Transferrin support of stimulated lymphocytes

Summary Transferrin was tested for its ability to replace serum in supporting mitogen and allogeneic cell stimulated human lymphocyte proliferation. Although transferrin, at concentrations greater than 5 μg/ml, was incapable of completely replacing the serum used to support phytohemagglutinin, Concanavalin A, and pokeweed mitogen, stimulated human lymphocytes, in the absence of serum it significantly augmented the proliferative responses observed for mitogen, yet not allogeneically-stimulated cells. Augmentation is not due to a nonspecific protein effect and appears to be independent of the metal content of transferrin. The mechanism of growth support appears to involve an effect of transferrin following the G₁ phase in the initial cell cycle. This work was supported by NIH Grants AM-17554 and AI-05155.     

Non-histone chromatin proteins of B lymphocytes stimulated by lipopolysaccharide.

The synthesis of non-histone chromatin proteins and nucleoplasmic proteins has been followed during lipopolysaccharide-induced division and differentiation of murine B lymphocytes. Synthesis was measured by pulse labelling with [3H]leucine, extraction of proteins was under conditions designed to prevent proteolysis and analysis of labelled proteins was by polyacrylamide gel electrophoresis. The average specific activity of non-histone chromatin proteins increased 3-fold, to a maximum, after culture for 24 h with lipopolysaccharide. Comparison of the relative synthesis of individual proteins (stimulation index) reveals three distinct responses: (1) those in the largest group show low stimulation indices, generally less than two; (2) a group of four proteins have indices between 4 and 5; (3) two proteins (molecular weights 21 000 and 22 000) both show an index of 5 at 24 h rising to between 7 and 8 by 48 h when the average specific activity is falling, coinciding with the period of rapid differentiation to high rate IgM secretion. Additionaly at this time, a newly labelled protein (Mr = 36 500) appears in the nucleoplasm followed by a second protein (Mr = 63 000) appearing between 48 and 72 h. The patterns of change are consistent with an overall increase in non-histone chromatin proteins synthesis, necessary for cell division, with superimposed specific changes in synthesis of non-histone chromatin proteins which could be related to regulation of cell differentiation.     

DNA polymerases from non stimulated and phytohemagglutinin stimulated normal human lymphocytes

The presence and some properties of DNA polymerases isolated from normal human lymphocytes, non stimulated and stimulated by phytohemagglutinin, are described. In the non stimulated lymphocytes two cytoplasmic DNA polymerases are found, one eluting from DEAE cellulose at 0.07 M NaCl (CI_n) and the other at 0.13 M NaCl (CII_n). In the nuclear soluble fraction only one enzyme activity is found (NI_n) which does not adsorb to DEAE cellulose. In the cytoplasm of stimulated lymphocytes only one enzyme activity is detected (CI_s) which elutes from DEAE cellulose at 0.12 M NaCl. The nuclear soluble fraction contains two activities, NI_s, which does not adsorb to DEAE cellulose, and NII_s, which elutes from DEAE cellulose at 0.07 M NaCl. Some properties of the different enzymes are described which indicate that NI_n and NI_s enzymes are clearly different from the others.     

Cytidine 5''-diphosphate reductase activity in phytohemagglutinin stimulated human lymphocytes.

The optimal conditions and the effect of deoxyribonucleoside triphosphates were determined for CDP reductase activity in PHA-stimulated lymphocytes. The enzymatic reaction showed an absolute requirement for ATP. In the absence of ATP, only dATP showed a minor stimulation of the reduction of CDP to dCDP. During transformation the CDP reductase activity reached a maximum at the same time as the four deoxyribonucleoside triphosphate pools, corresponding to mid S-phase at about 50 h after PHA addition. The DNA polymerase activity reached a maximum at 57 h.     

Non-histone chromatin proteins of B lymphocytes stimulated by lipopolysaccharide. II. Phosphorylation.

Stimulation of mouse lymphocytes with the B lymphocyte specific mitogen lipopolysaccharide results in an increased rate of phosphorylation of non-histone chromatin proteins. An initial small increase in phosphorylation occurs during the first 2 h and a much larger increase after 24 h of culture with mitogen. The phosphorylated nuclear and cytoplasmic proteins were analysed by polyacrylamide gel electrophoresis and the stimulation index of each prominent peak measured. It was inferred that selective stimulation of the phosphorylation of individual proteins had occurred from: (1) the range of stimulation indices for different proteins, and (2) the appearance, after 8 h stimulation of an apparently newly phosphorylated non-histone chromatin protein of molecular weight 115 000. The pool size of ATP was monitored and showed only small changes during the first 24 h of exposure to lipopolysaccharide. Phosphatase activity was found to be associated with lymphocyte chromatin and nucleoplasm and may help to regulate the level of phosphorylation of non-histone chromatin proteins in vivo. To preserve phosphorylated proteins during their isolation phosphatase activity was inhibited by Na2MoO4. The selective changes in phosphorylation of nuclear proteins precede, and continue during, the stimulation of immunoglobulin and DNA synthesis. Our results are thus consistent with the hypothesis that phosphorylation of non-histone chromatin proteins plays a role in the regulation of gene expression in B lymphocytes.     

X-irradiation of equine peripheral blood lymphocytes stimulated with phytohaemagglutinin in vitro.

Small lymphocytes were isolated from the peripheral blood of horses and incubated at 37 degrees C in Eagle's medium supplemented with 20 per cent foetal calf serum. The addition of phytohaemagglutinin (PHA) to the cultures resulted in: increased RNA and protein synthesis; the enlargement of the small lymphocyte into a lymphoblast-like cell; the initiation of DNA synthesis, and cell division. When survival was measured 24 hours after X-irradiation by means of phase-contrast microscopy, the lymphoblast-like cell was much more radio-resistant (D0 = 250 rad) than the small lymphocyte (D0 = 20 rad). This increase in radioresistance, however, was not observed until 12-24 hours after PHA treatment. To investigate which of the changes occurring during the transformation of the small lymphocyte was responsible for the increased resistance to irradiation, the percentage of cells surviving irradiation was compared with the percentage of cells incorporating significant amounts of 3HTdR, 3H-UR, or 3H-leucine at the time of irradiation. For this comparison, a dose of 100 rad was used because 100 rad killed essentially all of the small lymphocytes, but less than 35 percent of the cells which had become radioresistant from the PHA treatment. The results indicated that the increase in radioresistance was not associated with DNA synthesis, but instead correlated with the increase in RNA and protein synthesis which the cells had attained at the time of irradiation.     

Radiation protection of stimulated human lymphocytes by nicotinamide

Nicotinamide (NA) when added to human lymphocytes in vitro together with a mitogen, protected against the inhibition by gamma and UV radiation of stimulated cell growth. When stimulated by phytohemagglutinin (PHA), concanavalin A (Con A) or pokeweed mitogen (PWM) maximum protection has been observed with approximately 1 mM NA (dose reduction factor of 2-3). To obtain protection the cells had to be stimulated immediately after irradiation in the presence of NA. It is suggested that the intracellular level of NAD+ may be rate limiting for excision repair in human lymphocytes irradiated in the G0 phase. This level is presumably increased by exogenously supplied NA, leading to enhanced repair of DNA damage and increased survival.     

Protein synthesis in resting and stimulated human lymphocytes

Summary The ribosomal profiles in lysates from resting and phytohemagglutinin stimulated human lymphocytes have been analyzed by sucrose gradient centrifugation. The percentage of polyribosomes increased during lymphocyte transformation reaching a maximal value of 60 to 70% of the total ribosomes after 72 hours of mitogen addition. This time period coincides with maximalin vivo protein synthesis. On the other hand, in nonstimulated lymphocytes, about 25% of the ribosomal particles appeared as aggregates, independently of the incubation period.Experiments performed with homologous cell free systems containing ribosomes and supernatant fluids prepared from unstimulated or activated lymphocytes demonstrate that the mixtures containing both components from stimulated lymphocytes are several fold more active in polypeptide synthesis than the systems which contain ribosomal particles and cell sap from resting cells. Assays carried out with mixtures combining the components from both sources indicate that the increased activity depends on ribosomes as well as on the supernatant fractions.Dedicated to Professor LUIS F. LELOIR on the occasion of his 70^th birthday.     

Immunofluorescence of histone H1 in stimulated lymphocytes measured by flow cytophotometry.

The modification of histones or their redistribution during the transition from actively transcribing chromatin to the heterochromatic chromosomes seems to play a major in regulation of gene expression. The purpose of this study was to monitor the change in immunofluorescence of histone HI during phytohemagglutinin stimulation in peripheral lymphocytes. The histone antigens were prepared from pig thymus, proven to be pure by gel electrophoresis and repeatedly injected as RNA-complexes into rabbits. The antihistone HI antiserum titer was 1:4000, and there was no cross-reactivity with other histone fractions as shown by microcomplement fixation tests. Affinity chromatography purified antibody after being labeled with fluorescein isothiocyanate was able to differentially stain HeLa cells as controls and those, where histone HI had been extracted by perchloric acid treatment. The measurements were done on a Los Alamos Scientific Laboratories-flow cytophotometer cell sorter. Staining peripheral lymphocytes resulted in a bimodal distribution. The increase in number of cells with high fluorescence intensity had its maximum about 20 hr before the maximum proliferative activity of the lymphocytes as measured by number of cells in S phase with the DNA-stain mithramycin.     

Uptake of alpha-aminoisobutyric acid in pig spleen lymphocytes stimulated by sodium periodate.

The effect of sodium periodate on the ability of pig spleen lymphocytes to transport the nonmetabolizable amino acid, α-aminoisobutyric acid, was studied. NaIO₄-treated cells exhibited a lowered rate of uptake of α-aminoisobutyric acid in contrast to phytohemagglutinin- and concanavalin A-treated cells. However, when periodate-treated cells were preincubated with untreated cells for 2 h, the mixed cells exhibited twofold stimulation in the uptake of α-aminoisobutyric acid as compared to untreated cells. The increased uptake of α-aminoisobutyric acid in mixed cells was due to a change in the V but not in the K_m. The observed increased uptake of α-aminoisobutyric acid in mixed cells was inhibited (24%) by ouabain, although the level of uptake in untreated and NaIO₄-treated cells was not affected. Na⁺,K⁺-ATPase activity in mixed cells, which was ouabain sensitive, was stimulated 56%. Studies also showed that there was a decrease in the fluorescence polarization (P value) of diphenyl hexatriene in mixed cells (P = 0.21) as compared to untreated cells (P = 0.24). These results demonstrate that NaIO₄ treatment induces a change in the lymphocyte cell membrane and transport of α-aminoisobutyric acid. Incubation of NaIO₄-treated cells with untreated cells is required for the stimulatory effect in the uptake of α-aminoisobutyric acid, and the stimulation appears to be due to changes in Na⁺,K⁺-ATPase activity and membrane fluidity.