Search

The chromatin structure of a diploid precursor B-cell line (REH), in vitro-stimulated normal B-lymphocytes, and reactive and malignant lymph node B-lymphocytes was studied by staining formaldehyde-fixed, permeabilized cells with the DNA-specific fluorophore 7-aminoactinomycin D (7-AMD) and measuring single-cell fluorescence by flow cytometry. Resting peripheral blood B- and T-lymphocytes (G0 cells) bound low amounts of 7-AMD (7-AMD- phenotype), while G1 REH cells and purified B-cells stimulated with anti-mu + B-cell growth factor bound nearly twice as much 7-AMD (7-AMD+ phenotype). 7-AMD binding increased up to threefold and the differences in binding between G0 and G1 cells were nearly abolished when nuclei were isolated prior to fixation or when fixed whole cells were treated with DNase 1. 7-AMD binding increased in parallel with autofluorescence and approximately linearly with time during the G0-G1 transition of in vitro stimulated B-cells, as was determined by simultaneous measurements of 7-AMD fluorescence and autofluorescence or fluorescence of fluorescein isothiocyanate-labeled antibodies to the early activation antigen 4F2 and to the transferrin receptor. In cell suspensions from lymph node biopsies, the 7-AMD+ phenotype was a property of tumor cells in patients with high grade non-Hodgkin's lymphoma (H-NHL, Kiel classification, 5/5); cells with this phenotype were only found in one of nine low grade non-Hodgkin's lymphoma samples (L-NHL, 1/9). The other (8/9) L-NHL samples and the reactive lymph node contained only 7-AMD- cells. All tumors were diploid. The correlation observed between 7-AMD binding and DNase 1 susceptibility of DNA in chromatin (P less than 0.001) suggests that 7-AMD binding is a marker of general transcriptional activity. Surprisingly, the percentage of tumor cells in S phase did not correlate significantly with 7-AMD stainability (P = 0.07), while the light scattering (cell size) of G0/G1 cells was highly correlated to 7-AMD binding (P less than 0.001).

The blastogenic response of human lymphocytes in vitro to hyperoptimal concentrations of concanavalin A (Con A) has been studied by means of volume spectroscopy (measuring cellular and nuclear volume), flow cytofluorometry (measuring cellular DNA content) and incorporation of [3H]thymidine ([3H]dThd). The optimal Con A dose with respect to [3H]dThd incorporation was about 30 micrograms/ml. In cultures given hyperoptimal doses, e.g. 100 micrograms/ml, [3H]dThd incorporation was strongly inhibited, whereas the number of cells entering S-phase and significantly increasing their cellular and nuclear volume was considerably larger than with 30 micrograms/ml. With 200 micrograms/ml Con A, which induced negligible [3H]dThd incorporation, the percentage of responding cells was even larger. Hence, doses of Con A, which were hyperoptimal with regard to [3H]dThd incorporation, induced blastogenic response, including DNA synthesis, in a larger percentage of the cells than did the optimal dose. However, in cultures with hyperoptimal Con A doses, the progression of the cell cycle stagnated mainly during S- and G2-phase and few cells completed mitosis. Thus, the blocking effect of hyperoptimal doses was not confined to any particular point of the cell cycle. The reduced [3H]dTd incorporation, seen with hyperoptimal doses, is attributed partly to a failure of this assay under such conditions.

The blastogenesis of human blood lymphocytes has been studied by measuring the distribution of cellular and nuclear volumes in concanavalin A (Con A)-stimulated leucocyte cultures by means of a modified Coulter counter. Agglutinated and adherent cells were dispersed by 3 h of incubation with alpha-methyl-mannoside and EDTA. Fresh suspensions contained 5--35% monocytes with an average cellular volume 2--2.5 times that of small lymphocytes. The nuclear volume of monocytes was similar to that of small lymphocytes within 10% and remained constant upon stimulation. In cultures given the optimal dose of Con A, 30 micrograms/ml, measurable cell growth commenced within 8--12 h, whereas nuclear growth began some 6 h later, which is approximately 6--8 h before increased 3H-thymidine incorporation could be detected. The average volume of responding cells grew by a factor of about 4 until mitosis, which started after around 48 h, whereas the average nuclear volume increased by a factor of about 3. The percentage of cells responding by volume growth increased with mitogen concentration even for hyperoptimal concentrations (150 micrograms/ml) which strongly suppressed 3H-thymidine incorporation. The results exemplify the type of information obtained by volume spectroscopy of leucocyte cultures. This method appears to have several advantages over conventional assays of immune response.

The effect of various concentrations of rat-liver macrophages on the Con-A-induced blastogenesis of syngeneic spleen lymphocytes has been studied by means of flow-cytometric methods and by measurement of the uptake of 3H-thymidine. By measuring the distributions of cellular and nuclear volume and cellular DNA to characterize the progression of responding lymphocytes through the cell cycle, we have distinguished a promotive and an inhibitory effect of macrophages. The promotive effect is on the number of cells initiating blastogenesis and on their rate of progression through the cell cycle. The degree of promotion increased strongly with the concentration of macrophages even for concentrations that suppressed the incorporation of 3H-thymidine almost completely from about 30 h of culture. The inhibition observed for macrophage concentrations +/- 10% was a late effect causing stagnation of cell cycle and reduced viability only from about 24 h of culture.

The importance of substrate adhesion and cell aggregation for the Con A-induced blastogenesis of human peripheral lymphocytes was studied by flow cytometric methods. The percentage of lymphocytes responding to the mitogen by growth and DNA synthesis was approximately the same among non-adherent, single cells as among substrate-adherent and aggregated cells. However, the response of the former cells was delayed by about 10 h as compared with that of adherent and aggregated cells. This delay increased to about 20 h in monocyte-depleted cultures. Stimulation with succinyl-Con A, which caused negligible aggregation, produced a significant response. Blast cells appeared to be largely non-adherent. It is concluded that neither substrate adhesion nor cell aggregation are prerequisites for Con A-induced blastogenesis, although substrate adhesion seems to have a significant promotive effect that is probably associated with a direct contact of the lymphocytes with monocytes.