectins, with special reference to the Phaseolus vulgaris agglutinin (phytohaemagglutinin, PHA), were studied in growing rats. The orally administered PHA elicited a dose-dependent accumulation of liquor with elevated pH in the proximal small intestine. Although the concentration of alpha-amylase activity did not change, total alpha-amylase activity slightly, but significantly increased in the gut. When a panel of plant lectins with different carbohydrate binding specificities was tested at the dose of 100 mg/kg body weight, most of them stimulated the secretion of liquor, but the total alpha-amylase activity was increased only by PHA, ConA or WGA.     

Modulation of suppressor activity by thymosin

Modulation of suppressor cell activity by thymosin was evaluated in vivo in a murine tumor system, and in vitro on human lymphocytes. We showed that splenocytes from tumor bearing mice were able to enhance tumor growth in a syngeneic system. This enhancement was T dependent and disappeared by Thymosin treatment. A decrease of Tumor size associated with injection of bone marrow cells treated by thymosin was observed. In the human system we showed that ConA stimulated lymphocytes were able to suppress the response of normal lymphocytes to PHA, PWM and ConA, and in MLC. This effect was significantly blocked in presence of thymosin fraction 5.     

Effects of lectins on cAMP production and steroidogenesis in cultured adrenal tumor cells

The plant lectins, concanavalin A (conA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA) stimulate steroidogenesis in cultured adrenal tumor cells. ConA maximally stimulated steroidogenesis at 100 μg/ml following an approximate 4 h lag phase. ConA stimulation was completely inhibited by α-methyl-d-mannopyranoside and the WGA effect was prevented by N-acetyl-d-glucosamine. It was also found that conA alone did not cause a measurable increase in either intra- or extracellular cyclic adenosine 3′5′-monophosphate (cAMP) production. In addition, conA when added simultaneously with adrenocorticotropin (ACTH) doubled the intra- and extracellular cAMP production over controls treated with ACTH alone. This enhancement effect was dose dependent. When Y-1 cells were preincubated with conA and then treated with either ACTH or cholera enterotoxin (CT) there was a dose- and time-dependent inhibition of induced cAMP production. In the case of CT, the inhibitory effect occurred even with simultaneous addition of conA and CT. This effect was reversed by addition of both α-methyl-d-mannopyranoside and washing with Eagle's minimal essential medium (MEM) 1 h after CT had bound to its receptor. This reversal was not apparent for the inhibitory effect of conA on ACTH-induced cAMP production which occurred after 2 h of preincubation with conA. These results demonstrate that conA, as well as the other plant lectins, interact with specific membrane receptors to reversibly stimulate steroid production as well as enhancing or inhibiting ligand-induced cAMP production in cultured adrenal tumor cells.     

Application of Concanavalin A during immune responsiveness skin‐swelling tests facilitates measurement interpretation in mammalian ecology

The skin‐swelling test is a simple and widespread method used in field ecological research to estimate cellular immune responsiveness in animals. This immunoecological test is based on measuring the magnitude of tissue swelling response at specific times following subcutaneous application of an experimental pro‐inflammatory stimulant. In the vast majority of studies across vertebrate taxa, phytohemagglutinin (PHA) is used as a universal stimulant. Given the complexity of immune response activation pathways of PHA, however, interpretation of test results can be ambiguous. Goal of this study was to improve methodology of the skin‐swelling test to decrease this ambiguity. Here, we present an alternative protocol aimed at facilitating interpretation of skin‐swelling data for mammals. Based on previous evidence suggesting that mammalian T cells are readily activated by Concanavalin A (ConA) in vitro, we compared cellular immune responses in vivo to PHA and ConA as an alternative pro‐inflammatory stimulant in mice. We measured magnitude of tissue swelling and compared it with intensity of blood cell infiltration into tissue over a 72‐hour interval. Our results corroborate that PHA and ConA show important differences in both dynamics and response amplitude in rodents. ConA induces stronger swelling with a distinct leukocyte activity pattern and higher pro‐inflammatory cytokine (interleukin 6 [IL‐6] and interferon gamma[IFN‐γ]) expression than PHA during peak response (24‐h post‐treatment). Furthermore, unlike PHA, magnitude of swelling was positively associated with cellular activity (number of neutrophils infiltrating tissue) following ConA injection. We conclude that ConA is the more suitable stimulant for skin‐swelling tests in mammals. This is because of the molecular binding specificity in the two lectins, that is, ConA specifically activates T cells while PHA also triggers erythroagglutination. We propose that ConA be used in all future ecological testing in mammals as it exhibits better performance and its application facilitates immunological interpretation of skin‐swelling test results.     

Inhibition of ConA erythroagglutination by alkali-treated lipopolysaccharide

Bacterial lipopolysaccharide treated by hydrolysis in basic solution (hLPS) and then added to suspensions of human erythrocytes markedly inhibited erythroagglutination by concanavalin A (ConA), soybean agglutinin (SBA), and Phaseolus vulgaris phytohemagglutinin (PHA). Likewise, red cells presensitized by exposure to hLPS and then suspended in hLPS-free buffer were not agglutinated by ConA. However, when hLPS was not added to buffer both SBA and PHA strongly agglutinated erythrocytes presensitized by hLPS. Also, the binding of ³H-labeled ConA to red cells was unaffected by presensitization of the cells with hLPS. It appears, therefore, that membrane-associated hLPS localizes in the ConA receptor regions of erythrocyte membranes and inhibits ConA erythroagglutination by disruption of receptor responses to bound ConA or alteration of the active subunit structure of bound ConA.     

Effects of mitogens on sodium-potassium transport, H-ouabain binding, and adenosine triphosphatase activity in lymphocytes

The effect of various mitogens was studied on sodium (Na⁺) potassium (K⁺) transport, ³H-ouabain binding, and adenosine triphosphatase (ATPase) activity in human and sheep peripheral lymphocytes. Concanavalin A (ConA), phytohemagglutinin (PHA), horse anti-lymphocyte serum (ALS), and anti-IgG antisera, in order of decreasing potency, stimulated in particular the ouabain-sensitive K⁺ pump influx, while the cardiac glycoside-insensitive K⁺ leak flux was only slightly affected. Sheep lymphocytes primed in vivo with human IgG as antigen also responded with K⁺ pump flux activation when exposed to the antigen in vitro. Both PHA and ConA also stimulated active Na⁺ efflux in human lymphocytes. Apparently these mitogens activate the Na⁺K⁺ pump system in the lymphocyte membrane—an assumption supported by the finding of a significant activation of the ouabain-sensitive Na⁺K⁺-ATPase. From rate studies of ³H-ouabain binding carried out at 37 °C in presence and absence of sodium azide, and at 0 °C, it is concluded that PHA alters the rate of ouabain uptake to these cells. Thus PHA may alter the affinity of the pump for ouabain, equivalent to an increased cation turnover per pump site. However, our findings do not completely discount the possibility that PHA also increases the total number of ouabain molecules bound and therefore of Na⁺K⁺ pumps.     

Relationship between the capacity to be stimulated of lymphocyte subpopulations and the RAI staging in patients with chronic lymphocytic leukemia]

By means of the incorporation rate of 3H thymidine into the lymphocytes of patients with chronic lymphatic leukaemia the possibility of stimulating them by using different mitogens was checked and compared with normal persons. The examination covered 11 patients treated with extracorporeal irradiation of the blood (ECIB), 5 patients treated with a chlorambucil therapy, and 10 untreated patients who were classified according to the staging system proposed by RAI. The lymphocytes of the peripheral blood were stimulated as mixed and isolated T and B-lymphocytes in the microculture by using the mitogens PHA, PWM, ConA, and LPS. In all CLL patients there was a diminished stimulation rate of a mixed lymphocyte population. A relation existed between the seriousness of the stage and the diminution of the incorporation rate of 3H thymidine. A corresponding correlation could not be identified in untreated CLL patients. Isolated T-lymphocytes revealed better results of stimulation than the total population. As to their function B-lymphocytes showed a dependance on the kind of therapy. In the mixed lymphocyte culture of normal persons the best findings could be observed after stimulation with PHA, that is also valid for CLL patients. PHA, PWA, ConA, and LPS were suitable as substances stimulating B-lymphocytes with different efficacy in normal persons and CLL patients. Both collectives showed the best results in the T-lymphocyte culture after stimulation with LPS.     

Mitogen response of peripheral blood and splenic lymphocytes and effect of 2-mercaptoethanol in tumor-bearing mice

Summary A murine osteosarcoma in which the number of tumor cells can be continually monitored by measuring the circulating plasma alkaline phosphatase levels was used to determine the effect of tumor burden on peripheral blood and splenic lymphocyte response to mitogens. In animals with tumors of different sizes, the pattern of response of the peripheral blood lymphocytes (PBL) to mitogens is different from that of splenic lymphocytes. PBL response to ConA, PHA, and LPS was initially depressed, but response to PHA and LPS recovered later, as the tumor burden exceeded 6%. However, the recovery of LPS response was not consistent, in that recovery was not seen when the tumor burden was 5%–6%. Response to ConA remained depressed. Splenic lymphocytes showed progressive decline of PHA response. Treatment of tumor-bearing mice with 2-mercaptoethanol (2-ME) restored the ConA response of PBL in 56% of mice. Treatment with 2-ME did not restore PBL response to PHA or LPS. Abbreviations used in this paper: PBL, peripheral blood lymphocytes; peripheral blood lymphocytes; ConA, concanavalin A; PHA, phytohemagglutinin; LPS, lipopolysaccharide; 2-ME, 2-mercaptoethanol; FCS, fetal calf serum; AP, alkaline phosphatase; OS, osteosarcoma     

Progressive loss in vitro immune response with tumor growth.

Peripheral blood lymphocytes from rats carrying a transplantable hepatoma were cultured in the presence of phytohemagglutinin (PHA), concanavalin A (ConA) or dextran sulfate (DS) at various times after tumor cell inoculation or after its surgical removal. Mitogen-induced lymphocyte transformation, measured by tritiated thymidine incorporation, declined as the tumor size increased, especially when cells were cultured in autologous serum. The response to PHA and ConA declined prior to the response to DS. This inhibition could not be removed by extensive washing of the cells, alteration of serum concentration, time of incubation or mitogen dose. Culture for 24 hr prior to the addition of high doses of mitogen resulted in partial restoration of the PHA and ConA, but not DS, responses. Previously inhibited responses also returned when the tumor was surgically removed. Spleen cells from animals with large tumors were also inhibited.     

Lectin-mediated stimulation of DNA synthesis in cultures of embryonic neural retina cells

Plant lectins and other agents which are mitogenic for lymphocytes and fibroblasts were tested for their effects on DNA synthesis in primary monolayer cultures of neural retina cells from 10-day chick embryos. Concanavalin A (ConA), phytohemagglutinin (PHA), wheat germ agglutinin (WGA), and anti-retina cell antiserum significantly stimulated [³H]TdR incorporation; the maximum increase was reached 15 h after exposure of the cultures to these agents. Cells stimulated by ConA to synthesize DNA subsequently divided. The divalent succinyl derivative of ConA had a considerably lesser effect than the native tetramer, suggesting that cross-linking of cell surface components may be an important aspect of the changes that lead to the stimulation of DNA synthesis in these cells.Using [¹²⁵I]ConA, the average number of ConA-binding sites per 10-day retina cell was estimated to be 1.7 × 10⁶ (under the culture conditions employed); binding of the lectin to 25–50% of these sites was sufficient to elicit the maximal stimulation of DNA synthesis. Continuous association of the lectin with the cell surface for up to 8 h was essential for the maximal effect, since removal of the lectin from the cell surface (with α-methyl mannose) prior to this time reduced or prevented the stimulation of DNA synthesis.The stimulation by ConA of DNA synthesis in these cultures was dependent on the cell density and was reduced or absent at lower than optimal densities. Examination of this effect suggested that the frequency of intercellular contacts or specific cell associations play a role in the responsiveness of these cells to stimulation of DNA synthesis by ConA.     

Endorphinergic modulation of immune function: potent action of the dipeptide glycyl-L-glutamine

Glycyl-L-glutamine (GLG), the carboxy terminal dipeptide of B-endorphin, inhibits brainstem neuronal activity. It also occurs along with B-endorphin in pituitary secretory vesicles suggesting a neurosecretory role for this dipeptide. We have evaluated potential immunoregulatory actions of this compound using the Phytohemaglutinin (PHA) blastogenesis and the concanavalin A (ConA) suppressor cell induction assays. GLG in low doses (10(-12) M) enhanced the response of human lymphocytes to PHA induced blastogenesis, however; with higher doses of the dipeptide (10(-7) M) immunosuppression was consistently observed. In the suppressor cell induction assay, when GLG was used together with ConA, we observed a dose-dependent inhibition of suppressor activity. These results clearly indicate that GLG produces a dose dependent bidirectional modulation of at least two indicies of immune function, and confirm the presence of a second pituitary peptide with the potential for potent immunomodulatory action.     

Leukocyte migration inhibitory factor (LMIF) production in unidirectional mixed lymphocyte cultures.

Puromycin treatment of lymphocytes was used to develop a one-way test for leukocyte migration inhibitory factor (LMIF) production in the mixed lymphocyte culture (MLC) reaction. Lymphocytes incubated with this protein synthesis inhibitor induced a vigorous mediator production by nontreated allogeneic cells, being themselves unable to respond to stimulator cells. When puromycin-treated cells were stimulated with the mitogens PHA, ConA, or PWM, overall protein and DNA synthesis were significantly decreased with concomitant abolishment of LMIF production. Viability of stimulator lymphocytes was found to be essential for generation of the mediator in MLC reaction.     

Membrane characteristics of old and Rauscher leukemia virus infected mouse red blood cells

Some membrane characteristics of normal and Rauscher leukemia virus (RLV)-infected mouse red blood cells (RBC) were compared, both with regard to total populations and young and old groups of cells. Osmotic fragility, density distribution of cells and agglutinability by poly- -lysine (pLys), concanavalin A (ConA), phytohemagglutinin (PHA) and soybean agglutinin (SBA), were examined. RBC from RLV-infected mice were agglutinated at a higher rate and to a higher degree than normal mice RBC by pLys and by the lectins PHA and ConA. These RBC were generally osmotically more resistant and contained a young cell population of unusually high specific gravity. Comparison of RBC from RLV-infected mice with old RBC from normal mice showed some common membrane characteristics. Similarly to old RBC, RBC from RLV-infected mice have a high specific gravity and high agglutinability by pLys. However, they differ in that the RBC from RLV-infected mice are osmotically more resistant and are agglutinated by ConA; they are also agglutinated at a higher rate by PHA.