F. Kristensen

Mechanisms of serotonin-induced lymphocyte proliferation inhibition

When human peripheral blood lymphocytes were stimulated with phytohemagglutinin in the presence of serotonin, inhibition of [3H]thymidine incorporation occurred, the most marked inhibition occurring at high (10(-3)M) serotonin concentrations. This effect could not be reversed by the addition of Interleukin 2 (IL-2)-containing supernatants. Cytofluorometric analysis showed that virtually all of the cells remained in the G0 phase (unactivated) at 24 hr while some of the cells entered the G1a and G1b phases of the cell cycle by 42 hr. The cellular production of IL-2 was not affected by serotonin, as supernatants of treated cultures contained essentially the same IL-2 titers as did control cultures. Serotonin seemed to primarily affect cell activation and had little or no effect on proliferating cells. This was further confirmed by the lack of effects of serotonin on a variety of established proliferating lymphocyte, macrophage, and fibroblast cell lines. By contrast, dose-dependent inhibition of IL-2-dependent CTLL cells occurred. Serotonin was not toxic even at 10(-3) M concentrations. A marked decrease in IL-2 receptors and a change in their distribution on responder cells was seen when treated cultures were examined with the anti-Tac monoclonal antibody. At 24 hr this effect was contrastingly not seen for the OKT-8 marker, although a slight decrease in OKT-4-positive cells was seen. Serotonin thus produced an inhibition of lectin-stimulated lymphocyte proliferation via a mechanism independent of IL-2 production, and caused a decrease in the expression and distribution of IL-2 receptors on the surface of responder cells.

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).

Thymocyte activation by cytokines: Direct assessment of G0-G1 transition by flow-cytometry

Abstract Using mouse thymocytes, mitogen-induced [ 3 H]thymidine incorporation was compared with a recently developed flow-cytometric technique, based on acridine orange staining of cells, which differentiates the G 0 and G 1 phase of thymocytes. PHA induces a transient but considerable G 0 -G 1 shift without any substantial proliferation. On the other hand, crude supernatants derived from Con A-stimulated human peripheral blood mononuclear cells induce only a minor G 0 -G 1 shift and no proliferation. However, PHA in the presence of this supernatant induced an increased [ 3 H]thymidine uptake in thymocytes and a shift from G 1 to S. These results support the current hypothesis that a factor present in Con A-activated supernatants in conjunction with PHA stimulation indeed facilitates the entrance of G 1 cells into the S phase. The flow-cytometric technique might be used in the study of the interaction of endogenous mediators with exogenous mitogenic agents in activating lymphocytes to proceed through the initial G 0 -G 1 phases of the cell cycle.

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.

Canine lymphocyte cultures in vitro: Evaluation of peripheral blood lymphocyte response to mitogens

Lymphocytes from dog peripheral blood have been stimulated in vitro with 3 different mitogens (Con A, PHA and PWM). Culture medium was RPMI 1640 enriched with either autologous plasma, fetal calf serum of a newly described defined serum substitute. In such cultures the number of surviving and activated cells was measured by cytofluorometry and the proliferation was assessed by thymidine incorporation. In unstimulated cultures, up to 70% of all cells had disappeared (died) during the first 42 hours of incubation, whereas the number of viable cells was reduced to 50-60% in mitogen stimulated cultures. Of the surviving lymphocytes, between 25-40% of the cells appeared to have an elevated RNA-content (activated or G1 cells). By comparison between thymidine incorporation and number of mitogen induced G1 cells, a very high correlation was found (r=0.92). However, the Slope of the regression line was much lower than expected. The low thymidine incorporation per activated cell was primarily related to the high cell death and a resulting dilution of tritiated thymidine. Indeed, preliminary results suggested that the same thymidine incorporation per G1b cells could be obtained if peripheral blood lymphocytes were washed immediately before pulsing as could be obtained with lymph node cells without washing.

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.

Interleukin 1- and 2-like activities in the dog

Canine adherent and non-adherent peripheral blood leukocytes and spleen cells were examined for their ability to produce soluble factors with Interleukin 1- and 2- (IL-1 and IL-2) like activities. For this purpose, three conventional assay systems were used: (a) proliferation on an IL-2-dependent murine cytotoxic T-lymphocyte cell line, (b) enhancement of PHA-induced murine thymocyte proliferation and (c) proliferation of lectin-primed canine peripheral blood lymphocytes (PBL). Only the latter two types of cells respond to IL-1, whereas all three types respond to IL-2. Both types of factors were produced and the kinetics of their release/production were found to be identical to those of human PBL. Results suggested that species-related differences existed. Canine interleukin-containing supernatants had higher titers than murine interleukin-containing supernatants when analyzed on canine lymphocytes, and the reverse was found if murine target cells were used.

Lymphokine regulation of human lymphocyte proliferation: Formation of resting G0 cells by removal of interteukin 2 in cultures of proliferating T lymphocytes☆

The question of whether lymphocytes which have once been activated and have completed one or several cell cycle(s) can return to the G0 phase and stay ready for a new activation (G0-G1 transition), rather than simply die, was investigated. To do so interleukin 2 (IL-2) was removed from cultures of continuously proliferating human T lymphocytes and the formation of resting (G0) cells was measured. Kinetic analyses in freshly prepared peripheral blood lymphocytes (PBL) revealed that the onset of detectable RNA synthesis and the appearance of structures binding the anti-Tac antibody occurred simultaneously. This allowed the expansion of the definition of G0 T lymphocytes as cells having a low RNA (and DNA) content, and no Tac antigen. When cultured human T cells proliferating continuously by means of IL-2 were characterized in terms of their distribution in the cell cycle, 7 days after the initial PHA stimulation, it could be demonstrated that very few cells were in the G0 phase, supporting the concept of direct S/G2/M-G1 transition. However, when IL-2 was removed from the cultures, the [3H]thymidine incorporation per 10(4) cells and correspondingly the number of cells in the S/G2/M and G1 phases were reduced drastically and during the following 72-hr period, the number of G0 cells increased markedly. Restimulation of such in vitro formed G0 cells, under conditions permitting observation of their shift from the G0 to G0 phase, demonstrated that most cells could respond normally. Based on these observations, it was concluded that IL-2 not only ensures T-lymphocyte survival and proliferation, but IL-2 starvation induces many continuously proliferating T lymphocytes to stop cycling and to return to the G0 phase of the cell cycle where they remain functional.

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.