Early Events in Lymphocyte Transformation by Phytohaemagglutinin

Synthesis and phosphorylation of three main nuclear protein fractions were studied in human lymphocytes stimulated by phytohaemagglutinin (PHA). The first fraction to be synthesized and phosphorylated after induction was that of the acidic proteins, followed by that containing the soluble proteins. Synthesis of histories commenced 24 h after exposure to PHA. Distinctive patterns of both synthesis and phosphorylation of the acidic proteins were recorded at different times in the cell cycle, which may reflect activation or suppression of specific cellular functions. Phosphorylation of the histones also occurred, as an early event during lymphocyte transformation and also much later, at the time of DNA synthesis.     

Lymphocyte response to phytohemagglutinin: Intracellular volume and intracellular [K+]

The effect of phytohemagglutinin (PHA) on lymphocytes was examined with respect to free intracellular water volume and intracellular [K⁺]. At a cell concentration of 30 × 10⁶ lymphocytes/ml in modified Hank's Buffered Salt Solution (HBSS) in the presence of 10% human AB serum, addition of PHA at 3 mg/ml resulted in a 24–27% decrease in free intracellular water space within 30 to 60 minutes and a return to control level after three hours. A larger change in intracellular water (44%) was observed under similar conditions in the absence of serum. The absolute intracellular K⁺ content did not change after PHA addition, but the cell water volume decrease arising from PHA addition resulted in a 29% increase in intracellular [K⁺] at 60 minutes. The decrease in lymphocyte water volume induced by PHA was also observed for concanavalin A which stimulates lymphocyte proliferation, but not for wheat germ lectin, an agglutinating agent which is not mitogenic. Thus, volume regulation may be closely associated with the mitogenicity of these compounds.     

In vivo evolution of the Aeromonas punctata polyhydroxyalkanoate (PHA) synthase: isolation and characterization of modified PHA synthases with enhanced activity

In vivo random mutagenesis of the polyhydroxyalkanoate (PHA) synthase gene from Aeromonas punctata was performed employing the mutator strain Escherichia coli XL1-Red. About 200,000 mutants were screened on Nile red-containing medium and five mutants with enhanced fluorescence were selected. Four of these mutants exhibited enhanced in vivo and in vitro PHA synthase activity. Mutant M1, which carried the single mutation F518I, showed a five-fold increase in specific PHA synthase activity, whereas the corresponding mediated PHA accumulation increased by 20%, as compared with the wild-type PHA synthase. Mutant M2, which carried the single mutation V214G, showed a two-fold increase in specific PHA synthase activity and PHA accumulation only increased by 7%. Overall, the in vitro activities of the overproducing mutants ranged from 1.1- to 5-fold more than the wild-type activity, whereas the amounts of accumulated PHA ranged over 107–126% of that of the wild type. Moreover, all mutants mediated synthesis of PHAs with an increased weight average molar mass, but the molar fractions of 3-hydroxybutyrate and 3-hydroxyhexanoate remained almost constant. In vivo random mutagenesis proved to be a versatile tool to isolate mutants exerting improved properties with respect to PHA biosynthesis. Electronic Publication     

Comparison of the responses of density gradient separated lymphocytes to phytohemagglutinin and histocompatibility antigens

Rat peripheral blood leukocytes were fractionated into 5–9 subpopulations by centrifugation on discontinuous density gradients of bovine serum albumin. Responses of the various fractions to phytohemagglutinin (PHA) in vitro were compared with their responses to alloantigens in the mixed lymphocyte interaction in vitro and in a graft versus host reaction in vivo. The results showed that: (a) Cells from each of the gradient fractions responded to alloantigens both in vitro and in vivo, (b) Only cells of intermediate density responded vigorously to PHA at a concentration which optimally stimulated unfractionated cells, (c) Low density lymphocytes could be stimulated by 3–9-fold lower concentrations of mitogen. (d) Cells from low and high density fractions, which alone responded poorly to PHA, showed enhanced responses when mixed. These findings may have a significant bearing on the use of the in vitro response to PHA for evaluating the overall function of thymus derived cells in clinically related studies.     

Regulation of self-renewal of human T lymphocyte colony-forming units (TL-CFUs)

Formation of human T lymphocyte colonies in semisolid medium from T lymphocyte colony-forming units (TL-CFUs) under stimulation of phytohemagglutinin (PHA) has been reported by several authors. These TL-CFUs were present in unsensitized lymphocyte populations. We report here that such TL-CFUs are capable of renewing themselves. This was observed when colony cells from primary T cell colonies that developed in the presence of PHA were replated in methylcellulose medium containing irradiated autologous leukocytes and PHA. We have also been able to demonstrate serial transfer of TL-CFU for up to six passages. At each passage, colony-forming frequency was determined from the proportional relationship between the number of new colonies obtained and the number of colony cells plated. Examination of the number of new colonies derived from each individual T cell colony ("burst size of TL-CFU") showed that most colonies contained very few new TL-CFU and only a very small number of colonies contained many new TL-CFU. The distribution of burst sizes could be well fitted to a gamma distribution, in agreement with prediction from a stochastic model. We have identified an activity that enhanced the mean TL-CFU burst size three to ten times. This work provides the first evidence in vitro that self-renewal of human T lymphocyte progenitor cells can be stimulated by specific regulatory proteins.     

Stimulation of phagocytic function by factors inhibiting leukocyte and macrophage migration in humans

Fractions containing macrophage migration inhibition factor (MIF) and leucocyte migration inhibition factor (LIF) were obtained using Sephadex G-200 filtration from supernatant fluids of human lymphocyte cultures stimulated by PHA. The fractions were tested for the ability to affect migration and phagocytic activity of target cells. Peripheral blood leucocyte migration capacity was inhibited by the fraction with the molecular mass of 60,000-70,000 D (LIF), while migration activity of mouse peritoneal exudate cells was suppressed by the fraction with the molecular mass of 20,000-30,000 D (MIF). MIF- and LIF-containing fractions increased almost three-fold Fc-receptor-mediated phagocytic activity of neutrophils.     

Mechanisms of phytohaemagglutinin (PHA) stimulation of normal human lymphocytes: ‘trigger’, ‘push’ or both?

Abstract. The growth in volume of human peripheral blood lymphocytes after stimulation with various concentrations of PHA was measured with an electronic particle counter. The percentage of growing cells and averaged values describing their growth rates during the elapsed period of culture were estimated by fitting to the observed data the volume distributions derived from a mathematical model. With sub-optimal doses, the percentage of cells stimulated, and their incremental growth rate, increased with increasing dose of PHA, but the time-course of recruitment into the G₁-phase was similar with all PHA concentrations studied. The results provide strong support for the 'trigger' hypothesis that there is a distribution of stimulation thresholds within the lymphocyte population: consequently, increasing mitogen concentration will be expected to result in increased numbers of responding cells within the suboptimal concentration range.     

Partial characterization of immunosuppressive proteins from bovine uterine luminal secretions

Unfractionated and fractionated uterine luminal protein (ULP) secretions collected from nonpregnant and pregnant beef cows on Day 17 post-breeding were tested in vitro for suppression of lymphocyte blastogenesis to the mitogen phytohemagglutinin (PHA). In replicated experiments, ULP from nonpregnant and pregnant cows was separated into five molecular weight (M_r) fractions with Sephacryl S-200. Unfractionated (25 to 400 μg/ml) and fractionated (25 to 100 μg/ml) ULP was added to cultures containing 5 × 10⁵ bovine lymphocytes and 0.4 μg of PHA in a complete culture medium. At 48 hr, 0.5 μCi of ³H-thymidine was added to cultures, and cells were harvested at 60 h by automation. Thymidine incorporation data were expressed as percentage of control (no ULP) values. Unfractionated and all S-200 ULP fractions from nonpregnant and pregnant cows suppressed (P<0.05 to 0.001) lymphocyte blastogenesis to PHA, but to varying degrees of suppression. Unfractionated ULP was more suppressive (P<0.05) for pregnant than nonpregnant cows, which was likely due to the greater (P<0.05) immunosuppressive activity of S-200 fractions I (>219,000 M_r) and V (∼14,000 M_r) from the pregnant cows. At 25 μg ULP/ml, mean (± SEM) % of control values for fraction I from pregnant and nonpregnant cows were 9.1 ± 3.3 and 36.6 ± 8.5%, respectively (P<0.05). Values for fraction V were 15.7 ± 6.5 and 46.6 ± 6.1%, respectively (P<0.01). Within each reproductive class, fractions I and V were more suppressive (P<0.05) than fractions II, III and IV. Immunosuppression was not mediated by lymphocyte cytotoxicity.     

The response of human peripheral blood lymphocytes to phytohemagglutinin: determination of cell numbers.

Optimization and definition of conditions for studying lymphocyte function in vitro resulted in exponential proliferation of lymphocytes from day 2 to day 5 with an average doubling time of 20 hr. The number of cells in culture on day 5 was 5–10 times as great as the number initially planted and 10–20 times as great as the number surviving in culture on day 2. An improved pronase-cetrimide technique was used to determine the number of viable lymphocytes as a function of time after addition of PHA. The volume changes in nuclei, obtained after cetrimide treatment, were quantitated using a curve-fitting computer program.The response could be described in terms of an induction phase (0–2 days) characterized by a decrease in cellularity and an increase in nuclear volume, a proliferation phase (2–5 days) characterized by an exponential proliferation and a continued increase in the number of cells having a large nuclear volume, and a lysis phase (5–14 days) characterized by a decrease in cellularity and a decrease in nuclear volume. The results reported here suggest that the ratio of the number of cells cultured to the volume of culture medium was crucial for optimal transformation and proliferation, 10⁵ cells/ml producing far better responses than 10⁶ cells/ml.     

PHA and endotoxin stimulation of human lymphocytes separated by albumin density gradient centrifugation.

Venous blood from eight healthy subjects was divided into four fractions on a discontinuous albumin density gradient. The percentage recovery of lymphocytes was 82.3%; the purity of the lymphocyte fractions was 83.6%. The lymphocytes were cultured with PHA and Endotoxin, and the samples were analysed after 24, 48, and 72 hours. After PHA stimulation immunoblasts appeared up to 59.3% in the cultures from the 19-21% albumin fraction. After Endotoxin stimulation the maximum (75.8%) was reached in the heavy (25-27% albumin) fraction. Thus, it is concluded that the lymphocytes which can be stimulated with both the mitogens have different densities, the PHA-stimulable T lymphocytes being ligther than the Endotoxin-stimulable B lymphocytes. It is also concluded that as a mitogen Endotoxin is equal to PHA.     

Changes in the proliferative response of human lymphocytes in vitro on exposure to subcellular components of Bordetella pertussis]

Different components of B. pertussis were found to have a similar inhibitory effect on thymidine-3H incorporation caused by phytohemagglutinin (PHA) in the culture of lymphocytes taken from donors immunized with tetanus toxoid. However, the same fractions of B. pertussis produced differential effects on the reaction of lymphocyte proliferation in response to tetanus toxoid: murein-"ontaining membranes enhanced thymidine-3H incorporation, RNA-containing fractions reduced it and water-soluble components of disintegrated B. pertussis produced no effect on lymphocyte proliferation.     

Specific Role of Each Human Leukocyte Type in Viral Infections: II. Phytohemagglutinin-treated Lymphocytes as Host Cells for Vesicular Stomatitis Virus Replication In Vitro

The mitogenic agent, phytohemagglutinin (PHA), added to human mixed leukocyte cultures and to lymphocyte cultures converted small lymphocytes into lymphoblasts and increased lymphocyte susceptibility to vesicular stomatitis virus (VSV). Maximum virus yields were 30- to 1,000-fold higher in PHA-treated than in control cultures. VSV replicated to peak titers before lymphocytes were morphologically transformed by PHA, and virus titers fell as lymphoblast destruction began. PHA neither induced significant VSV replication in polymorphonuclear leukocyte cultures, nor increased the large virus yields in monocyte cultures. The treatment of PHA with heat, digestive enzymes, rabbit anti-PHA serum and serial dilutions failed to dissociate that portion of the PHA extract responsible for the conversion of lymphocytes into virus-susceptible cells from those components responsible for leukoagglutination or lymphocyte transformation.     

Effect of colchicine, vinblastine and cytochalasin B on human lymphocyte transformation by phytohemagglutinin

The effect of colchicine, vinblastine, and cytochalasin B has been investigated on the phytohemagglutinin (PHA) induced transformation and DNA synthesis of human lymphocytes. The three drugs produced, at an appropriate concentration, inhibition of DNA synthesis and lymphocyte transformation, if added prior to PHA. Inhibitory concentrations of cytochalasin B were no longer effective in preventing DNA synthesis if added 2 h after exposure to PHA; on the other hand, colchicine and vinblastine were effective even if they were added 16 h after PHA. Studies of lymphocyte aggregation to beads of Sepharose with chemically bound PHA suggest that the effects of these drugs do not seem to lie primarily on blocking PHA binding to the lymphocyte membrane, but rather on a subsequent step(s).     

Separation of human lymphocyte subpopulations. I. Transformation characteristics of rosette-forming cells

Lymphocytes were isolated from human peripheral blood by carbonyl-iron treatment and Ficoll-Isopaque centrifugation. The lymphocytes were allowed to form rosettes with sheep erythrocytes, either uncoated (E) or coated with antibody and complement (EAC).In 32 experiments E rosettes were separated from free lymphocytes on a Ficoll density gradient. Thus, depleted (non-E) and enriched (E) fractions were obtained. It was found that in the original suspension 24 ± 7.2% of the lymphocytes formed rosettes with EAC and 56 ± 8% with E. In fraction non-E these values were 56 ± 11.4 and 22 ± 8.9%, respectively; in fraction E 8 ± 3.8 and 79 ± 8.8%. Moreover, the percentages of Ig-bearing cells among the fractions were found to follow closely those of CRL.In a series of lymphocyte culture experiments these fractions were compared with the original suspension and a control suspension (rosetted, not separated), as well as with a recombined population (non-E + E). It was found that fraction non-E showed an increased response to PHA and PWM, and an enhanced MLC stimulatory capacity, whereas fraction E was decreased in these respects. Moreover, fraction E displayed significantly reduced spontaneous DNA synthesis.It is concluded that the responses to PHA or PWM, as well as the capacity to stimulate allogeneic cells, are not solely dependent on the cells forming rosettes with E.     

Immunosuppressive alpha globulin from bovine thymus. I. Preparation and assay

DEAE chromatography at pH 5.0 of the saline-soluble proteins from bovine thymus glands yields a protein fraction similar in activity to the immunosuppressive alpha-2 globulins previously described from bovine and human serum. Of 32 preparations 16 had consistent and reproducible suppressive activity to DNA synthesis in phytohemagglutinin (PHA)—stimulated lymphocytes in concentrations ranging from 50–600 μg/ml in vitro. In vivo immunosuppression, not directly related to the degree of in vitrolymphocyte suppression, occurred in doses of 4–5 mg per mouse, and was assessed by the hemagglutinin response to sheep red blood cells. A variety of other protein and nucleic acid fractions were not suppressive in these assays; in particular, fractions A and B, which precede the immunosuppressive Fraction C in the elution from DEAE-cellulose, are neither suppressive, nor do they significantly alter the effects of Fraction C.     

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

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

Growth of human T lymphocyte colonies from whole blood: culture requirements and applications

Growth of human lymphocyte colonies from whole blood following stimulation with PHA, Con A, or PPD is described. Individual colony cells were identified as T lymphocytes on the basis of surface marker and enzyme cytochemical characterizations. Colony formation increased as a power function over a wide range of cell concentrations above a critical minimal concentration. The whole blood culture system eliminates possible selective effects of lymphocyte colony techniques utilizing gradient-enriched lymphocyte fractions and more closely approximates the in vivo milieu. The whole blood colony method is more sensitive for the detection of low-level radiation effects on lymphocytes than widely used tests that measure 3H-thymidine incorporation. In preliminary studies, we used the whole blood method to determine the relative radiosensitivity of lymphocytes from humans with various hematopoietic disorders, and observed abnormalities in mitogen responsiveness and colony formation in some of the patient groups. This method has wide application for studies in cellular and clinical immunology.     

Brief Communication Commercial Pha Preparations Contain Different Mitogenic Components

Three commercially available purified phytohaemagglutinin (PHA) preparations have been examined both chemically and biologically. Marked differences were observed in both composition and mitogenic effect on lymphocytes; the existence of at least two distinct mitogenic fractions in PHA was confirmed. A number of commercial preparations of PHA have become available for use as mitogens in lymphocyte cultures. Since there is no cross-standardization of biological activity between these preparations, workers have used their own discretion about which to use and whether further purification is desirable. Three commercial purified PHA preparations have been examined, and marked biological and chemical differences have been found.     

PHAMCL biosynthesis systems in Pseudomonas aeruginosa and Pseudomonas putida strains show differences on monomer specificities

The production of PHA from plant oils by Pseudomonas species soil isolated from a sugarcane crop was evaluated. Out of 22 bacterial strains three were able to use efficiently plant oils to grow and to accumulate PHA. Pseudomonas putida and Pseudomonas aeruginosa strains produced PHA presenting differences on monomer composition compatible with variability on monomer specificity of their PHA biosynthesis system. The molar fraction of 3-hydroxydodecanoate detected in the PHA was linearly correlated to the oleic acid supplied. A non-linear relationship between the molar fractions of 3-hydroxy-6-dodecenoate (3HDdΔ₆) detected in PHA and the linoleic acid supplied was observed, compatible with saturation in the biosynthesis system capability to channel intermediate of β-oxidation to PHA synthesis. Although P. putida showed a higher 3HDdΔ₆ yield from linoleic acid when compared to P. aeruginosa, in both species it was less than 10% of the maximum theoretical value. These results contribute to the knowledge about the biosynthesis of PHA with a controlled composition from plant oils allowing in the future establishing the production of these polyesters as tailor-made polymers.     

Human chorionic gonadotropin: effects of crude and purified preparations on lymphocyte responses to phytohemagglutinin and allogenenic stimulation.

The factors that prevent maternal immunologic rejection of the histoincompatible fetus are not understood. High levels of human chorionic gonadotropin (HCG) are present in the placenta, and several reports have noted suppresion of mitogen-induced lymphocyte transformation when cultures were supplemented with crude preparations of HCG. Purified HCG and multiple lots of crude HCG obtained from different suppliers were examined for their ability to suppress lymphocyte transformation produced by phytohemallutinin (PHA) or allogeneic stimulation. Crude preparations of HCG produced suppression of the lymphocyte stimulation induced by low doses of PHA, but the suppression could be overcome completely by increasing the PHA dose. The purified preparations of HCG produced no suppression of lymphocyte responses, even at the lower PHA dose. Purified HCG did not give a dose-related suppression of allogeneic lymphocyte responses, and crude lots of HCG gave highly variable results. One lot of crude HCG produced spontaneous stimulation of lymphocytes. Isoelectric focusing of HCG preparations demonstrated multiple bands, and lymphocyte suppression may be secondary to these additional unidentified proteins. The failure of pruified HCG to suppress lymphocyte responses makes it unlikely that the absence of maternal rejection of the fetus is due to high placental levels of HCG.