Dissociation of Phospholipids “Flip-Flop” and CD16 Downregulation in Stimulated Neutrophils with FMLP and TNFα in Relationship with ROS Production.

There are 3 kinds of PMN apoptosis: spontaneous, Fas or TNFα mediated system and ROS induced. The purpose of this work is to investigate the mechanisms of modulation of the CD16 decrease and the phospholipids “flip-flop” which are classically described as simultaneous events in apoptosis in relationship with the intracellular production of H2O2. Incubations during different times of PMN suspensions from healthy controls were performed in presence of Phorbol Myristate Acetate (PMA) at priming (10⁻⁹ M) and stimulating (2 10⁻⁶ M) concentrations, of fMLP 5 10⁻⁶M, of TNFα at 200ng/ml and finally of agonistic anti-Fas antibodies at 1 μg/ml. Inhibitors of PKC (staurosporin 10⁻⁵ M) and of the oxidase (DPI 400 μM) were tested with stimulating PMA. Standard two-colour flow-cytometry was performed for detection on one side of FITC-Annexin-V fixation and membrane impermeability to Propidium Iodide and on the other side of DCFH-DA for the intracellular H2O2 detection and CD16-PECy5.

Results after 3 hours incubation are given in Table 1.

Table 1.

Stimulating PMA induced both intracellular H2O2 production and apoptosis with increase of Annexin V fixation and CD16 down-regulation. Partial prevention of both features was observed when the ROS production was completely inhibited in presence of staurosporin. DPI had an effect on Annexin V fixation but no effect on CD16 decrease dissociating the 2 apoptotic features. Neutrophils activated with fMLP generated mainly extra-cellular H2O2 and did not undergo apoptosis measured thanks to Annexin V fixation, but CD16 down-regulation was observed. In presence of TNFα, a very slight stimulation of intracellular H2O2 production was observed with an inhibition of the Annexin V fixation and a CD16 down-regulation.

The Fas mediated signalling system induced moderately both Annexin fixation and CD16 down-regulation without ROS production.

Discussion: CD16 down-regulation seems to be present with fMLP and TNFα without other apoptotic features. Moreover, TNFα at 200ng/ml offers a protection for the phospholipids “flip-flop”. Dissociation of the classical PMN apoptotic features CD16 down-regulation and Annexin-V fixation with TNFα and fMLP demonstrates the complexity of the transduction signals used by these stimulants, and for the first time the independence of CD16 down-regulation from the phospholipids “flip-flop” which appears to be strongly dependent of intracellular ROS or more moderately of Fas stimulation and is never present without CD16 decrease. Only the presence of both events demonstrates apoptosis with certitude. On the opposite, it appears that the CD16 decrease alone cannot reflect the presence of the cell apoptosis. This decrease in absence of phospholipids “flip-flop”could be induced by extra-cellular ROS.