F. Joncourt

Age-related changes of mitogen responsiveness in different lymphoid organs from outbred NMRI mice

Lymphocytes from spleen, thymus and lymph nodes from individual young adult (3--4 months) and aged (26--30 months) NMRI mice were stimulated with the mitogens Con A, PHA and LPS. 24 hours later, the number of cell with increased RNA-content (G1 cells) was determined by cytofluorometry. In parallel the 3H-thymidine incorporation after 48 hours was measured for the same cell samples. Aged animals in average produced less G1 cells and incorporated less 3H-thymidine as compared to young adults. By calculating the 3H-thymidine incorporation per G1 cell, the proliferative capacity of mitogen-induced G1 cells can be estimated. These ratios are lower in aged mice as compared to young adult, suggesting that in these animals not only less cells can be activated as measured by cytofluorometry, but also from these activated cells again fewer continue the cell cycle by initiating DNA-synthesis. In response to Con A and PHA, aged mice in average produce less G1 cells in all of the three lymphoid organs tested. In response to LPS, however, the young adult produced only few G1 cells in lymph nodes and practically none in thymus, whereas in aged animals a considerable number of G1 cells was found in both organs. Corresponding results were found for the 3H-thymidine incorporation. These results indicate that in addition to the reduction of the mitogen-response an age-related change in the distribution of mitogen-responsive cells in the different lymphoid organs takes place.

Assessment of interleukin 1 and interleukin 2 effects on cycling and noncycling murine thymocytes

Abstract When mouse thymocytes are stimulated with PHA, the proliferative response is very low, unless the culture medium is enriched with interleukin 1 (IL-1)- or interleukin 2 (IL-2)-containing supernatants. Cytofluorometric analyses show, however, that PHA stimulation generates a significant number of cells with increased RNA content (transition from the G 0 to G 1 phase of the cell cycle). If IL-2 is added to such cultures, the activated cells complete their process of RNA synthesis and then enter the S phase. The use of IL-2-containing culture medium thus permits one to obtain a high correlation between the number of g 1 cells and [ 3 H]thymidine incorporation ( r = 0.97). Enrichment with IL-1-containing supernatants also results in a statistically significant correlation ( r = 0.68), but the regression lines are markedly different for the two interleukins ( s = 20.3 for IL-2 and s = 9.2 for IL-1), when analyzed after 48 hr of incubation. These observations suggest that the G 1 phase must be divided into two subcompartments, G 1a and G 1b , the G 1a -G 1b transition being an IL-2-dependent event. If the number of G 1b cells is used to establish correlations with [ 3 H]thymidine incorporation, all values fall on the same regression line, regardless of culture conditions and of the addition of interleukins. It is concluded that IL-2 regulates lymphocyte proliferation at the level of RNA synthesis (G 1a -G 1b transition) rather than that of DNA synthesis (G 1 -S transition).

Lymphocyte Proliferation, Lymphokine Production, and Lymphocyte Receptors in Ageing and Various Clinical Conditions

ConclusionThe combination of cytofluorographic analysis of human or murine lymphocytes moving through various phases of the cell cycle following lectin stimulation, determination of DNA synthesis by 3H-thymidine uptake, quantitative assessment of lymphokines produced in culture supernatants (e.g. IL 2), and evaluation of various cellular receptors (e.g. IL 2, insulin, corticosteroid) by fluorescent antireceptor monoclonal antibodies or binding of radiolabeled ligand permit one to develop a more comprehensive picture of the defects in lymphocyte functions which are associated with aging and various other clinical conditions.

Aging and Immunity: Decrease in Interleukin-2 Production and Interleukin-2-Dependent RNA-Synthesis in Lectin-Stimulated Murine Spleen Cells

The RNA-content of G1 cells in lectin-stimulated spleen cell cultures of young and aged NMRI mice was determined by flow cytometry. In spleen cells of aged mice a preferential decrease of G1 cells with a high RNA-content, so-called G1b cells, was found. Since, as shown in a previous report, only cells with a high RNA-content are able to proliferate and the passage of low (G1a) to high (G1b) RNA-content is interleukin-2(IL-2)-dependent, the ability of young and old spleen cells to produce IL-2 was tested. In old spleen cells a diminished production of IL-2 was found. Addition of external IL-2, however, did not increase the proliferative capacity of old spleen cells, nor did it induce more G1b cells. Thus spleens of aged mice contain cells, which can be activated by lectin, but then fail to respond to IL-2. Both decrease in IL-2 production and receptivity for IL-2 may contribute to the diminishing immune response in aging individuals.

Human lymphocyte proliferation. I. Correlation between activated and proliferating T-lymphocytes

The response of human peripheral blood lymphocytes to Con A and PHA has been analyzed by [3H]thymidine incorporation and cytofluorometry. Using the latter method, it is possible to quantitate the number of cells in the G0 phase (normal RNA and DNA content) and in the G1 phase (elevated RNA, but normal DNA content). A very high correlation is found between numbers of Con A or PHA-induced G1 cells and [3H]thymidine incorporation in healthy donors. This high correlation is found when culture medium is enriched with 10% autologous plasma or 10% AB-serum. The use of a recently developed defined serum-free medium (RPMI 1640 with albumin, alanine, transferrin, sodium selenite and zinc chloride), however, suggest that donors can be divided into two groups according to different medium requirements for PHA-stimulated lymphocytes. Because several immunoregulatory mechanisms at the level of T-lymphocytes take place in the G1 phase, it can therefore be expected that cytofluorometric analyses of lymphocytes in the various cell cycle phases may improve the interpretation of altered lymphocyte response to lectins and antigens.

Age-Related Changes in the Formation of Glucocorticoid and Insulin Receptors during Lectin-Induced Activation of Human Peripheral Blood Lymphocytes

The influence of age on the number of receptors for insulin and glucocorticoids on human T cells was examined. The specific binding of 125I-insulin and 3H-dexamethasone to phytohemagglutinin-(PHA)-stimulated T cells was found to decrease with age. Populations of PHA-stimulated T cells, however, are heterogeneous with respect to cell cycle phases, and cells in different cell cycle phases have been shown to bear different numbers of receptors. Therefore, it was not clear whether the measured decrease in specific binding in cultures from aged donors was due to a decreased number of activated cells or to a decreased number of binding sites per cell. In parallel to measurements of receptors, performed at different times following stimulation (20 and 44 h), numbers of cells in the different early cell cycle phases G0, G1a and G1b were quantitated by flow cytometry. In this way the receptor number per cell in each phase of the cell cycle could be determined. Receptor numbers on resting cells and in the earliest phase of activation (G1a) were found not to be influenced by age. A decreased receptor density, however, was apparent on G1b cells from aged donors.

Cell cycle-related changes in number of T-lymphocyte receptors for glucocorticoids and insulin

Enriched human peripheral T-lymphocytes were stimulated with PHA and examined for variations in insulin and glucocorticoid (dexamethasone) receptor numbers during the early phases of the cell cycle. Cells in G0, G1a and G1b phases, where the G1a - G1b transition is an Interleukin 2 dependent event, were quantitated by flow cytometry. Few but significant numbers of glucocorticoid receptors (2700/cell) and no insulin receptors (-1/cell) were found in the resting (G0) phase. As cells entered the G1a phase the specific binding of dexamethasone increased and of insulin took place. Although the specific binding further increased as T-cells entered the G1b phase (as measured at 44 h of incubation and using hydroxyurea-treated cells), the major changes in the specific binding of dexamethasone took place during the period 16 - 20 h after stimulation. Based on these findings, it is concluded that both receptor types (cell membrane and cytoplasmic receptors) are being formed and increased at G1 phase prior to cell proliferation, indicating the importance of G1 phase in immunoregulation.

Age-Related Changes in G₀-G1 Transition and Proliferative Capacity of Mitogen-Stimulated Murine Spleen Cells

The early kinetics of mitogen-stimulated of mitogen-stimulated spleen cells from young adult and aged NMRI mice were investigated. Using RNA synthesis as measured by cytofluorometry by cytofluorometry and 3H-leucine and 3H-thymidine incorporation as parameters, it was apparent that age-related changes occur at different levels during mitogen stimulation. First, in aged mice fewer cells per 10(6) spleen cells are being activated by mitogen, and, second, fewer of the activated lymphocytes will proliferate. However, the cells which initiate synthesis of RNA, protein, and DNA apparently behave in identical manners, chronologically, as cells from young adult mice.

Lymphocyte Proliferation, Lymphokine Production, and Lymphocyte Receptors in Aging

Lymphocyte proliferation is one of the basic functions of the immune system, since it appears required as well for the acquisition of immunological memory as for clonal expansion of specific lymphocyte populations. In recent years, the combination of several techniques, such as analysis of the cell cycle by cytofluorometry (Darzinkiewicz et al., 1976; Stadler et al., 1980), measurement of [3H]-TdR uptake, detection of membrane receptors by immunofluorescence or ligand binding (Munck and Vira, 1975), and quantitative assessment of various lymphokines produced by proliferating lymphoid cells, has permitted the establishment of an integrated picture of the various events associated with the proliferation of lymphocytes. Although this picture is still fragmentary, the analysis of lymphocyte functions which it makes possible has already been found relevant and informative in several clinical situations, where a dysregulation of lymphocyte functions is apparent. The purpose of this paper is to review briefly the current possibilities to analyze lymphocyte proliferation in clinical situations and their application to the aging process.

Type of inducing signal regulates transactivation by p53.

The tumor suppressor gene p53 is expressed in the contrasting cell fates apoptosis and proliferation. We examined whether the transactivation of the p53 target genes, waf1 and mdm2, is dependent on the cause of p53 induction in human peripheral blood mononuclear cells (PBMC). Both apoptosis triggered by the purine analog 2-chlorodeoxyadenosine (CdA) and growth stimulation by the mitogen phytohemagglutinin (PHA) induced a comparable level and time course of p53 mRNA expression. Both stimuli led also to an increase of p53 protein levels. The cytotoxic agent, but not the mitogen, led to transactivation of waf1 and mdm2 within 18 h. Transactivation was followed by apoptosis of 89% of the PBMC within 48 h. The c-myc oncogene and poly(ADP-ribose)polymerase (PARP), which also have a dual function in proliferation and apoptosis, showed an early induction by both CdA and PHA. These results add further evidence that growth stimulation and DNA damage-induced apoptosis share early gene activation pathways in normal cells. However, since p53 does selectively translate into transactivation of target genes depending on the cause of induction, this function of p53 seems to be regulated by additional factors, which are closely related to the ultimate fate of the cell.