Abstract
Activation of human thymocytes and pre-B cells via the CD3/T cell receptor (TCR) complex or the IgM/B cell receptor complex, respectively, results in apoptotic cell death. Similarly, cross-linking of the activation marker CD69, which belongs to the natural killer complex, causes apoptosis of lipopolysaccharide-preactivated monocytes. Here we show that pertussis toxin (PTX) inhibits the activation-induced apoptosis of these three cell types, though it fails to prevent the programmed cell death that follows exposure of cells to the synthetic glucocorticoid dexamethasone (thymocytes, pre-B cells) or to interleukin 4 (monocytes). The capacity of pertussis toxin to suppress activation-induced death is not due to quenching of the activation signal, because thymocytes exposed to PTX are still capable of mobilizing Ca2+ after TCR-alpha/beta cross-linking and proliferate in response to costimulation with PTX and CD3/TCR ligation. The apoptosis-inhibitory effect of PTX depends on the presence of an intact adenosine diphosphate (ADP)-ribosylating moiety, since a mutant pertussis toxin molecule that lacks enzymatic activity, but still possesses the membrane translocating activity, fails to interfere with activation-induced cell death. A toxin that induces a different spectrum of ADP ribosylation than PTX, cholera toxin, fails to inhibit apoptosis. To suppress apoptosis, the intact PTX holotoxin must be added to cells before the lethal activation step; its addition 30 min after initial activation remains without effect on apoptosis. These data unravel a PTX sensitive signal transduction event that intervenes during an early step of activation-induced cell death of immune cells.
Highlights
To induce apoptosis in thymocytes, cells were exposed to plasticbound anti-CD3 (Leu 4b; Serotec Ltd., Oxford, UK; 20/~g/ml in 50/~l/well PBS containing 0.1% BSA incubated for 4 h at 37~ followed by three washes with PBS). 4 h after starting cell cultures, anti-T cell receptor (TCR)-o~//~mAb (TA 1043, 2/zg/ml; T Cell Diagnostics, Cambridge, UK) was added
Using a system in which purified thymocyte subpopulations were exposed to immobilized anti-CD3ol during a 4-h culture period, followed by addition of a mAb specific for the TCR-oJ~8 [10], we observed that CD4+CD8 + and CD4+CD8 - thymocytes are more susceptible to apoptosis induction than CD4CD8 + or C D 4 - C D 8 - cells (Fig. 1 A)
Preincubation of these thymocyte subpopulations with pertussis toxin (PTX) for a minimum of 30 rain completely prevents the CD3/TCR-mediated induction of apoptosis (Fig. 1, A and B) and apoptosis-associated DNA fragmentation (Fig. 1 C). This apoptosis-inhibitory effect is specific for activation-induced death, because PTX does not block the glucocorticoid (DEX)-induced death of thymocytes (Fig. 1 A)
Summary
Activation of human thymocytes and pre-B cells via the CD3/T cell receptor (TCR) complex or the IgM/B cell receptor complex, respectively, results in apoptotic cell death. We show that pertussis toxin (PTX) inhibits the activation-induced apoptosis of these three cell types, though it fails to prevent the programmed cell death that follows exposure of cells to the synthetic glucocorticoid dexamethasone (thymocytes, pre-B cells) or to interleukin 4 (monocytes). The intact PTX holotoxin must be added to cells before the lethal activation step; its addition 30 min after initial activation remains without effect on apoptosis These data unravel a PTX sensitive signal transduction event that intervenes during an early step of activationinduced cell death of immune cells. We show that pretreatment with pertussis toxin (PTX) inhibits the subsequent activation-induced apoptosis of three cell types that are notoriously prone to apoptosis induction: thymocytes, pre-B leukemia ceils, and monocytes. The inhibitory effect of PTX on activation-induced death depends on the presence of intact ADP-ribosyltransferase activity, thereby implying G protein(s) in the regulation of apoptosis of immunologically relevant cells
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