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Pertussis toxin-sensitive GTP-binding proteins regulate activation-induced apoptotic cell death of human natural
J Carracedo1, R Ramírez, P Marchetti
1Unidad de Investigación, Hospital Reina Sofía, Córdoba, Spain.
Abstract:
Apoptosis of natural killer (NK) cells can be induced by non-specific physical damage (UV irradiation, heat shock) or by simultaneous ligation of the CD16 and the interleukin-2 receptor (IL-2R) molecules, but not with either anti-CD16 or IL-2 alone. Whereas blockade of GTP-binding protein (G protein)-mediated signal transduction using ADP-ribosylating bacterial toxins or the GTPase-resistant GTP analog guanosine 5'-0-(3-thiotriphosphate (GTP gamma S) does not affect non-specific induction of NK cell apoptosis, such interventions do inhibit induction of apoptosis by anti-CD16/IL-2. The G proteins involved in the regulation of activation-induced NK apoptosis are sensitive to pertussis toxin (PTX) and to the non-specific GTP analog GTP gamma S but not to cholera toxin, Pseudomonas exotoxin A or diphtheria toxin. A pertussis toxin mutant that lacks ADP-ribosylating activity, but conserves the membrane translocating and T cell-mitogenic effects of the native molecule, fails to inhibit NK apoptosis. To exert their apoptosis-inhibitory effect, PTX and GTP gamma S must be employed before cells are activated. Later addition has no effect, suggesting the implication of G proteins in the transmission of apoptosis-inducing signals, but not in the effector stage of apoptosis. Pre-incubation with PTX or GTP gamma S does not affect the activation of NK cells by CD16 cross-linking, IL-2 stimulation- or both, as assessed by the induction of CD69 expression, protein tyrosine phosphorylation and calcium mobilization. Moreover, neither PTX nor GTP gamma S compromise the effector function of NK cells or the susceptibility of target cells to NK-mediated lysis. These data suggest apoptosis as a novel mechanism by which NK responses may be controlled in vivo, as well as an experimental and therapeutical strategy to counteract endogenous down-regulation of NK responses.
Insights
Natural killer (NK) cell apoptosis is regulated by G proteins, which are sensitive to pertussis toxin (PTX) and GTPγS. These G proteins are involved in transmitting apoptosis-inducing signals, not in the effector stage, suggesting a novel control mechanism for NK cell responses.
Area of Science:
- Immunology
- Cell Biology
- Molecular Signaling
Background:
- Natural killer (NK) cells undergo apoptosis through physical damage or combined CD16 and interleukin-2 receptor (IL-2R) ligation.
- The role of G protein-mediated signal transduction in NK cell apoptosis induction was previously unclear.
Purpose of the Study:
- To investigate the involvement of G proteins in the regulation of NK cell apoptosis.
- To determine the specific G proteins and signaling pathways involved in activation-induced NK cell apoptosis.
Main Methods:
- NK cells were treated with ADP-ribosylating bacterial toxins (pertussis toxin, cholera toxin, Pseudomonas exotoxin A, diphtheria toxin) or GTP analogs (GTPγS).
- Apoptosis induction was assessed following stimulation with anti-CD16 and/or IL-2.
- NK cell activation markers (CD69 expression, tyrosine phosphorylation, calcium mobilization) and effector functions were evaluated.
Main Results:
- Blockade of G protein signaling with pertussis toxin (PTX) or GTPγS inhibited NK cell apoptosis induced by combined anti-CD16/IL-2 ligation, but not non-specific apoptosis.
- PTX and GTPγS were effective only when applied before cell activation, indicating their role in signal transmission.
- PTX and GTPγS did not affect NK cell activation, effector function, or target cell lysis.
Conclusions:
- G proteins, specifically those sensitive to PTX, play a crucial role in transmitting apoptosis-inducing signals in NK cells.
- Apoptosis represents a novel mechanism for controlling NK cell responses in vivo.
- Targeting these G protein pathways offers a potential strategy to counteract NK cell response down-regulation.