Sensitivity of S49.1 cells to anti-CD95 (Fas/Apo-1)-induced apoptosis: effects of CD95, Bcl-2 or Bcl-x transduction.

T lymphocytes have variable sensitivity to anti-CD95 which does not correlate closely with the level of CD95 expressed. To investigate this phenomenon, we screened murine T lymphocyte cultures for their sensitivity to anti-CD95. Subclones of the S49.1 cells showed widely variable sensitivity to anti-CD95 but similar levels of CD95. The resistant clones became sensitive after treatment with actinomycin D suggesting that they expressed resistance protein(s) with a high turnover relative to the CD95 apoptosis induction machinery. Our data suggest that the resistance protein(s) are not Bcl-2, Bcl-x, Fap-1 or Bag-1. Forced, increased expression of CD95 made most of the resistant cells more sensitive, but some remained resistant suggesting that the expression of the resistant protein(s) is heterogeneous and that increased CD95 levels does not always overcome the resistance.

Expression of Bcl-2, Bcl-x, and Bax after T cell activation and IL-2 withdrawal.

Bcl-2, bcl-x, and bax genes code for proteins that affect the susceptibility of cells to apoptosis. In general, the expression of bcl-2 or bcl-x inhibits apoptosis while bax promotes apoptosis. We examined the levels of these proteins by immunoblotting in resting and activated T cells and in thymocytes. Bcl-2 and Bax proteins vary coordinately, but Bcl-x varies independently: Bcl-2 and Bax are higher in splenic T cells than in thymocytes, and their levels increase even more after T cell activation. In contrast, Bcl-x is almost undetectable in splenic T cells but is manyfold greater in thymocytes and in activated splenic T cells. When CTLL-2 cells or activated T cells are starved of IL (IL-2), the level of Bcl-x but not Bcl-2 protein drops before the onset of apoptosis. Stable transfection of either bcl-2 or bcl-x expression plasmids promotes the survival of CTLL-2 cells in the setting of IL-2 withdrawal. Over 70 to 90% of the transfected cells remain viable at 48 h after IL-2 withdrawal when all of the control transfected cells are apoptotic. These findings suggest that a decrease in Bcl-x protein levels precedes apoptosis after IL-2 withdrawal in T cells and that transfected bcl-2 promotes survival after IL-2 withdrawal by functionally masking this drop in Bcl-x.

Control of CD4 effector fate: transforming growth factor beta 1 and interleukin 2 synergize to prevent apoptosis and promote effector expansion.

The signals that determine the size and duration of the primary T cell immune response are not well defined. We studied CD4 T cells at an important checkpoint in their development: when they have become effectors and are ready to rapidly mediate effector functions, both via direct interaction with antigen (Ag)-presenting cells and via cytokine production. We determined the effects of specific Ag and the cytokines interleukin (IL) 2 and transforming growth factor (TGF) beta 1 on T helper cell type 2 (Th2) effector apoptosis versus expansion. Th2-polarized effector cells were generated in vitro from naive CD4 T of T cell receptor transgenic mice, and then restimulated with or without peptide Ag plus Ag-presenting cells and cytokines. In the absence of added cytokines, effector cells cultured without Ag died of apoptosis after 4-7 d. Paradoxically, Ag both induced proliferation and high levels of cytokine synthesis and accelerated effector cell death. IL-2 directly induced proliferation of effectors, supported and prolonged Ag-induced proliferation, and partially blocked apoptosis. TGF-beta did not effect proliferation or influence cytokine secretion, but it also partially blocked apoptosis. Together, IL-2 and TGF-beta synergized to almost completely block apoptosis, resulting in prolonged effector expansion and leading to the accumulation of a large pool of specific effectors. When Ag and both cytokines were present, the effector population increased 10(4)-10(5) fold over 20 d of culture. The synergy of IL-2 and TGF-beta suggests that they interfere with programmed cell death by distinct mechanisms. Since Th2 effectors are specialized to help B cells develop into antibody-secreting plasma cells, these results suggest that the availability of Ag and of the cytokines IL-2 and TGF-beta is a key factor influencing the fate of Th2 effector cells and thus the size and duration of the primary antibody response.

Apoptosis and Bcl-2 expression in cultured murine splenic T cells.

To elucidate the mechanism by which bcl-2 affects apoptosis in post-thymic T cells, we investigated the expression of Bcl-2 protein in primary cultures of splenic T cells and in the interleukin-2 (IL-2)-dependent T-cell line CTLL-2. The overall level of Bcl-2 was determined by immunoblotting, and the variability in Bcl-2 expression was determined by flow cytometry. For a few days after concanavalin A (Con A) plus IL-2 activation, the overall level of Bcl-2 in T cells remains unchanged, but it becomes more heterogeneous. By 5 days after activation, the expression returns to a more homogeneous distribution, but it is increased up to threefold above pre-activation levels, depending upon the dose of IL-2 supplied. When Con A blasts or CTLL-2 cells are deprived of IL-2 for 24 hr, there is no change in their overall Bcl-2 levels which remain homogeneous even though almost half of the cells are apoptotic. However, when bcl-2 transfected CTLL-2 cells are deprived of IL-2, they do not undergo apoptosis, and their endogenous Bcl-2 protein level slowly decreases relative to their total protein. These data document the IL-2-dependent expression of Bcl-2 in activated T cells, confirm the ability of deregulated bcl-2 to inhibit the onset of apoptosis after IL-2 withdrawal, but suggest that, after IL-2 withdrawal, a drop in Bcl-2 levels relative to total protein levels does not precede apoptosis.

Recombinant follicle-stimulating hormone induces ovulation and tissue plasminogen activator expression in hypophysectomized rats.

Ovulation in mammals is preceded by surges of the two pituitary gonadotropins, LH and FSH. Although previous studies have shown that purified FSH induces ovulation when administered to hypophysectomized rats, proof that FSH has inherent ovulatory potential is lacking because all FSH preparations have varying degrees of residual LH. To determine if FSH alone can induce ovulation, we generated LH-free recombinant FSH (RCFSH) by culturing eukaryotic cells transfected with the human common alpha- and FSH beta-subunit genes. Immature hypophysectomized rats were implanted with estrogen and then primed with PMSG (15 IU, sc). Fifty-two hours later, either RCFSH or hCG was injected (sc) to induce ovulation. A dose-dependent increase in the ovulation rate was stimulated by RCFSH, reaching 100% ovulation at 18 IU/rat, comparable to that achieved with 12 IU hCG. The maximum number of oocytes ovulated per ovary was similar for both groups. Ovulation induced by either RCFSH or hCG was time dependent and associated with a periovulatory increase in the ovarian activity and message levels of tissue-type plasminogen activator, a protease important in the preovulatory degradation of the follicle wall. Because PMSG has inherent LH-like activity in rats, we also implanted hypophysectomized rats with a minipump (sc) that released RCFSH (4 IU/day) to induce follicle growth. Fifty-two hours later, a single sc injection of a surge dose (20 IU) of RCFSH also induced ovulation, further indicating the ability of FSH alone to induce both follicle growth and ovulation. To test whether FSH can also induce ovulation in adult animals, rats were hypophysectomized on proestrous morning and treated with increasing doses of RCFSH (ip) to induce ovulation. At 7.8 IU RCFSH, all rats ovulated, with about 10 oocytes/rat. These results demonstrate that RCFSH is capable of inducing ovulation in hypophysectomized immature and adult rats, with associated increases in ovarian tissue-type plasminogen activator gene expression. Thus, FSH may be involved in follicular rupture in addition to its role in follicle recruitment and maturation. The preovulatory surges of both LH and FSH may represent a protective mechanism to ensure an optimal ovulatory stimulus. The present finding also serves as the basis to formulate new ovulation induction protocols.