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.     

Aliphatic aldehydes inhibit the proliferative response of human peripheral blood lymphocytes to phytohemagglutinin and alloantigens

The effect of methyl glyoxal (MG) and various 4-hydroxyalkenals on the response of human peripheral blood lymphocytes (PBL) to phytohemagglutinin (PHA) or allogeneic cells has been investigated. Pretreatment of PBL with aldehydes significantly reduced the percentage of blast-transformed cells and [3H]thymidine incorporation into DNA in both PHA- and alloantigen-stimulated cultures, hydroxyalkenals being more effective than MG. Further experiments showed that these aldehydes also affected the proliferation of pre-activated lymphocytes. The percentage of blasts as well as [3H]thymidine incorporation into DNA were significantly decreased when the aldehydes were added until 72 h after application of the mitogenic stimulus.     

Establishment of dose-response relationships between doses of Cs-137 gamma-rays and frequencies of micronuclei in human peripheral blood lymphocytes

It has been suggested that the yield of micronuclei in human peripheral blood lymphocytes could be used as a biological dosimeter in cases of radiation exposure. In the present study micronuclei were induced in lymphocytes by exposing human blood samples in vitro to various doses of Cs-137 gamma-rays. The blood samples were then cultivated using the cytokinesis block method. Coded programs were employed to establish the relationships between the frequencies of micronuclei and various doses of gamma-rays. The best fit was obtained by the linear-quadratic model, Y = c + aD + bd2, where Y is the yield of micronuclei, D is the dose in Gy and c, a, b, are constants. It seems there is a correlation between the yields of MN in mononuclear cells and the corresponding doses of radiation. Therefore an attempt was made to include these MN in the calculation of the dose-response relationship.     

Initial changes in ouabain-dependent and ouabain-resistant potassium fluxes in human peripheral blood lymphocytes exposed to phytohemagglutinin

The early changes in potassium fluxes in PHA-treated human lymphocytes were studied. The increase in both ouabain-sensitive potassium influx and ouabain-resistant potassium efflux at the optimal mitogenic concentration of PHA was observed. No change in potassium content was found.     

Mitogenic receptors on human peripheral blood lymphocytes: the interaction of Phaseolus vulgaris erythroagglutinating phytohemagglutinin and anti-thymocyte globulin on the human peripheral blood lymphocyte membrane.

A horse anti-human thymocyte antibody (ATG) obtained from the Upjohn Company was shown to stimulate DNA synthesis in human lymphocytes with a time course and magnitude of radioactive thymidine uptake comparable to that seen with phytohemagglutinin (E-PHA) and concanavalin A (Con A). Low mitogenic or nonmitogenic concentrations of intact ATG or its Fab fragments inhibited E-PHA-induced mitogenesis, whereas the response to Con A was unaffected. Competitive binding studies with ATG and E-PHA revealed mutual inhibition of binding to lymphocytes suggesting that E-PHA and the ATG share a common receptor site on the cell surface. ATG binding was unaffected by Con A. From the analysis of the binding data and the inhibition of mitogenesis, it appears that at least part of the E-PHA response in human lymphocytes involves receptors that are not acted on by Con A.     

Non-mitogenic binding of phytohemagglutinin to lymphocytes

³H-PHA is bound to lymphocytes both at 37°C and at 0°C. However, the lectin is rapidly shed from cells which have been pulsed at 0°C and no DNA-synthesis takes place even if the pulsed cells are kept at 37°C for 64 hours. Cells pulsed at 37°C and subsequently at 0°C do not shed the bound lectin. They are induced to synthesize DNA when subsequently incubated at 37°C for 64 hours.     

Subpopulations of human peripheral blood lymphocytes.

Multiple staining protocols have been developed for the classification of subpopulations of human peripheral blood lymphocytes. Of the non-T (E^?) cells, roughly half (10–20% PBL) have receptors for complement components as detected with complement-coated zymosan particles, but do not show Fc receptors as detected with Ripley IgG-coated human RBC. The other half are C^?, Fc⁺, with a small percentage possessing both receptors. The C⁺, Fc^? cells can be subdivided into cells which are IgM⁺ (75%) or IgM^?. Cells with Fc receptors detected with aggregated IgG were IgM⁺.     

Premature chromosome condensation : Conformational changes of chromatin associated with phytohemagglutinin stimulation of peripheral lymphocytes

Phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes were shown to be distinguishable from unstimulated lymphocytes by the technique of premature chromosome condensation (PCC). Greater than 70% of the PCC from lymphocytes stimulated by incubating with PHA for 18–22 h showed greatly extended PCC as compared with only 30% in the unstimulated cultures. This decondensation pattern of the PCC paralleled with the previously reported increase in the template activity of chromatin. The PCC method can be useful in determining the proliferative potential of bone marrows of leukemic patients during and after chemotherapy.     

The transport and distribution of monovalent cations during the blast transformation of human peripheral blood lymphocytes activated by phytohemagglutinin]

Potassium (rubidium) influx, sodium and potassium contents, as well as size distribution, DNA and protein contents and synthesis have been examined in PHA-activated human lymphocytes within 0.5-72 h. A complex set of ionic events was found to include at least two stages of the increase in potassium and sodium contents per g cell protein and in ouabain-sensitive potassium influx which are preceded by a decrease in potassium content by almost 17% within the first 2-5 h. The kinetics of potassium and sodium changes has own pattern for each of cations, thus indicating definite changes in the ouabain-resistant transport of potassium and sodium during the G0----G1----S progression. The late increase in potassium content per g cell protein was found to correlate with the growth in cell size. This finding confirms the rule which was stated earlier for other animal cells, i. e. cells that prepare to proliferate are to raise their potassium per g cell protein up to the level of 0.8-1.0 mmole (Vereninov. Marakhova, 1986).     

Nonpermissivity of human peripheral blood lymphocytes to adenovirus type 2 infection.

The capacity of freshly explanted human peripheral blood lymphocytes (PBL) to support the replication of human adenovirus type 2 (Ad2) was investigated. Unlike other types of human cells, PBL were found to be highly nonpermissive. Ad2 adsorbed 30 to 40% of both T and non-T cells. Virus uncoating was very slow and inefficient, resulting in a 40-fold reduction compared with HEp-2 cells. On a population basis, viral DNA synthesis was reduced 460-fold and infectious virus production was reduced 10(6)-fold. Only 0.35% of PBL produced infectious centers, yielding 0.8 PFU per infected cell. Phytohemagglutinin stimulation increased DNA synthesis 23-fold, infectious centers 11-fold, and virus yield 14-fold. We conclude that resting human PBL are highly nonpermissive to Ad2 infection and that phytohemagglutinin can only marginally lift this nonpermissiveness.     

Increase of fructose-1,6-diphosphatase activity in cultured human peripheral lymphocytes and its suppression by phytohemagglutinin.

The specific activity of fructose-1,6-diphosphatase in freshly isolated human peripheral lymphocytes is usually less than 10% of what can be found in normal animal livers. The enzyme activity is strongly inhibited by AMP and is also inhibited by its substrate, fructose-1,6-diphosphate, at concentrations higher than 20 μM. In cultured lymphocytes the enzyme activity gradually increases so that by day 3 of the incubation the specific enzyme activity could be 15 to 80 fold higher than the preincubation level. The increase would continue for at least 10 days if the culture medium is periodically renewed. When phytohemagglutinin is present in the culture medium, the increase of the enzyme activity is completely suppressed. No free soluble enzyme inhibitor could be detected in phytohemagglutinin-treated cells by mixing and dialysis experiments.