MRGPRX2-mediated mast cell response to drugs used in perioperative procedures and anaesthesia

Abstract

The study of anaphylactoid reactions during perioperative procedures and anaesthesia represents a diagnostic challenge for allergists, as many drugs are administered simultaneously, and approximately half of them trigger allergic reactions without a verifiable IgE-mediated mechanism. Recently, mast cell receptor MRGPRX2 has been identified as a cause of pseudo-allergic drug reactions. In this study, we analyse the ability of certain drugs used during perioperative procedures and anaesthesia to induce MRGPRX2-dependent degranulation in human mast cells and sera from patients who experienced an anaphylactoid reaction during the perioperative procedure. Using a β-hexosaminidase release assay, several drugs were seen to cause mast cell degranulation in vitro in comparison with unstimulated cells, but only morphine, vancomycin and cisatracurium specifically triggered this receptor, as assessed by the release of β-hexosaminidase in the control versus the MRGPRX2-silenced cells. The same outcome was seen when measuring degranulation based on the percentage of CD63 expression at identical doses. Unlike that of the healthy controls, the sera of patients who had experienced an anaphylactoid reaction induced mast-cell degranulation. The degranulation ability of these sera decreased when MRGPRX2 was silenced. In conclusion, MRGPRX2 is a candidate for consideration in non-IgE-mediated allergic reactions to some perioperative drugs, reinforcing its role in mast cell responses and their pathophysiology.

Figure 1

Determination of mast cell degranulation by NMBAs, iodinated contrasts, opiates, non-steroidal anti-inflamatory diclofenac and antibiotics. β-hexosaminidase assays of several NMBAs (A) iodinated contrasts (B) the opiates morphine and remifentanil (C) the non-steroidal anti-inflammatory diclofenac (D) and several antibiotics (E) on LAD2 mast cells. Bars show the mean ± SEM of at least 3 replicates of the experiment. First bar (CTL−; negative control) correspond to unstimulated cells and last bar (I + P; positive control) correspond to cells stimulated with Ionomycin and PMA. Statistical significance (^#p > 0.99; *p < 0.05; ****p < 0.0001; unpaired ANOVA with Bonferroni post-hoc test) is relative to CTL−.

Figure 2

Determination of the expression of the MRGPRX2 receptor. (A) Western blot of mast cells silenced for MRGPRX2 (MRGPRX2-shRNA) or control cells (Non-target-shRNA). Percentage of MRGPRX2 expression on MRGPRX2-shRNA or NT-shRNA cells (Non-target-shRNA). Data is the mean of six experiments. (B) FcεRI (C) and KIT (E). Dot lines correspond to isotype control, grey lines correspond to Non-target shRNA cells and black lines correspond to MRGPRX2-shRNA cells. Positive cells from a representative experiment are indicated in the histogram. Bar charts represent the percentage of FcεRI (D, n = 3) and KIT (F, n = 4) positive cells. Data show the mean ± SEM. Statistical significance (***p < 0.001) was determined using unpaired t-test with Welch’s correction and it is relative to non-target shRNA.

Figure 3

Morphine, vancomycin and cisatracurium responses are mediated through MRGPRX2. (A) β-hexosaminidase assays and (B) percentage of CD63 expression in Non-target and MRGPRX2-silenced cells tested with Cistracurium (50–100 µg/mL), Morphine (1–10 µg/mL) or Vancomycin (500 µg/mL). Data show the mean ± SEM. Statistical significance (*p < 0.05, **p < 0.01, ****p < 0.0001; unpaired t-test with Welch’s correction) is for non-target shRNA versus MRGPRX2 shRNA.

Figure 4

Skin test negative sera from patients are capable of inducing a degranulation response in mast cells. This degranulation capacity is reduced when MRGPRX2 is downregulated. (A) Percentage of CD63 expression of non-target or MRGPRX2 knockdown mast cells incubated with sera from healthy controls (control sera) or sera from patients (see Table 2). Data show the mean ± SEM. Statistical significance (*p < 0.05, **p < 0.01, ****p < 0.0001; unpaired t-test with Welch’s correction) is for non-target shRNA versus MRGPRX2 shRNA. (B) Percentage of CD63 expression in mast cells incubated with control sera or patient’s sera at different time points (0 h to 24 h versus long term collection). Data show the mean ± SEM. Statistical significance (*p < 0.05, **p < 0.01, ****p < 0.0001; unpaired t-test with Welch’s correction) is for 0–24 h versus long term collection. (C) Percentage of CD63 expression of non-target or MRGPRX2 knockdown mast cells incubated with sera from healthy controls and healthy patients who received several drugs. Data show the mean ± SEM. Statistical significance (*p < 0.05, **p < 0.01, ****p < 0.0001; unpaired t-test with Welch’s correction) is for non-target shRNA versus MRGPRX2 shRNA. All data is representative of three independent experiments.