Glatiramer Acetate Induces Mast Cell Degranulation via MRGPRX2, Implications for Local and Systemic Adverse Reactions

To the Editor:

Glatiramer acetate (GA) is an immunomodulatory drug that consists of a mixture of cationic peptides and is widely used for the treatment of relapsing-remitting multiple sclerosis (RRMS). When administered subcutaneously (20 mg), it causes injection site reactions (ISRs) such as erythema and pruritus in most patients. In addition, it causes immediate post-injection systemic reactions (IPISR) in ~10% of the patients and may lead to the discontinuation of its use.^{1, 2} The involvement of both IgE-dependent and IgE-independent pathways has been postulated to explain the adverse effects of GA.^{1, 2} However, the possibility that GA induces mast cell (MC) degranulation has not been reported.

Mas-related G protein-coupled receptor X2 (MRGPRX2) is predominantly expressed in MCs and likely contributes to adverse hypersensitivity reactions caused by clinically used drugs such as neuromuscular blocking agents, opiates, and antibiotics such as fluoroquinolones.^{3, 4} Flow cytometry analysis demonstrated that unlike RBL cells, cells stably expressing MRGPRX2 (RBL-MRGPRX2) express the receptor on the cell surface (Figure 1A). In RBL-MRGPRX2 cells, GA induced a dose-dependent degranulation, as measured by β-hexosaminidase release, with a maximal response of ~60% at 10 µg/mL (Figure 1B). By contrast, GA at concentrations of 10 and 30 µg/mL did not induce degranulation in native RBL cells even though they responded to antigen/IgE stimulation (Figure 1C). MrgprB2 is the mouse counterpart of human MRGPRX2 and compound 48/80, a classic MRGPRX2/B2 agonist, induced degranulation in mouse peritoneal MCs (PMCs) but GA, at concentrations up to 100 µg/mL, did not (Figure 1D). These findings suggest that GA selectively activates human MCs via MRGPRX2.

Figure 1. GA causes MC degranulation via MRGPRX2.

Receptor expression on (A) RBL-2H3 (RBL), RBL-MRGPRX2 and (E) LAD2 cells are represented by flow cytometry histograms. GA mediated β-hexosaminidase release was determined in (B) RBL-MRGPRX2, (C) RBL-2H3, (D) mouse PMCs and (F) LAD2 cells. (G) LAD2 cells were cultured in the presence / absence of PTx (100 ng/mL, 16 h) or preincubated with C7 or C9 (5 min) and degranulation in response to GA was determined. Data are represented as mean ± SEM, statistical significance was determined by One-way ANOVA (B-D, F) and Two-way ANOVA (G-H) at ****P<0.0001, **p<0.01, *p<0.05. N= 3–5.

To delineate the mechanism via which GA induces MC degranulation, we utilized LAD2 cells, a human MC line that endogenously expresses MRGPRX2.⁵ The receptor is expressed at a similar level in RBL-MRGPRX2 and LAD2 cells (Figure 1A and 1E) and GA induced similar dose-dependent degranulation with maximal responses of between 60–80 % in both cell types (Figure 1B and 1F). MRGPRX2 couples to Gαi family of G proteins and we found that their inhibition by pertussis toxin (PTx) resulted in almost complete attenuation of degranulation in response to GA (Figure 1G). A small molecule inverse MRGPRX2 agonist C9 selectively inhibits MRGPRX2-mediated degranulation in human MCs.⁵ As shown in Figure 1H, C9 caused significant inhibition of degranulation in response to GA but its inactive analog C7 did not.

To determine the relevance of the findings in RBL-MRGPRX2 and LAD2 cells, we performed selected experiments with primary human skin-derived MCs obtained from 3 healthy donors. Flow cytometry analysis demonstrated that MCs from all 3 donors express cell surface MRGPRX2 but at levels less than those in RBL-MRGPRX2 and LAD2 cells (Figure 2, A–C). Furthermore, GA (10 µg/mL) induced 20 – 40 % degranulation as measured by β-hexosaminidase (Figure 2, D–F) and tryptase (Figure 2, G–I) release in skin MCs derived from all three donors. GA induced MC degranulation was substantially inhibited by the inverse MRGPRX2 agonist C9, but not its inactive analog C7 (Figure 2, D–I). Furthermore, treatment of skin MCs with PTx resulted in almost complete inhibition of degranulation as measured by both β-hexosaminidase and tryptase release (Figure 2, D–I). In total, these findings demonstrate that GA induces degranulation of human skin-derived MCs via MRGPRX2, which signals via Gαi-family of G proteins and suggest that RBL-MRGPRX2 cells and LAD2 cells could be utilized for future signaling studies.

Figure 2. GA induces degranulation in primary human skin-derived MCs via MRGPRX2.

(A, B, C) MRGPRX2 expression on skin MCs from 3 healthy donors are represented by flow cytometry histograms. MCs were preincubated in presence of PTx for 16 h or with C9 or C7 for 5 min and GA-induced β-hexosaminidase (D, E, F) and tryptase (G, H, I) release were determined. Data are represented as mean ± SEM, statistical significance was determined by Two-way ANOVA with Tukey’s multiple comparisons (D-I) at ****P<0.0001, ***p<0.001, **p<0.01, and *p<0.05, N= 3–4.

GA likely modulates RRMS via the inhibition of myelin basic protein-mediated T cell proliferation and cytokine secretion.⁶ However, we found that concentrations of GA (1 – 10 µg/mL) that inhibit T cell responses induce MC degranulation via MRGPRX2. This could explain why RRMS patients or healthy volunteers with no previous exposure to GA display positive ISR to the drug.¹ However, the reason why ~10% of the RRMS patients develop IPISR following GA treatment is not known but could result from increased MRGPRX2 expression or the presence of gain-of-function mutation. Regardless, MRGPRX2 inhibitors could be utilized in RRMS patients to prevent discontinuation of GA use. It is noteworthy that MC-derived mediators are elevated in MS patients, which can activate T cells. Therefore, the use of MRGPRX2 inhibitors in RRMS patients could result in the attenuation of T cell-mediated inflammation and thus providing additional benefit.

Abbreviations

C48/80

compound 48/80

FcεRI

high-affinity immunoglobulin E receptor

GA

Glatiramer acetate

GPCR

G Protein-coupled receptor

IPISR

immediate post-injection systemic reactions

ISR

injection-site reaction

LAD2

laboratory of allergic diseases type 2

MCs

mast cells

MrgprB2

Mas-related G protein-coupled receptor B2

MRGPRX2

Mas-related G protein-coupled receptor X2

PE

phycoerythrin

PTx

Pertussis toxin

PMCs

peritoneal mast cells

RBL-2H3

rat basophilic leukemia-2H3 cells

RBL-MRGPRX2

RBL-2H3 stably expressing MRGPRX2

RRMS

relapsing-remitting multiple sclerosis

rhSCF

recombinant human stem cell factor

SCF

stem cell factor