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F Bettens

Publications and source records attributed to F Bettens.

44 records · Page 3Linked to original sources

Lymphokine regulation of activated (G1) lymphocytes. II. Glucocorticoid and anti-Tac-induced inhibition of human T lymphocyte proliferation.

The regulation of the first cell cycle of human, activated (G1) PBL was analyzed by flow cytometry and [3H]thymidine incorporation. Endogenous IL 2 production was blocked in situ by pharmacologic concentration of DEX (100 to 1000 nM), resulting in an 80 to 90% reduction of thymidine uptake. Although T lymphocyte activation (G0-G1a transition) by PHA was unaltered, cells remained in the G1a phase of the cell cycle due to insufficient RNA synthesis for proliferation. The addition of IL 2-containing supernatants reversed this inhibitory effect of DEX by allowing the cells to synthesize more RNA (G1a-G1b transition). Such cells could enter the S phase and proliferate. Similar studies were performed on cells treated with a monoclonal antibody (anti-Tac) against the IL 2 receptor. In these studies, IL 2-induced RNA synthesis, and subsequent proliferation of DEX-treated and PHA-stimulated cells was inhibited by anti-Tac. Anti-Tac did not, however, inhibit the effect of endogenous IL 2 (PHA-stimulated PBL without DEX treatment), although it did bind equally well to such cells. Thus, IL 2 directly or indirectly regulates human T cell proliferation at the level of RNA synthesis. Furthermore, anti-Tac can inhibit the mitogenic signal given by endogenous IL 2, but not by in situ produced IL 2, an observation of importance to further investigations of the mechanisms by which IL 2 interacts with specific receptors to elicit proliferation.

Adult↗

Lymphokine regulation of activated (G1) lymphocytes. I. Prostaglandin E2-induced inhibition of interleukin 2 production.

PGE2-induced inhibition of the proliferatory response of PHA-stimulated human PBL and Con A-stimulated murine thymocytes was analyzed by flow cytometry. It was found that the activation process (G0-G1a transition) was not influenced by PGE2 over a wide range of concentrations (10(-10) to 10(-6) M), nor was the formation of IL 2 receptors inhibited. Similarly, the viability of human lymphocytes was practically unaltered. In contrast, the IL 2-dependent cell cycle event (G1a-G1b transition), which is required for proliferation, was inhibited in a dose-dependent fashion. The addition of IL 2-containing supernatants to such cultures prevented the PGE2-mediated block in the G1a phase and reconstituted a normal lymphocyte proliferation. Furthermore, lower IL 2 titers were measured in supernatants from PHA-stimulated human PBL treated with PGE2. These findings strongly suggest that PGE2 primarily exerts its inhibitory effect on lymphocyte proliferation through an inhibition of IL 2 production.

Adult↗

Human lymphocyte proliferation. II. Formation of activated (G1) cells.

The kinetics of phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes in the early phases (G0, G1a, G1b) of the first cell cycle have been analyzed in vitro by cytofluorometry and [3H]thymidine incorporation. Cells were detected in the G1a, G1b and S phases 12-14, 20-22 and 24-26 h after stimulation, respectively. The total number of PHA-induced G1 cells reached a plateau after 32 h of incubation, upon which a second increase followed, 10 h later. The latter increase was considered to be a result of cells initiating their second cell cycle, since it could be abolished by hydroxyurea. By examination of supernatants, interleukin 2 (IL 2) activities could be recovered 6-10 h after PHA stimulation. In contrast, the monokine IL 1 was already detected after 2 h. Both interleukins were present in the culture medium before the major G1a cell formation took place. The titer of free IL 2 increased to a maximum after 18-22 h, whereafter a decline was observed. This decline was less pronounced if cultures were treated with hydroxyurea. Finally, among individual donors, the total number of lymphocytes entering the G1 phase and the time at which the G1a-G1b transition took place varied. Lower numbers of G1 cells and delayed G1a-G1b transition coincided with lower IL 2 titers and delayed occurrence of maximal titers in the supernatants.

Adult↗

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.

Cell Cycle↗

Effect of macrophages on the go-gl and gl-S transition of thymocytes.

Thymocytes freed of adherent cells and stimulated by mitogens yield only a poor proliferative response ([3H]-thymidine uptake). However, the activation process assessed fluorocytometrically (RNA-synthesis) is not reduced to the same extent. Addition of adherent cells caused full restoration of both activation and proliferation, whereas 2-mercaptoethanol (2-ME) restores activation completely, but proliferation only partially. Cell viability is not influenced by the absence or presence of adherent cells or of 2-ME during the first 24 hr of incubation, which is the time period used to assess cell activation. Macrophages on the other hand, can be replaced completely by the addition of 2-ME and Interleukin (IL-1), or IL-2 containing supernatants. The addition of these substances even appears to be superior, suggesting that macrophages can as well suppress as enhance the thymocyte response to mitogens. The results presented further support the current concept of the multifunctional role of macrophages: depending on the selected mitogen, it appears that only the IL-1 production is an absolute, but indirect requirement for mitogen-stimulated thymocytes, in order to proceed through the cell cycle.

Animals↗

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.

Aging↗

Cytokine gene expression in atopics: effect of IL-4 on IL-1 beta and IL-6 mRNA levels.

Mononuclear cells from atopic blood donors showed increased IL-3 steady state mRNA levels. This finding complemented our earlier observations that cells from atopics also showed increased IL-4 but decreased IFN-gamma, IL-1 beta and IL-6 mRNA levels. Therefore, we investigated the effect of human recombinant IL-4 on cytokines mRNA levels in mononuclear cells from normals and atopics. In the presence of IL-4 steady state levels of IL-1 beta and IL-6 mRNA were decreased even if cells were co-stimulated with polyclonal activators such as PMA, PWM or PHA. No influence of IL-4 on granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3 or IFN-gamma mRNA levels was observed with the exception of a decreased IFN-gamma mRNA level in PWM stimulated cells.

Gene Expression Regulation↗