Mechanism of phosphatidylserine inhibition of IgE/FcεRI-dependent anaphylactic human basophil degranulation via CD300a.

To the Editor: The process that leads to histamine release from human basophils constitutes a continuum that starts with piecemeal degranulation and eventually converts to anaphylactic degranulation (AND). These degranulation events are accompanied by mobilization of distinct cellular compartments and expression of surface molecules that are not or barely expressed on resting basophils. Two different histamine-containing compartments, which show distinct early signaling requirements and kinetics, have been described, namely, the CD203c compartment and the CD63 compartment. The appearance of CD63 is invariably associated with AND.Mechanisms that regulate basophil activation likely relate to activation conditions (IgE/FcεRI-dependent and -independent), mode of degranulation (piecemeal degranulation vs AND), and changes in functional properties of inhibitory receptors. The inhibitory receptor CD300a (IRp60) is constitutively expressed on human basophils and rapidly upregulated in response to IgE/FcεRI cross-linking; its engagement selectively suppresses AND without a significant effect on CD203c. Moreover, recently CD300a has been shown to be a receptor for phosphatidylserine (PS) and phosphatidylethanolamine expressed on the membrane of apoptotic cells. This study aimed at investigating the effect of PS recognition by CD300a on IgE/FcεRI-dependent basophilic activation. Three experimental models addressed (1) the effect of apoptotic K562 cells on IgE/FcεRI-mediated activation of basophils in whole blood, (2) the putative role of PS, and finally (3) the effect of the PS-binding molecule Annexin Vand CD300a-blocking antibody on inhibitory signals delivered by apoptotic PBMCs on separated basophils, excluding interference of other cells or humoral factors. For detailed information on experimental design, see the Methods section in the Online Repository at www.jacionline.org. Aliquots of whole blood from 5 patients and 5 controls were preincubated with serial dilutions of viable or apoptotic K562 cells (from 20 3 10/mL to 20 3 10/mL; 378C, 30 minutes) before stimulation with buffer (negative control) or with antiIgE (positive control) and, in patients, rBet v 1, the recombinant major allergen from birch (Betula verrucosa) pollen (for detailed information, see the Methods section). This resulted in a significant dose-dependent inhibition of CD63 upregulation (Fig 1, A) that was more pronounced in healthy subjects than in allergic patients (Fig 1, B; P < .05). Similar results were obtained with patients’ basophils stimulated with rBet v 1 (Fig 1, C). There was no significant effect of viable or apoptotic K562 on the spontaneous expression of CD63 and CD203c on basophils (data not shown). Fluorescein-stained liposomes, noncoated or coated with PS or phosphorylcholine, were added to magnetic bead–isolated basophils of 2 healthy donors (48C, 20minutes) and immediately assessed by flow cytometry (for detailed information about liposomes preparation, see the Methods section). Next, the functional effect of noncoated and PSand phosphorylcholine-coated liposomes was assessed by preincubating different concentrations of nonstained liposomes with isolated basophils at 378C for 30 minutes before anti-IgE stimulation. These experiments revealed that only PS-coated liposomes bind basophilic membranes (Fig 1, D) and inhibit IgE/FcεRI-mediated activation of the cells in a dose-dependent manner, starting with a ratio liposomes/basophil 20/1. As with apoptotic K562 cells, this effect was more pronounced on the CD63 compartment (Fig 1, E; see Fig E1 in this article’s Online Repository at www.jacionline.org). To study the effect of the PS-binding molecule Annexin Vand CD300a-blocking antibody (clone TX49), apoptotic autologous PBMCs (ratio PBMCs/basophil 20/1) were used (n5 3). To bind PS on the membrane of apoptotic PBMCs (aPBMCs), cells were incubated with different concentrations of recombinant human Annexin V at 378C for 30 minutes; thereafter, basophils were added for 30minutes at 378C. Subsequently, cells were stimulated with buffer or anti-IgE. Inversely, to block CD300a receptor on isolated basophils, cells were exposed to different concentrations of the monoclonal anti-human CD300a-blocking antibody TX49 at 378C for 30 minutes before adding aPBMC followed by anti-IgE activation. These experiments showed that blocking CD300a resulted in dose-dependent reversal of the magnitude of inhibition up to 77% (Fig 2, A and C; P 5 .05). Preincubation with anti-CD300a alone did not result in any significant effect on basophil activation markers (data not shown). This is not in contrast with our previous study, in which a CD300a receptor-activating antibody (clone MEM-260) was used, resulting in an inhibition of CD63 upregulation. Similarly, preincubating the cells with recombinant human Annexin V reversed the magnitude of aPBMC-induced inhibition up to 82% (Fig 2, B and D; P 5 .04). Taken together, these results led us to conclude that the interaction between PS and CD300a inhibits IgE/FcεRIdependent anaphylactic basophil degranulation. Nevertheless, the biological or clinical relevance of basophilic response/ inhibition in IgE-mediated hypersensitivity needs to be elucidated. The externalization of PS in the absence of apoptosis as a consequence of granular membrane fusion with the cell membrane involving inversion of membrane leaflet polarity and upregulation of the inhibitory receptor CD300a during basophilic degranulation on a single cell level may contribute to self-terminating mechanisms through cis recognition. Whether the finding of increased apoptosis of monocytes on day 3 of venom immunotherapy suggests the involvement of PS-CD300a interaction on the early suppression of basophils and mast cells is a hypothesis that deserves investigation. Finally, the modulation of basophilic phagocytic activity through CD300a-PS interaction seems difficult to postulate because these cells express low phagocytic activity. In conclusion, we have for the first time demonstrated that the apoptosis-related signal PS suppresses human basophil anaphylactic degranulation via the inhibitory receptor CD300a (IRp60). These data provide interesting input to study the role of phospholipids recognition in modulation of basophilic responses and endorse the role of CD300a as a pharmacologic target for the treatment of allergic disease.