Reply to Sabato V et al “Surface expression of MRGPRX2 expression on resting basophils: An area of controversy”

Abstract

Finally, we conclude that the highly interesting role of MRGPRX2 on resting and activated basophils and eosinophils and its relation to IgE-and non-IgE-mediated hypersensitivity warrants further study.

Principles, potential, and limitations of ex vivo basophil activation by flow cytometry in allergology: A narrative review

“…However, evidence of usefulness of BAT in IDHRs from IgE/FcεRI-independent pathways is limited and should be interpreted carefully. The reporting that basophils constitutively express the MRGPRX2 receptor 30 contradicts earlier observations, 3,137 and it is difficult to align with the observation that various MRGPRX2 agonists do not trigger basophil degranulation in control individuals. 121,128,129…”

“…25 Although modern purification protocols are designed to avoid cell loss and activation (eg, by using low temperatures, Ca2 1 /Mg2 1 -free buffers [with added EDTA], and using negative immunomagnetic selection), cell loss and background activation can occur. 30,31 Alternatively, basophils can be primed intentionally with IL-3 to optimize analytical sensitivity (ie, responsiveness to allergen 1,32,33 and autoreactive antibodies 34 ).…”

“…Other explanations for false-negative results are a nonresponder's cell status (as is observed in about 5% to 15% of the patients), poor storage conditions for the blood sample (analyses are best performed within 4 hours 33 ), use of cytotoxic (concentrations of) allergens, degraded allergens, 58 (pharmacologic) interference with surface receptors, 59 inhibition by cross-reactive compounds, 60 and blunted responses because of a preactivated status of the cell. 30 Therefore, BATs should always include a negative control setting to assess spontaneous expression of the readout, a positive control to verify responsiveness of the cell on cross-linking of surface sIgE-FcεRI complexes. N-formylmethionyl-leucyl-phenylalanine, which acts independently from IgE-FcεRI can be of benefit to include such a positive control to verify cell viability.…”

Basophil and mast cell activation tests by flow cytometry in immediate drug hypersensitivity: Diagnosis and beyond

“…Moreover, quantitative PCR studies have shown that MCs from the skin show high expression of the receptor 18 . Whether MRGPRX2 is also constitutively expressed by human basophils, as claimed recently by Wedi et al 19 is an ongoing subject of controversy [20][21][22][23] and is addressed below. Its presence, or absence, will ultimately determine whether or not these cells will respond to MRGPRX2 agonists, including many drugs 5 .…”

“…The reasons for these divergent results are diverse and largely relate to the applied protocol and setting of sometimes arbitrarily chosen decision thresholds. The low sensitivity of the traditional CD63-based BATs as reported by various authors 6,52,53 most likely results from the fact that resting whole blood basophils, unlike isolated basophils 19,22 , barely express the MRGPRX2 receptor 19,20,22,23 .…”