Phytohemagglutinin effects on cultured human neural crest tumors

Summary Two human melanoma lines, RPMI-7931 and HS-294, respond to mitogenic stimulation by PHA. A dose-response curve for these lines can be demonstrated with maximal stimulation at 16 and 32 μg per ml and inhibition at 75 to 250 μg per ml PHA. The mitogenic effects of PHA were inhibited byn-acetyl-d-galactosamine. Neuroblastoma cells also exhibit a similar but less important dose-response to PHA. These data indicate that human melanomas and neuroblastomas may have PHA receptors or mechanisms for mitogenic stimulation which are analogous to those observed with normal lymphocytes.     

Pertussis toxin-sensitive G-proteins are not involved in activation of T-lymphocytes.

Multiple effects of pertussis toxin (PT) on Jurkat T-cells can be distinguished on the basis of their dose-response and their kinetics. High concentrations of PT deliver to cells an activating signal resulting in a rapid rise in [Ca2+]i followed by IL-2 synthesis. This activation is accompanied (within 2 h) by a down-regulation of the CD3/TCR complex from the cell surface. Cells then become refractory towards stimulation by CD3 mAb or PHA. All these effects, referred to as 'mitogenic effects', present the same dose-response curves with an EC50 of 0.5 micrograms/ml. Short term effects (PT-induced Ca2+ movements, down-regulation of CD3/TCR complex and inhibition of PHA and CD3-induced Ca2+ signal) are observed under conditions where no PT-induced ADP-ribosylation can be detected. In contrast, ADP-ribosylation of the 40,000 alpha-subunit of G-proteins requires a sustained (18 h) incubation of intact cells in the presence of low concentration (EC50 = 0.3 ng/ml) of PT. Dose-response curves for PT-dependent ADP-ribosylation and mitogenic effects are separated by three orders of magnitude. Covalent modification of G-protein has no effect on CD3-induced increase in [Ca2+]i and IL-2 synthesis induced by a combination of phorbol ester and either CD3 mAb, PHA or calcium ionophore. These data indicate that transduction of the mitogenic signal does not involve a PT-sensitive G-protein. Furthermore, inhibition of mitogenic signals following PT treatment results from a PT-induced activation leading to a down-regulation of the CD3/T cell receptor complex.     

Inhibition of phytohemagglutinin-stimulated lymphocyte transformation by neutral SH-dependent proteinase of the spleen

The effect of neutral SH-dependent proteinase of the spleen on transformation of human peripheral blood lymphocytes has been studied. Proteinase (1--10 micrograms/ml) inhibits phytohemagglutinin-stimulated (PHA) lymphocyte transformation and has no effect on spontaneous transformation. Proteinase does not alter the mitogenic properties of PHA and possesses no cytotoxicity. Therefore, inhibition of stimulated lymphocyte transformation is either resultant of proteinase direct action on the cell or associated with the inhibitor factor formation.     

Effects of arachidonic acid and other unsaturated fatty acids on mitogenesis in human lymphocytes.

The effect of fatty acids and other lipids on mitogenic responses in cultured human peripheral blood lymphocytes was studied. Several-fold enhancement of tritiated thymidine incorporation was observed at 0.1 to 5.0 micrograms/ml concentrations of arachidonic acid. Other unsaturated fatty acids produced less marked changes. Increased responsiveness was demonstrable in a variety of media including RPMI 1640 supplemented with 10% fetal calf serum. Changes were also observed in uridine incorporation, total cell number, and blast transformation, indicating that the effect was not on thymidine transport or pool size per se. Arachidonic acid failed to affect PHA binding, indicating that the lectin-cell interaction was not altered. Higher concentrations of fatty acids were inhibitory.     

Delayed hypersensitivity and migration inhibition in two lines of mice genetically selected for high or low responsiveness to phytohemagglutinin

Delayed-type hypersensitivity (DTH) and cell migration inhibition (MI) were studied in two lines of mice genetically selected for the high (Hi/PHA) or low (Lo/PHA) in vitro response of their lymphoid cells to phytochemagglutinin (PHA). A rapid photoelectric procedure for reading cell migrations enabled the study of MI over a wide range (10 log) of antigen concentrations in vitro. Hi/PHA mice required immunization with a 10 times higher dose of ovalbumin (OVA) in Freund's complete adjuvant (FCA) than Lo/PHA mice for a comparable response in DTH (footpad swelling) and MI of their induced peritoneal exudate cells (PEC). Lo/PHA spleen showed marked bizonal MI on Day 5 after immunization with low doses (0.1 and 0.5 micrograms) of OVA in FCA, one peak being obtained in presence of in vitro concentrations of 10(-3) or 10(-2) micrograms/ml OVA and another peak at 1 or 10 micrograms/ml, whereas Hi/PHA spleen showed stimulation of migration. In contrast, MI in Lo/PHA spleen failed to persist beyond Day 19, whereas it appeared progressively in Hi/PHA spleen, being maximal by Day 27. Low-zone inhibition in Hi/PHA spleen and PEC was lacking or poor even after immunization with higher doses of OVA in FCA. The implications of these findings are discussed.     

The effect of butylated hydroxytoluene, with and without cortisol, on stimulated lymphocytes.

In the present work we undertook to ascertain whether butylated hydroxytoluene (BHT), which is used in food as an antioxidant, is capable of either inhibiting human lymphocyte stimulation or acting synergistically with cortisol and prednisolone to the same end. BHT cytotoxicity was observed at concentrations higher than 100 micrograms/ml. In the concentration range of 0.0 to 60.0 micrograms/mL, BHT showed no effect on the uptake of 3H-thymidine by PHA stimulated lymphocytes. However, at 50 micrograms/mL BHT suppressed mixed lymphocyte reaction (MLR). A synergistic effect with regard to suppression of PHA stimulated lymphocytes was observed when the cells were incubated with BHT in the presence of either cortisol or prednisolone.     

Genetic control of T-lymphocyte mitogenesis in chickens

The in vitro mitogenic response to PHA and Con A was determined in three inbred lines of chickens. Lymphocytes from one line consistently showed a greater stimulation by PHA than the other two lines. Analysis of F₁ crosses and backcrosses indicated that this quantitative difference was controlled by more than one gene. More substantial differences in Con-A stimulation were also observed between the three lines, and the data indicated that separate genetic systems were controlling the variation in PHA and Con-A stimulation. Analysis of F₁ crosses, backcrosses and assortative matings between backcrosses revealed that the variation in Con-A stimulation was controlled by at least two major genes, one of which may be linked to the major histocompatibility complex. Surprisingly, one line appeared to be segregating for Con-A stimulation in spite of an inbreeding coefficient greater than 0.98.     

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.     

Total and exchangeable calcium in lymphocytes: Effects of PHA and A23187

Calcium has been suggested as an internal second messenger when lymphocytes are stimulated by mitogens to enter the cell cycle. We have assessed the effect of 2 lymphocyte stimulants, the plant lectin phytohemagglutinin (PHA) and the calcium ionophore A23187, on human lymphocyte nucleic acid synthesis, total cell calcium content, and ^{4 5}Ca labeling. We have used an ultrasensitive method for the measurement of total cell calcium in the same samples used for radiolabeling. Mitogenic concentrations of A23187 (～ .25 μ mole/liter) caused an increase in both total cell calcium and ^{4 5}Ca labeling. These increases were almost completely blocked by inhibitors of mitochondrial respiration, suggesting that the calcium increment after ionophore treatment was located in the mitochondria. In contrast, total cell calcium was not altered at optimal mitogenic PHA concentrations (0.1 μg/ml and above). However, at the minimum PHA concentrations that caused stimulation (0.025 to 0.1 μg/ml), the dose response of ^{4 5}Ca uptake was very similar to that of DNA sysnthesis. Importantly, we could not stimulate DNA synthesis with PHA without increasing lymphocyte ^{4 5}Ca labeling. Thus, an increase in total cell calcium is not essential for mitogenesis; however, an increase in ^{4 5}Ca exchange is closely associated with the mitogenic effects of A23187 and PHA.     

Functional properties of the 50 kd protein associated with the E-receptor on human T lymphocytes: suppression of IL 2 production by anti-p50 monoclonal antibodies

Monoclonal antibody 9.6 is specific for a 50 kd T cell surface protein (p50) associated with the sheep erythrocyte (E)-receptor on human T lymphocytes. This antibody interferes with many T cell functions. We have examined the effect of antibody 9.6 on lymphocyte proliferation and interleukin 2 (IL 2) production triggered by mitogens, soluble antigens, and alloantigens to elucidate the mechanism(s) of its immunosuppressive action. At concentrations as low as 50 ng/ml, 9.6 suppressed lymphocyte proliferation and the elaboration of IL 2 by T cells stimulated by PHA, alloantigens, or low concentrations of the phorbol ester TPA (less than or equal to ng/ml). Furthermore, in cultures stimulated by a combination of PHA plus TPA, 9.6 did not inhibit the acquisition of IL 2 receptors but inhibited proliferation and IL 2 production. Immunoaffinity-purified IL 2 completely restored lymphocyte proliferation in cultures inhibited by 9.6. Studies of kinetics of inhibition by 9.6 showed that this antibody inhibited lymphocyte proliferation induced by PHA, alloantigen, and PPD even when added at 24, 48, and 72 hr, respectively, after the initiation of these cultures, suggesting that 9.6 does not block lectin binding or antigen recognition by T cells and that it can inhibit lymphocyte proliferation even after cells have undergone one or more rounds of cell division. A dose-response analysis of lymphocyte proliferation induced by PHA or by TPA demonstrated that the degree of inhibition by 9.6 decreased with increasing concentrations of these mitogens. Antibody 9.6 did not inhibit lymphocyte response induced by optimal concentrations of PHA (50 to 100 micrograms/ml; PHA-M) but inhibited proliferation of maximally induced lymphocytes by using a synergistic combination of low concentrations of PHA (5 micrograms/ml, PHA-M) plus TPA (1 ng/ml). Taken together, these findings indicate that 1) 9.6 inhibits lymphocyte proliferation by affecting IL 2 production, 2) 9.6 does not inhibit the acquisition of 9.6 receptors induced by a synergistic combination of PHA plus TPA, and 3) p50 molecules may be involved in multiple pathways of T cell activation.     

Involvement of proteolytic acivity in early events in lymphocyte transformation by phytohemagglutinin

The stimulation by phytohemagglutinin (PHA) of DNA synthesis in cultured blood lymphocytes of guinea pig was markedly inhibited by addition of leupeptin, a well-characterized, powerful protease inhibitor of tripeptide nature. About 30 to 40 per cent inhibition was observed at 40 μg/ml of leupeptin when leupeptin was added 30 min prior to or together with PHA. Per cent inhibition by the appropriate amount of leupeptin was proportional to the amount of PHA added in the range of 0.6 to 3.0 μg of PHA at which the per cent inhibition reached maximum. This inhibitory effect of leupeptin on PHA stimulation was abolished when the lymphocytes were preincubated with PHA for more than 10 min before addition of leupeptin or preincubated with leupeptin for more than 60 min prior to PHA addition.     

Analysis of the direct effects of prostaglandins on human sperm function

Time-exposure photomicrography and interspecies in-vitro fertilization procedures have been used to examine the influence of prostaglandins on human sperm function. An analysis of variance indicated that the presence of PGs in the incubation media was associated with a significant increase in sperm velocity and the frequency of sperm head rotation, although there were no differences between individual PGs in the degree of stimulation observed. Changes in the penetrating ability of human spermatozoa were detected after exposure to PGs, particularly PGE-1 and PGE-2. PGE-2 induced a sustained increase in penetration rates at all doses of greater than 8.4 micrograms/ml, while exposure to PGE-1 gave a bell-shaped dose-response curve which exhibited a peak between 8.4 and 33.3 micrograms/ml and progressively fell to reach control levels at the maximum concentration tested of 270 micrograms/ml. A combination of PGE-2 and PGE-1 produced a dose-response curve similar to that for PGE-1 alone, while exposure to PGF-2 alpha was without effect. Seminal extracts containing predominantly 19-hydroxy PGE-1, or equal amounts of 19-hydroxy PGE-1 + 2 induced a slight, but significant, rise in penetration rates while a combination of PGs representing the major components of human seminal plasma was without significant effect. We conclude that certain prostaglandins may have a direct action on the functional competence of human spermatozoa.     

Ascaris suum: human lymphocyte mitogenic factor content

Ascaris suum extract was found to contain a mitogenic factor which stimulates human lymphocytes. The extract (100 micrograms protein/ml) induced an increase in [3H]thymidine incorporation into human lymphocytes at a level similar to that obtained with pokeweed mitogen (11 micrograms/ml). Stimulation of mitosis appeared to be more effective with T lymphocytes than non-T lymphocytes. The mitogenic activity of the extract was reduced by only 27% when treated at 56 C for 30 min or when immersed into boiling water for 1 min. The extract was fractionated into four protein peaks by Sephacryl S-200 column chromatography. Lymphocyte mitogenic activity was observed in the first half of the first protein peak. Allergenic activity, assessed by the passive cutaneous anaphylaxis test in rats, was observed in the latter half of the same peak. These results suggest that A. suum contains both an allergen and a mitogenic substance.     

Cyclic nucleotide levels in activated human T lymphocytes

Phytohemagglutinin (PHA)-MR69 was used to activate human peripheral blood lymphocytes. The PHA concentration in the range of 1 to 4 micrograms/ml was optimal for lymphocyte stimulation. Cell activation occurred only in the presence of Ca ions and 5 min after it was followed by an increase in cGMP but not in cAMP. Immunomodulator, methylene bisphosphonic acid (10(-7) M and 4.10(-5) M), did not influence in culture. The cAMP and cGMP levels in PHA activated cells. Methylene bisphosphonic acid similar to 1-hydroxyenthylidene-1,1-bisphosphonic acid, aminomethylene bisphosphonic acid and phosphoneacetic acid on its addition to the culture (in the range from 10(-8) to 10(-4)M) 60 min before PHA or 24 or 48 hours after PHA administration produced no effect on the [3H]-incorporation into PHA-activated human blood lymphocytes.     

Direct evidence that the insulin receptor mediates a mitogenic response in cultured human fibroblasts

To define the role of the insulin receptor in mediating a mitogenic response in cultured human fibroblasts, the effects of specific monoclonal antibodies against the insulin and the type I IGF receptor on insulin-stimulated [3H]thymidine incorporation were investigated. Insulin stimulated [3H]thymidine incorporation in a biphasic fashion. In the first phase, a half-maximal effect was observed at 20 ng/ml, and a seemingly maximal effect was obtained at 100-1000 ng/ml. With 10 micrograms/ml insulin, a secondary increase in [3H]thymidine incorporation was seen which was similar to the maximal effect of IGF-I. These [3H]thymidine incorporation results were corroborated with cell replication studies. MC-51, a highly specific monoclonal antibody for the insulin receptor, inhibited the stimulation of [3H]thymidine incorporation by 25 ng/ml of insulin. AlphaIR-3, a monoclonal antibody specifically directed against the type I IGF receptor, had no significant effect on insulin-stimulated [3H]thymidine incorporation at low (10-1000 ng/ml) concentrations of insulin. However, alpha IR-3 interfered with the incremental increase in [3H]thymidine incorporation observed at 10-100 micrograms/ml insulin. These data demonstrate that insulin, at low concentrations, is capable of stimulating DNA synthesis and replication of human fibroblasts through interaction with its own receptor, while at supraphysiological concentrations, much of insulin's mitogenic effect is mediated through the type I IGF receptor.     

Wheat germ agglutinin and concanavalin A inhibit the response of human fibroblasts to peptide growth factors by a post-receptor mechanism

The effect of plant lectins on amino acid uptake and DNA synthesis in cultured human skin fibroblasts stimulated by various peptide mitogens was studied. Wheat germ agglutinin (WGA), at a concentration of 5 micrograms/ml, which by itself had little effect on 3H-aminoisobutyric acid (AIB) uptake, markedly inhibited stimulation of 3H-AIB uptake by somatomedin-C, insulin, epidermal growth factor (EGF) and platelet-derived growth factor. This inhibition could not be overcome by increasing the concentration of peptide added. Neither WGA nor concanavalin A (Con A) significantly affected basal 3H-thymidine incorporation. However both lectins, at concentrations of 5-20 micrograms/ml, decreased EGF- and insulin-stimulated DNA synthesis while succinyl Con A, a divalent lectin derivative, did not. The inhibitory effects of lectins on mitogenic stimulation were reversed by alpha-methyl mannose (Con A) or N-acetylglucosamine (WGA), and were not due to a reduction in the binding of growth factors to their receptors. It is concluded that certain lectins noncompetitively inhibit the response of human fibroblasts to multiple peptide mitogens at the post-receptor level, possibly by interfering with lateral mobility and aggregation of mitogen-receptor complexes.     

Lectin-induced actin polymerization in human lymphocytes: A possible signal for mitogenesis

Employing the DNase I inhibition assay, a decrease in G-actin is demonstrated in human mononuclear cells following stimulation with mitogenic lectins concanavalin A (Con A) and phytohemagglutinin (PHA), as well as a nonmitogenic lectin, wheat germ agglutinin (WGA). The decrease in G-actin can be prevented by pretreatment of cells with cytochalasin E, indicating that the decrease is likely due to conversion to F-actin. Thus, the receptor-mediated actin polymerization is common to both the mitogenic as well as the nonmitogenic lectins. The maximal decrease in G-actin with Con A and PHA occurs at the same concentrations of the lectins that give optimal mitogenic responses. It is a distinct possibility that actin polymerization could be one of the signals necessary for the initiation of mitogenesis. The difference between a mitogenic and a nonmitogenic lectin may lie in the fact that a second signal (or signals), derived from macrophages, may not be generated by a nonmitogenic lectin such as WGA.     

Lectin extracts of champedak seeds demonstrate selective stimulation of T lymphocyte proliferation.

The effect of extracts of champedak (Artocarpus integer) seed lectin on the proliferation of normal human lymphocyte was investigated. The IgA1 binding lectin was demonstrated to stimulate the proliferation of human peripheral blood mononuclear cells. Action of the lectin on enriched T and B cell populations demonstrated T lymphocyte specificity. The lectin was not mitogenic to B lymphocytes. Optimal stimulation of proliferative response was achieved when cells were subjected to 5 days exposure to the crude lectin at 20 micrograms/ml.     

Enhanced production of human interferon-gamma by the use of a combination of inducers--phytohemagglutinin, picibanil and tumor promoting agent

Phytohemagglutinin (PHA), picibanil (OK432) and tumor promoting agent (TPA) were tested in various combinations for optimal induction of human interferon-gamma (HuIFN-gamma). It was found that the use of a mixture of all 3 inducers resulted in IFN production 2-3 times higher than either PHA (10 micrograms/ml) in combination with TPA (5 ng/ml) or picibanil (10 micrograms/ml) alone. The IFN was produced by T lymphocytes and could be neutralized by specific IFN-gamma antiserum. It was pH 2.0 labile and species specific.     

Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture

A medium consisting of a rich basal nutrient mixture supplemented with bovine insulin (10 micrograms/ml), human transferrin (10 micrograms/ml), human cold-insoluble globulin (5 micrograms/ml), and ethanolamine (0.5 mM) supported the growth of the A431 human epidermoid cell line in the absence of serum with a generation time equal to that of cells in serum-containing medium. Addition of epidermal growth factor (EGF) to this culture medium at concentration mitogenic for other cell types resulted in a marked inhibition of A431 cell growth. Inhibitory effects of EGF were observed at 1 ng/ml and near-maximal effects were observed at 10 ng/ml. The inhibitory effect of EGF could be reversed by the omission of EGF in subsequent medium changes and could be prevented by the addition of anti-EGF antibody to the culture medium. Inhibition of A431 cell growth by EGF also could be demonstrated in serum-containing medium.