The pseudo-allergic/neurogenic route of mast cell activation via MRGPRX2: discovery, functional programs, regulation, relevance to disease, and relation with allergic stimulation

Abstract

Mast cells (MCs) form operating units with sensory nerves and can contribute to sensations of itch and pain. However, it remained enigmatic for decades how MCs are actually activated in the absence of atopy. MRGPRX2 was discovered only recently but has already changed our view of MC biology. As the receptor of multiple endogenous and exogenous ligands, including substance P and various drugs, MRGPRX2 can be viewed as the missing link underlying clinically relevant MC degranulation in the context of drug-triggered pseudo-allergy and autonomous (eg, neuronal) MC activation in disease. Its existence explains previous findings that remained inexplicable for a long time. The confinement of MRGPRX2 to MCs, and even only to the subgroup of MC_TC-type MCs, makes research in this field exciting from a theoretical as well as from a translational or pharmacological perspective alike. In this review, I will first give a brief overview of MCs, their subsets and modes of activation, then briefly touch on the history of MRGPRX2 discovery, summarize some recent advances regarding ligands, functional aspects and regulation by extracellular cues, and recapitulate the emerging role of the MRGPRX2 system in health and disease. Finally, a concise comparison between MRGPRX2 and FcεRI will be presented, contrasting key characteristics of the 2 dominant ways of MC activation. There is a huge disproportion in our understanding of FcεRI-triggered versus MRGPRX2-triggered events, but since research into the latter is in full swing, some of the missing pieces of the puzzle are likely to be filled in soon.

Mast cells (MCs)

MCs are tissue-resident cells of hematopoietic origin that appear early in evolution and are strategically located at the interface of host and environment in tissues that are susceptible to infiltration by micro-organisms and noxious agents^1,2. In particular, MCs are known as principal effector cells of IgE-mediated type I hypersensitivity, encompassing allergic rhinitis, asthma, food allergy, subsets of urticaria and eczema, and in the most severe case, anaphylaxis^3,4. In the skin, MCs are also associated with itch sensations through operating units with sensory neurons^5,6. In addition, MCs have been implicated in various physiological processes and as linkers between innate and adaptive immune responses. In this regard, several of their activities are in principle beneficial to the host like tissue homeostasis and repair, protection against pathogens, and detoxification of potentially harmful substances, processes by which MCs safeguard health. In contrast, MC activities can be detrimental even outside of typical allergic constellations, where the cells can drive incontrollable inflammation, in particular through various cytokines, chemokines and growth factors. MCs thereby contribute to complex disorders and conditions like fibrosis, autoimmunity, and the progression or metastasis of certain tumors^1,7–9. In contrast, MCs have homeostatic roles, aid in wound healing and perform multifaceted immunoregulatory functions, which can reduce or disrupt ongoing inflammation^10,11. In addition, MCs are well-recognized for organizing host defenses, not only against bacterial, but also against several viral infections^12–16.

MCs are unique cellular elements that form a separate clade within the hematopoietic network, being well-separated from other lymphoid and myeloid lineages, including basophils in mice and men^17–19. In accordance, several surface receptors and intracellular molecules constitute distinctive and (in part) highly selective MC characteristics. Receptors encompass the FcεRI or IgE receptor (IgER) itself, which triggers allergic MC activation (more on this below), the IL-33 receptor component ST2 (gene IL1RL1) and the receptor tyrosine kinase KIT^1,9,20,21.

KIT is of particular significance to the lineage, as the stem cell factor (SCF)/KIT axis is implicated in the expansion of MCs from their progenitors and controls nearly every aspect of MC biology throughout lifespan, including proliferation, chemotaxis, adhesion, mediator generation and release, as well as survival in their natural habitats^1,20,22.

The most salient features of MCs are their storage granules (secretory lysosomes) filled with an array of preformed bioactive molecules²³. This property endows MCs to respond swiftly and efficiently by releasing the prestored mediators into the extracellular milieu in a process termed degranulation or granule exocytosis, of which different subtypes exist^24,25 (see forthcoming text and Fig. 1).

Figure 1: Preformed skin MC mediators and modes of exocytosis by the allergic vis-à-vis pseudo-allergic/neurogenic route. Resting MCs with intact granules, filled with various mediators explained in the legend below (left). Allergic degranulation by aggregation of heterotetrameric FcεRI (center). This pathway is fairly slow and characterized by intracellular granule-granule fusion known as compound exocytosis. Degranulation elicited by GPCR signaling, leading to very rapid exocytosis of single granules (right) (modelled on Gaudenzio et al²⁵).

Mediators stored in MC granules comprise heparin (or other sulfated proteoglycans), the biogenic amine histamine (synthesized by histidine decarboxylase), and a number of MC proteases, especially tryptase (TPSAB1/TPSB2), chymase (CMA1), and carboxypeptidase A3 (CPA3)^23,26 (Fig. 1); their composition varies across MCs in different tissues (as described in the next paragraph).

Enzymes involved in the production of arachidonic acid mediators, in particular prostaglandin D2 (PGD2) and leukotriene C4 (LTC4) are also highly enriched in MCs (genes: HPGDS, LTC4S) in accordance with MCs’ potential to generate vast amounts of eicosanoid mediators¹⁸. As a result, metabolites of PGD2 and LTC4 are well suited as serum biomarkers to diagnose anaphylaxis, as reported previously^27,28.

MC heterogeneity

MCs from disparate tissues are distinguished on the basis of the neutral protease composition of their granules. One type, so-called MC_T (tryptase⁺) contains only tryptase, its appearance typically depends on T lymphocytes and it is the principal subtype in respiratory and intestinal mucosa¹. MC_TC (tryptase⁺, chymase⁺), on the other hand, contain both tryptase and chymase and are predominant in skin, and some other locations like intestinal submucosa and myocardium. The distinction between MC_TC and MC_T has a correlate in the mouse where connective-tissue MCs (CTMCs) and mucosal MCs (MMCs) can be distinguished¹¹.

The typical MC granules, and especially the MC proteases contained therein are lineage-defining features^23,26. Accordingly, MC chymase, followed by tryptase was basically confined to MCs in the comprehensive FANTOM5 atlas, in which both protease genes were expressed ≈10,000-fold higher over the mean on non-MCs, making them the most selective features of the lineage (together with the newly uncovered MRGPRX2, described below)^18,29,30.

The skin is a particularly MC-rich organ^31,32 and dermal MCs are key to systemic phenomena like anaphylaxis and systemic mastocytosis^20,33 aside from local involvement in skin disorders. In fact, cutaneous symptoms develop in the majority of anaphylaxis cases, while manifestations in other organs are more variable³³. In the skin, virtually all MCs are of the MC_TC phenotype (95%–100%³⁴). Apart from chymase, which lent its name to the classification, MC_TC and MC_T cells exhibit further diversity manifested by differential expression of receptors, ultrastructural features, secretory functions, and pharmacological manipulation³⁵. Unique MC_TC traits, not expressed by MC_T, include carboxypeptidase A (CPA3), cathepsin G (CTSG) as well as CD88 (C5AR1) and C3AR1³⁶. The latter receptors are present in MC_TC but less prominent than in other immune cells. For example, while C5AR1 is expressed by MC_TC-type MCs, expression is manifold higher in monocytes, macrophages, neutrophils, eosinophils and basophils^29,30. In contrast, the newly uncovered receptor MRGPRX2 represents not only an MC_TC-specific trait absent from MC_T but it is confined to these cells altogether¹⁸. It can therefore be considered a novel signature molecule of the MC_TC-type MC³⁷.

Of particular interest are the differing abilities of MC_T-type and MC_TC-type cells to respond to a rich variety of endogenous and exogenous stimuli. The reason for this distinction remained enigmatic for decades. The discovery of MRGPRX2 as the shared receptor for diverse secretagogues can now excellently explain the dichotomy, as will be further detailed below.

MC activation

Acute effects or “immediate hypersensitivity reactions” result from the rapid release of inflammatory mediators from activated MCs, and underlie diseases like rhinoconjunctivitis, asthma, urticaria, food allergy, and anaphylaxis. Activation can proceed through the allergic route, which comprises 3 components: the high affinity IgE receptor (FcεRI) on the MC surface, IgE, directed against a specific antigen bound to FcεRI, and the antigen itself which aggregates two or more FcεRI entities to ignite granule exocytosis^1,3,38.

FcεRI has remained the major receptor responsible for the clinical manifestations of MC activation for decades and is still a premier research focus in MC biology. On the MC surface, FcεRI is comprised of 3 different subunits: an IgE-binding α subunit, a signal-amplifying β subunit, and 2 disulfide-linked signal-initiating γ subunits (Fig. 1)^38,39. Aggregation elicits a largely determined tyrosine kinase cascade, culminating in granule exocytosis with its release of histamine, heparin, and proteases (Fig. 1). Most of the mediators acutely secreted from MCs exert their effects on end-organ targets like epithelial and endothelial cells, airway smooth muscle cells and nerves by activating G-protein-coupled receptors (GPCRs), including histamine (HRH1; HRH2; HRH3; HRH4), tryptase (PAR2=F2RL1), LTC4 (CYSLTR1; CYSLTR2), and PGD2 (PTGDR; PTGDR2=CRTH2)⁴⁰. The secreted substances induce the typical signs and symptoms of allergic reactions like vasodilation, vascular permeability, stimulation of sensory nerves, and smooth muscle contraction. In the skin, symptoms like wheal and flare reactions and pruritus are typical. In addition to histamine, MC tryptase is a key mediator of (nonhistaminergic) itch through direct activation of PAR-2 on sensory nerves or indirectly via keratinocytes^41,42. Atopic dermatitis (AD) itch seems to be particularly dependent on tryptase⁴².

Other than exteriorization of granule contents, MCs can generate a number of other bioactive substances de novo. In fact, MC mediators have been classically divided into 3 groups, that is (a) preformed, granule-associated (as described above), (b) rapidly generated from membrane lipids (especially PGD2 and LTC4), and (c) newly produced by transcription/translation^3,39. The latter subset comprises multiple cytokines, chemokines, and growth factors. The allergic route elicits all 3 classes of mediators, though the third category is heterogeneous and MC-subset dependent. In skin MCs, for example, FcεRI evokes robust TNF-α, IL-5, IL-31 responses and several chemokines, but it does not enhance IL-6^18,43–45, while IL-6 regulation differs in other MC subtypes⁹.

MC cytokines are believed to orchestrate acute antimicrobial defense programs but can also contribute to chronic inflammation and pain, for example by facilitating neurogenic inflammation^6,37. In other instances, they can have predominantly anti-inflammatory functions¹⁰. Thus, in contrast to early mediators, late-phase mediators have more heterogenous roles, whose functions are context-dependent, and co-determined by other cells and microenvironmental cues in addition to type and stage of pathology. Together, dysregulated MC cytokine synthesis can mediate a variety of physiological processes and is believed to underlie complex chronic diseases, as has been extensively reviewed^1,3,46,47.

In addition to FcεRI-aggregation multiple other receptors can evoke MC cytokine responses without degranulating MCs directly, including SCF/KIT, Toll-like receptors, and IL-33/ST2¹. Conversely, substantially fewer receptors trigger granule exocytosis. However, only receptors that elicit degranulation can precipitate acute hypersensitivity reactions and be the root of allergic (or the clinically indistinguishable pseudo-allergic) symptoms from instantly released mediators (Fig. 1). Several GPCRs can induce exocytosis, especially C5AR1, and C3AR1. Although their clinical significance remains to be fully established, complement receptors seem to contribute to some types of food allergic and anaphylactic responses, for example to contrast media or dialysis membranes^48,49.

Collectively, MC mediator release constitutes a pivotal event in anaphylaxis and milder manifestations of acute MC degranulation, and it can also lead to the initiation of more complex inflammatory circuits beyond allergy and “purely” MC driven disorders. MRGPRX2, a GPCR highly effective at MC degranulation, recently entered the scene and can explain clinically relevant hypersensitivity in the absence of allergy, as will be described in the next paragraph.

Gateways to nonimmunologic MC activation: MRGPRX2 takes center stage

As pointed out, MC_TC differ from MC_T in their responses to endogenous and exogenous secretagogues. Generally speaking, MC_TC are degranulated by many substances to which MC_T are unresponsive^50,51. Nonallergic MC stimulation in the skin has a long history and was initially observed by skin tests and later also in dermal MC containing suspensions ex vivo^50–52. Common histamine liberators known early on are neuropeptides like substance P (SP), vasoactive intestinal peptide (VIP), cortistatin, somatostatin and basic secretagoues like codeine, morphine, and compound 48/80 (c48/80)^50,51. Fjellner and Hägermark⁵³ observed already in 1982 that “… histamine responses were induced by (…) morphine. The putative neurotransmitters substance P and vasoactive intestinal polypeptide (VIP)—which moreover are potent histamine liberators—had no additive effect.” In 1985, Piotrowski and Foreman⁵⁴ remarked: “It is concluded that the receptors mediating histamine release and the flare response are similar, and that SP, Som (Somatostatin), and VIP are acting at a similar receptor to produce these effects. It is probable that this receptor is also the site of action of compound 48/80.” A similar observation was published by yet another laboratory: “The similar characteristics of histamine release induced by substance P, VIP, somatostatin, compound 48/80, poly-L-lysine and morphine suggest that they share a common pathway of activation-secretion coupling distinct from that of IgE-dependent activation”⁵⁵. Because responses were saturable by a single stimulus, these early observations hinted at similar or identical mechanisms of action. However, it took several decades to comprehend the underlying basis.

Since the early 1950s, c48/80 has been employed as “histamine liberator” in various species, triggering secretion from skin (but no other) MCs also in humans^50–52. Hundreds of reports had used c48/80 to degranulate MCs (or to “deplete skin histamine”), but it was not until 2006 that Tatemoto and colleagues reported on a specific receptor for this basic compound using not only transfected HEK293 cells but also MCs⁵⁶. Beforehand, it was believed that basic-amphipathic secretagogues interact directly with pertussis toxin-sensitive G proteins (Gαi) in MCs to propagate downstream signaling in a receptor-independent manner (reviewed in Ali³⁷). So, while participation of G proteins in nonimmunologic MC activation was suspected early on, it was a long and slow process to realize that most of these ligands converge on the very same receptor, namely MRGPRX2.

Tatemoto and colleagues also provided the first demonstration that MRGPRX2 is confined to CTMC/MC_TC and is barely expressed by MMC/MC_T (difference of ≈500-fold at mRNA level)⁵⁶. Despite this seminal report, MRGPRX2 still attracted fairly little interest by the MC community and it took several more years for the field to really take off.

Together with the FANTOM5 consortium we described MRGPRX2 as a new signature molecule of MCs in 2014, finding its expression to be 10,000-fold enriched over the mean of non-MCs from all across the body, a ratio otherwise only observed for the MC specific proteases tryptase and chymase¹⁸.

In 2015 McNeil and colleagues finally demonstrated that MRGPRX2 acts as the receptor for a multitude of drugs, which cause pseudo-allergic reactions in susceptible individuals, including icatibant, neuromuscular blocking agents, and fluoroquinolone antibiotics^57,58.

This was seen in cultured human MCs in vitro, but also in the mouse in vivo⁵⁷. In fact, the group had succeeded in identifying Mrgprb2 as the murine MC-expressed ortholog of MRGPRX2. Mrgprb2 deficient mice and MCs cultured from these mice showed dramatic reductions in MC degranulation to c48/80, SP and paw edema used as an in vivo readout. Conversely, Mrgprb2 deficient mice had no defect in MC numbers and Mrgprb2-lacking MCs responded normally to FcεRI aggregation. Because the data were so convincing, with pseudo-allergic reactions being basically abolished in the absence of Mrgprb2/MRGPRX2, this paper provided the major spark fueling research into this novel pathway and helped renew interest in the basic principles underlying pseudo-allergic and neurogenic MC activation.

Meanwhile MRGPRX2 had also been identified as the receptor for endogenous host defense peptides (HDPs), including cathelicidin (LL-37) and β-defensins, raising the interesting possibility that MCs could organize anti-microbial responses in part by the activation of MRGPRX2^59,60.

The discovery of MRGPRX2 can explain a number of puzzling results inexplicable in the past, making it a true paradigm changer in MC biology. One example is MC activation by SP, that is not inhibitable by NK-1R/NK-2R antagonists (eg, in LAD2 MCs) or occurs even in the complete absence of NK-1R/NK-2R^61,62. It turns out that in accordance with other MC subsets^25,56,57, skin MCs chiefly utilize MRGPRX2, and that NK-1R contributes only modestly as SP receptor in these cells^63,64. Azimi et al⁶⁵ also reported that SP-mediated itch in vivo requires members of the Mrgpr cluster. Still more, the same authors reported that NK-1R antagonists have off-target effects at murine Mrgprb2, but not at human MRGPRX2, explaining the poor effectiveness of NK-1R antagonists in clinical trials compared with preclinical mouse models⁶².

The significance of Mrgprs to the transmission of SP signals is corroborated by recent findings in vivo obtained in models of hyperalgesia, in which SP signaling proceeded in an NK-1R independent, but MRGPRX2/b2-dependent manner⁶⁶. Although it remains to be elaborated in greater detail, chances are that brain MCs, at least in the rat, express the rat equivalent of MRGPRX2 (MrgprB3¹¹) and respond to SP via this route⁶⁷.

Further discrepancies concern other neuropeptides like pituitary adenylate cyclase-activating peptide (PACAP), which was known for years to degranulate MCs, but this response was also uncoupled from its canonical receptors^68,69. In fact, PACAP can activate MRGPRX2, which thus constitutes an alternative PACAP receptor^56,57. Now, that the receptor for a plethora of MC activating substances has been unveiled, a variety of previously inexplicable findings can finally be understood and put into context.

MRGPRX2’s role in disease versus health

As mentioned, MRGPRX2 is the key participant in pseudo-allergic reactions elicited by a number of drugs, including muscle relaxants, opioids, icatibant and fluoroquinolones^57,70. In addition, it is the receptor for a wide range of endogenous ligands, including neuropeptides and HDPs^56,57,59,60.

Besides acute hypersensitivity reactions, other clinical manifestations do not seem to have an allergic component (no culprit IgE is detectable) yet are precipitated by degranulating MCs all the same. Accumulating evidence suggests that MRGPRX2 deregulation may underlie many of the phenomena. One example is chronic idiopathic urticaria (CIU), the most common form of chronic urticaria, for which precipitating agents are unidentified. It was known for decades that CIU patients respond more vigorously to intradermal SP and VIP injections despite comparable MC numbers and skin histamine¹¹. A recent report indicated that the reason may lie in MRGPRX2 expression, as MCs in CIU skin display higher levels of MRGPRX2 compared with healthy skin⁶³. In fact, the discovery of MRGPRX2 has helped to revive the issue of SP’s contribution to CIU pathology⁷¹. The finding that Mrgprb2/MRGPRX2 mediates NK-1R-independent MC activation in vitro and in vivo, has provided further support for the theory that SP and MCs are associated with pathologies and conditions like hyperalgesia and neurogenic inflammation⁶⁶. Mrgprb2 on MCs could be complemented by Mrgpra1 on specialized neurons in the mouse, since Mrgprb2 and Mrgpra1 both exhibit topological similarities with MRGPRX2 and may thus function as its orthologs^65,72. Mrgpra1 may be crucial in the response to SP, as Mrgprb2 has a much lower affinity for SP than its human counterpart^56,57,70.

Other clinical conditions elicited by MC mediators in the absence of allergy comprise injection-site reactions, for example to icatibant^37,73, “idiopathic” MC activation in patients with mastocytosis or in mast cell activation syndrome, many of whom not sensitized to typical allergens (or not in contact with precipitating allergen when the reaction commenced), as well as idiopathic anaphylaxis^48,73.

Furthermore, contributions of MRGPRX2 to pain and itch are currently being elucidated in mouse models of postoperative hyperalgesia, and nonhistaminergic itch in the context of contact dermatitis^66,74.

While most basic scientists in the field are convinced by now that the discovery of MRGPRX2 represents a true paradigm shift in MC biology, clinicians tend to be more skeptical, especially when long-held ideas are challenged (such as that anaphylaxis is typically triggered by FcεRI-mediated activation of allergy effector cells). A recent study reporting on a patient with 5 severe episodes to hymenoptera venoms and ciprofloxacin in the absence of specific IgE, however, indicates that the importance of MRGPRX2 starts to be recognized by clinicians treating anaphylaxis patients, as well^75,76. Clearly, the non–IgE-mediated, pseudo-allergic/neurogenic pathway requires deeper insights in humans, including epidemiologic studies evaluating MRGPRX2’s significance in drug and idiopathic anaphylaxis (and milder forms triggered by acute MC mediator release) and a perception of the amplified MRGPRX2 “activity level” in affected patients. With more research conducted, MRGPRX2 will likely turn out the principal receptor involved in anaphylaxis in patients who do not suffer from type-I allergy and also help explain individual reactivity patterns.

In addition, MRGPRX2 is likely implicated in AD, especially in intrinsic forms of the disease, in which patients have no elevated IgE, as MRGPRX2 represents a receptor for staphylococcus δ-toxin critically implicated in AD pathogenesis⁷⁷. Another route of MC activation in AD may be by neuropeptides as intimate associations of MCs with nerves have been reported in AD, with degranulated MCs invading nerve bundles in lesional skin⁷⁸. Precipitation of the “Red Man Syndrome” by vancomycin has also been attributed to MRGPRX2 activation⁷⁷, and MRGPRX2 has been linked to the pathology of rosacea, a disease probably mediated, at least in part, by MCs following activation by neuropeptides or cathelicidin^37,79.

In a subgroup of patients with irritable bowel syndrome, a 10.7-fold change in MRGPRX2 expression was found in colon descendens biopsies⁸⁰, and the clinical relevance of this finding deserves further investigation. Similarly, Manorak and colleagues reported on hyperexpression of MRGPRX2 in lung MCs in patients with severe asthma compared with MCs in normal lung tissue, paralleling the increase in MC_TC MCs in an organ normally dominated by MC_T-type MCs; this likewise suggests that MRGPRX2 may contribute to diseases in organs, in which the MC_T-type normally prevails, including asthma⁸¹.

A selection of clinical manifestations potentially arising from aberrant MRGPRX2 function are summarized in Figure 2. Moreover, and as mentioned above, recent in vivo studies in the mouse have revealed an important role of Mrgprb2 in the development of hyperalgesia as well as nonhistaminergic itch and contact dermatitis^66,74, and it will be of interest to determine whether similar conditions depend on MRGPRX2 also in humans.

Figure 2: Potential implication of MRGPRX2 in mast cell (MC) dependent diseases. Summary of the current knowledge associating MRGPRX2 function with various diseases. Evidence is still rather weak in most instances, but due to intense research in this field, the connections are likely to be strengthened soon and additional ones discovered.

In contrast to the detrimental, disease-promoting functions of MRGPRX2, evidence is accumulating that the receptor can be beneficial and organize host-defenses by mobilizing antibacterial mechanisms. For example, MCs are activated by HDPs like β-defensins and cathelicidin in an MRGPRX2-dependent manner^59,60,82 as well as by bacterial products themselves like staphylococcus δ-toxin⁷⁷ and quorum-sensing molecules secreted by bacteria to signal population density⁸³. Moreover, exogenous activation of Mrgprb2 by mastoparan during bacterial infection turned out beneficial and accelerated bacterial clearance and wound healing in a model of Staphylococcus aureus induced dermonecrotic lesions through recruitment of leukocytes, most importantly neutrophils⁸⁴.

Collectively, although research is still at an early stage, MRGPRX2 may have plenty of roles in the body ranging from acute non-IgE dependent hypersensitivity and contribution to chronic diseases but also orchestration of host defenses and other advantageous processes at the other end of the spectrum.

MC-neuronal crosstalk

As MCs often reside near nerve fibers, they are prototypical candidates for modulating neural activity, nociception and pruritis, and an improved understanding of the mechanisms underlying peripheral and central sensitization under consideration of MCs is of elevated interest^5,6,40. As mentioned, MRGPRX2 may play a particularly active role in the MC—nerve communication, as recently reported in a seminal study of neurogenic inflammation and post-operative hyperalgesia in a murine model⁶⁶.

The close proximity at the neuroimmune interface also indicates that neuropeptides may reach sufficient concentrations to activate MCs via MRGPRX2, especially when the 2 constituents move even closer like in AD skin⁷⁸.

MC products may vice versa signal back to neurons, for example, via tryptase activating PAR-2^41,42. Interestingly, a recent study reported that MRGPRX2-triggering favors the release of tryptase over histamine⁷⁴. The role of tryptase in this pathway is noteworthy, not only due to its role as mediator of nonhistaminergic itch⁴², but also because tryptase seems to possess a built-in off-switch, potentially resulting in timely controlled patterns of activation and inactivation. For example, tryptase can degrade a number of substrates, including cytokines, neuropeptides and cathelicidin^26,85. As the latter 2 activate MCs via MRGPRX2, this suggests the existence of a negative feedback loop in the form of MRGPRX2-ligand >MC-MRGPRX2 activation >tryptase release -> MRGPRX2-ligand degradation.

Interindividual variability in MRGPRX2 function

So, if MRGPRX2 is such a universal receptor and so prominent on MCs (especially in the skin) and its ligands so numerous and omnipresent, encompassing not only drugs but also endogenous substances, the first question that pops into mind is “Why are serious reactions not (even) more frequent in the population?” One of the major reasons may be that MRGPRX2 is a low-affinity, nonselective receptor, a quality which sets it apart from the majority of other typical GPCRs^37,51.

Stimulation by drugs will thus require high concentrations precisely in those organs, in which MCs express MRGPRX2 and this may (fortunately) not be easily achieved in real life scenarios. Therefore, stimulation may only become clinically relevant in individuals who have a particularly active MRGPRX2 route, dictated by hyperexpression and/or hyperactivity of MRGPRX2 itself, or components of its downstream signaling machinery.

In fact, a study from our laboratory revealed differential propensity in the population to respond to MRGPRX2 ligands, whereby responses differed between 3.1 and 52.4% (net histamine release) for c48/80 and between 2.9 and 52.6% for SP in skin MCs from different individuals⁶⁴. A recent expansion of the datasets demonstrated an even wider range with a (current) maximum at 67.7% for c48/80 and 65.8% for SP (unpublished results). The different proneness to become activated via this route may, at least in part, stem from variable expression of MRGPRX2, as insinuated by our study⁶⁴. MRGPRX2 activity may be likewise regulated at the level of MRGPRX2 subtype expression, as demonstrated by the existence of several single nucleotide polymorphisms, reducing MRGPRX2 function⁸⁶.

Differences in signaling strength offer another explanation as to whether ligand binding to MRGPRX2 will elicit a stronger or weaker response. The signaling cascades transduced via MRGPRX2 are still poorly understood but coupling to Gi and Gq class G-proteins has been reported¹¹. G protein selection may depend on the cell type, as in transfected HEK293 cells only Gq coupling was noted^84,87, while Gi was (additionally) involved in MRGPRX2-transfected RBL-2H3 cells and in MCs (endogenously expressing MRGPRX2)¹¹. Possibly slightly altered patterns of G-protein activation exist not only among different cell types but also in the same cell type across subjects. The same applies to the activation of β-arrestins versus G-proteins. While this issue has been investigated for distinct ligands (i.e. biased vs. balanced entities⁸⁸), individual factors may partially dictate the outcome of the delicate balance between G-protein and β-arrestin activation even to the same ligand, but in MCs from different people. Collectively, inter-individual differences in signaling machineries may contribute to whether an individual’s MCs will react more or less strongly to MRGPRX2 ligands but further research will clearly be needed to actually prove or dismiss this theory.

Another possibility is at the level of ligands, including different uptake efficiency of exogenous agonists or overexpression of endogenous ligands that activate MRGPRX2. Neuronal MC activation may therefore differ across individuals not only as the result of different spatial arrangements between MCs and adjacent neurons⁷⁸, but also by differential propensity to express, store, and release neuropeptides that serve as MRGPRX2 ligands, including SP (on the connection with urticaria, see details above). With the discovery of MRGPRX2 as the major neuropeptide receptor on MCs, this line of research will sooner or later uncover patient-specific factors, that dictate why and under what circumstances and in which organs (or locations) individuals are prone to react to the pseudo-allergic/neurogenic route.

In addition, interindividual differences in overall MC load (not only as a result of mastocytosis but stemming from natural variability in the absence of disease) is yet another parameter that could potentially decide whether MC derived mediators will reach the critical threshold to precipitate a clinically noticeable event. The latter is also true for FcεRI-dependent processes, which likewise vary in strength across individuals by so far unidentified mechanisms^32,45,64,89.

Together, interindividual variation in overall MRGPRX2 expression and isoform pattern, complemented by qualitative or quantitative differences in signaling machineries may determine whether a person will easily suffer from MRGPRX2-triggered reactions. Moreover, low-affinity toward MRGPRX2 ligands combined with sub-threshold availability of such ligands in MRGPRX2-expressing organs (in particular skin) can potentially explain why MRGPRX2 activation does not constantly precipitate symptoms in the general population. Notwithstanding, the frequency of MRGPRX2-driven hypersensitivity reactions may rise in the future after reclassification of presumably IgE-dependent reactions as MRGPRX2-triggered.

Regulation of MRGPRX2 function by extracellular cues

MC phenotype and functional aspects are driven by multiple stimuli from the cells’ immediate microenvironment. A further level of regulation may therefore be achieved by influences from the micromilieu modulating responsiveness to MRGPRX2 ligands by adjusting receptor (or ligand) expression, MRGPRX2 signaling or both. In their natural habitats such as the skin, MCs are surrounded by diverse cell types which directly or indirectly control not only MC differentiation and maintenance but also adapt and fine-tune functional competence even after terminal maturation. There are well-established lineage supportive or influencing factors, and numerous mediators have been demonstrated over the last decades to regulate responsiveness to FcεRI-aggregation^9,39. Far less is known about the impact of extracellular cues on MRGPRX2 function. Therefore, we started out to elucidate this connection for some particularly important representatives, including SCF, IL-4 and, IL-33.

As mentioned, SCF is the best-defined MC expansion and survival factor, which in addition to maturation, survival, and proliferation regulates MC chemotaxis, adhesion, cytokine expression, and metabolism^1,20,39. Although SCF does not trigger degranulation on its own, it acts in synergy with FcεRI and is thus an important amplifier of allergic MC activation^39,90. Studying the priming effects of SCF on FcεRI-triggered and MRGPRX2-triggered histamine release in skin MCs, we surprisingly learnt that SCF attenuates degranulation induced by the MRGPRX2 route, while it augments the FcεRI-elicited process as expected⁶⁴. The negative effect of SCF after short preincubation (priming) was duplicated on chronic exposure: maintenance of tissue-derived MCs in culture requires the presence of MC supportive factors, most importantly SCF, while IL-4 can further increase MC yield and/or function^{43,89,91–95}. The altered in vitro milieu introduces several changes to skin MCs compared with their ex vivo equivalents^{18,43,45,96,97}.

With regard to MRGPRX2 biology, the in vitro environment dampens (but does not eradicate) pseudo-allergic responses as well as MRGPRX2 expression, while it does the opposite on the allergic route⁹⁸. As short withdrawal of SCF or IL-4 individually, but most potently of both collectively, was able to partially reestablish the MRGPRX2 pathway in our study, both SCF and IL-4 seem to make negative adjustments to MRGPRX2 function also on prolonged exposure⁹⁸. Under all conditions tested, the FcεRI-triggered route showed the inverse pattern of regulation, substantiating that allergic and pseudo-allergic MC activation can obey distinct and even opposite rules.

Retinoic acid (RA) is a critical hormone in the cutaneous environment⁹⁹ regulating different aspects of skin MCs, including adhesion, cytokine production, degranulation, survival, and proliferation^44,100–104. In fact, MCs may be among the most significant targets of the vitamin A metabolite in the skin milieu, as evidenced by the enriched expression of RA-network genes in MCs vis-à-vis all other major skin cell subsets⁴⁴. Interestingly, MC culture in the presence of RA was found to counter pseudo-allergic and to concurrently favor allergic degranulation¹⁰². Attenuation of the pseudo-allergic route was associated with downregulation of MRGPRX2 in RA-exposed MCs after 7 d of treatment, while FcεRI expression and function were concomitantly enhanced¹⁰².

Conversely, RA was recently reported to trigger acute MC activation via MRGPRX2/b2 and thereby to contribute to the frequent condition of RA-induced dermatitis¹⁰⁵.

The previously described findings for SCF, IL-4, and RA indicated a clear dichotomy between the 2 stimulatory systems, whereby strengthening of one seemed to automatically weaken the other. However, subsequent focusing on IL-33 revealed that this unifying pattern is actually too simplistic.

In fact, in a chronic setting (mimicking chronic inflammation) both routes were attenuated by IL-33, albeit to different degrees. While the impairment of allergic degranulation was donor-dependent and rather subtle¹⁰⁶, the MRGPRX2 pathway was basically abolished, resulting from the elimination of MRGPRX2 expression in IL-33 cultured cells¹⁰⁷. Downregulation of MRGPRX2 occurred at the transcriptional level, was rapid (2–4 h), and partially mediated by JNK activity (but apparently not by other MAPKs)¹⁰⁷. Conversely, exploring IL-33’s impact upon short-term priming, we found that both routes were promoted to a similar degree. IL-33 was thereby identified as the first mediator to uniformly affect allergic and pseudo-allergic/neurogenic degranulation. For both receptor systems, the positive effect of IL-33 depended on p38 activity. This was intriguing because even though p38 forms part of the signaling cascade elicited by FcεRI-aggregation, the kinase is more commonly associated with functions like cytokine production (especially TNF-α), migration, and adhesion, but not degranulation in the first place^108–111. We confirmed that p38 had no effect on degranulation elicited by the 2 routes in the absence of IL-33, so that p38 was only involved in the potentiation of degranulation by IL-33¹⁰⁷.

Taken together, the MC modulating factors investigated thus far show a clear-cut distinction and favor one route over the other with the exception of IL-33. Strikingly MRGPRX2 function is typically attenuated by MC supportive factors and drops when MCs are not embedded in their tissue of origin, whereas the FcεRI system can be boosted in surroundings of supraphysiological concentrations of selective factors, most importantly SCF^45,95. This raises the interesting question as to how MRGPRX2 expression and function is maintained in the skin environment.

Comparison between FcεRI and MRGPRX2

The 2 pathways of MC-dependent hypersensitivity show unique features not shared between them. One striking finding is the two patterns of granule discharge described by Gaudenzio et al²⁵ and graphically modelled in Figure 1. We reported that the allergic and pseudo-allergic/neurogenic routes are independently controlled, as evidenced by studies with skin MCs from a multitude of individual donors⁶⁴. The efficiency of one route is therefore not predictive of the other, and subjects with pronounced allergic or pseudo-allergic stimulability are not automatically equipped with a generally enhanced secretory competence⁶⁴. This makes an overlap in exocytotic signaling cascades unlikely, and also largely rules out commonalities in the late predegranulation steps (ie, assembly of proteins involved in traffic, priming, tethering and docking of secretory vesicles²⁴), at least the rate-limiting ones. Conversely, degranulation by calcium ionophore is highly correlated with the FcεRI-mediated process, suggesting that the rate-limiting event of allergic degranulation occurs downstream of Ca²⁺ mobilization³². This is notable given that calcium ionophores often serve as positive control in degranulation assays without considering that responses to these substances can vary substantially across MC subsets, including MCs from different individuals. While FcεRI-elicited degranulation is uncoupled from the MRGPRX2-triggered process, activation of the MRGPRX2 route by different ligands, like c48/80 and SP, gives a nearly perfect correlation and the pathway can be saturated by a single ligand alone⁶⁴.

Our findings showing independence of the routes are in accordance with the 2 patterns of granule exocytosis referred to above²⁵. In their study MRGPRX2-mediated degranulation was a rapid process, associated with a quick and transient peak of Ca²⁺ influx, followed by secretion of individual granules, whereas FcεRI-elicited secretion was delayed, but progressive, and characterized by granule-to-granule fusion, also termed compound exocytosis²⁵. Granules were more heterogenously shaped and of bigger volume in the allergen-triggered pathway, yet smaller and more spherical for SP. Only the FcεRI-route required IKK-β activation and encompassed SNAP23/STX4 complex formation typical of compound exocytosis²⁵.

Notably, a recent study reported that MRGPRX2-triggering favors the release of tryptase over histamine, while more histamine is liberated following allergic activation⁷⁴. Although this needs to be confirmed for other types of MCs (also in humans), it implies at least subtle differences in the repertoires of acute mediators being secreted after activation of distinct receptors, which should also be identifiable in clinical settings (allowing distinction between allergic and “pseudo-allergic” anaphylaxis on the basis of mediator signatures).

MCs are also potent producers of other inflammatory mediators beyond those prestored in granules. So far, there is controversy as to whether MCs activated via MRGPRX2 synthesize and release significant amounts of lipid mediators and cytokines. For eicosanoids, for example, Benyon and colleagues showed that LTC4 and PGD2 are generated at ≈20-fold lower level by SP as compared with anti-IgE in MC containing dermal fractions, and similar amounts were detected for other MRGPRX2 ligands⁵⁰. Varricchi et al¹¹² could not detect a significant increase in LTC4 or PGD2 after SP over baseline in skin MCs, while Fujisawa and colleagues reported on PGD2 responses of skin-derived cultured MCs to the same stimulus (in the range of 1 ng/mL). Another study detected PGD2 production by CBMC (cord-blood derived MCs)¹¹³, and some PGD2 was found by Gaudenzio and colleagues after SP stimulation in PBcMCs (peripheral-blood derived cultured MCs) (below 1 ng/mL as compared with over 30 ng/mL induced by the allergic route).

Together, the pseudo-allergic route obviously generates eicosanoids, especially PGD2, but the amounts are substantially lower compared with the classic allergic pathway. PGD2 production may also critically depend on the MC subset, purification process, micromilieu, and perhaps also on (epi-)genetic peculiarities of the MCs leading to donor-dependent activity patterns (relatively low numbers of donors were included in the studies carried out so far). It will be important to gain more detailed insights into this issue, also because metabolites of PGD2 and LTC4 can serve as serum biomarkers to diagnose an anaphylactic episode^27,28.

Whether MCs produce cytokines and chemokines on stimulation with MRGPRX2 ligands is likewise a matter of debate. A number of studies reported on diverse cytokines and chemokines being produced after MRGPRX2 triggering (including TNF-α, GM-CSF, IL-8, CCL2, CCL3, CCL4, IL-2, IL-3, IL-4, IL-6, IL-31, and nerve growth factor)^{75,84,114–118}. IL-2, IL-4, IL-6, nerve growth factor, and IL-31 were only detected after stimulation with cathelicidin and β-defensins¹¹⁵, so that the cytokine patterns may partially rely on the precise ligand.

The great majority of studies employed cells of the LAD2 cell line^{59,84,115–118}, and it remains unknown whether similar responses can be stimulated in non-transformed primary MCs. The focus on the LAD2 line is somewhat problematic because, apart from having various chromosomal aberrations¹¹⁹, LAD2 cells seem to have a particularly active MRGPRX2 route compared with other MC subsets (β-hexosaminidase release in the range of 70% at an optimal ligand concentration) (eg, Navines-Ferrer et al¹²⁰). We reported that SP does not only trigger degranulation but also cytokine responses in LAD2 cells, while only degranulation was detected in MCs isolated from human breast skin tissue ex vivo, even though the principal receptor was unknown at that time⁶¹. Equally without awareness of the receptor, SP, VIP, and c48/80 were found to be substantially more effective inducers of degranulation and cytokine responses in LAD2 cells compared with PBcMCs¹¹⁴. However, the latter study found at least some increase (over baseline) of MCP-1, RANTES, IL-8, TNF-α, GM-CSF and even IL-3 after VIP, SP, and c48/80 stimulation (IL-3 only after c48/80) in PBcMCs. In contrast, equally using PBcMCs as the MC subset, Gaudenzio detected low levels of VEGFA only yet no TNF-α, IL-13, GM-CSF, or MCP-1, although the parallel allergic stimulation resulted in the production of high levels of all 5 cytokines²⁵. More work is certainly needed to illuminate this aspect further, in particular for physiologically relevant MC subsets of different (epi-)genetic makeups studied in comparison with the allergic route.

As mentioned in the previous chapter, clear-cut differences between the routes can be found in terms of regulation, whereby MRGPRX2 and FcεRI functions are inversely regulated by SCF, IL-4, and RA (yet similarly by IL-33).

Figure 3 gives a brief overview of the allergic and the pseudo-allergic route and contrasts their key characteristics. Notably, MRGPRX2 activation potentially applies to all individuals, while triggering of FcεRI mainly occurs in atopic subjects (in a clinically relevant manner) due to the requirement of 3 components instead of 2 (ie, the intermediary “allergen-specific IgE” in addition to receptor and ligand)¹²¹.

Figure 3: Differences and commonalities between allergic (FcεRI-mediated) and pseudo-allergic/neurogenic (MRGPRX2-triggered) MC activation. See text for further details and references. Baso indicates basophils; CORT, cortistatin; hemat, hematopoietic; MC, Mast cell; SP, substance P; VIP, vasoactive intestinal peptide.

In line with its novelty there is a heavy disproportion in our perception of FcεRI versus MRGPRX2 with <130 records on pubmed dealing with MRGPRX2 (or the alternative MRGX2 or murine Mrgprb2, as of October 2019) vis-à-vis thousands addressing the IgE-mediated or allergic route of MC (and basophil) activation. That this is a rapidly growing research field, however, as indicated by the number published in the period from 2016 till now (over 70%).

Of note MRGPRX2 activation applies to all individuals (for instance, icatibant causes injection-site erythema and swelling in nearly every patient)^11,73. Even though clinically inconspicuous in the majority of subjects, it may explain why MC mediators are detectable systemically (in serum or plasma) in virtually all individuals, also at steady-state (eg, Nassiri et al²⁸).

Another aspect is specificity: MRGPRX2 is restricted to MC_TC-type MCs (and perhaps a small fraction of specialized neurons in dorsal root ganglia). It therefore exceeds FcεRI cell-specificity, since the latter is also expressed in MC_T-type MCs, basophils and (in distinct composition) several other blood cell types. During evolution, the limited expression of MRGPRX2 might have permitted development of (at least some) affinity toward a wide spectrum of substances, because in this way, the same receptor could be exploited for activation of a particular cell type by a multitude of danger signals from other cells or the outside without the risk of collateral damage resulting from parallel activation of other (immune and non-immune) cells. MRGPRX2 could thereby have developed as a master regulator of MC_TC activation responsive to a large number of endogenous and exogenous ligands which target other, more selective GPCRs on their own in other cells (including SP, VIP, cortistatin, somatostatin, and PACAP). In fact, the relatively nonselective and low-affinity binding distinguishes MRGPRX2 from other GPCRs, such as classical neuropeptide receptors, wherein the members of a particular ligand family have high degrees of structural similarity, while MRGPRX2 shows greater tolerance and flexibility in terms of ligand preference³⁷.

Current and future directions

Because of the strong evidence for MRGPRX2’s critical contribution to clinical hypersensitivity and its suspected implication in various other disorders yet also in activities beneficial to the host, MRGPRX2 is an area of highly intense research.

As the receptor is so versatile with tens of ligands already identified, the quest for additional ligands (that may explain clinical observations inexplicable beforehand, especially adverse reactions to drugs) is in full swing and has uncovered proteases like Cathepsin S¹²², further antimicrobials (including antibiotics and nonpeptide host-defense peptide mimetics)^116,118,123, perioperative drugs^120,124, phenothiazine antipsychotics¹²⁵, gold chloride¹²⁶, mucunain (the active principle in Cowhage)¹²⁷, radiocontrast media¹²⁸ as well as endogenous peptides like albumin-derived entities and hemokinin-1^81,116,129.

Other areas of research encompass the establishment of systems that assist in ligand identification in a high-throughput manner (eg, Lansu et al⁷⁰), exploration of MRGPRX2 inhibitors or antagonists^{70,113,117,130,131}, and engineering of activators in contexts in which MC degranulation is deemed protective¹³². In fact, c48/80 has been successfully used as an adjuvant in mice (summarized in Subramanian et al¹¹). Moreover, research into structure-activity relationships is being carried out, aiming to disclose the importance of selected amino acid residues for the binding of individual agonists^72,86.

As mentioned under “MRGPRX2 in health and disease” in vivo studies are currently underway to determine the involvement of MRGPRX2 in physiological and pathologic conditions. For example, using Mrgprb2-deficient mice Green and colleagues demonstrated a role of Mrgprb2/MRGPRX2 in the neurogenic route of MC activation, leading to mechanical and thermal hyperalgesia in a model of postoperative pain⁵⁹. Another study from the same laboratory found that Mrgprb2/MRGPRX2-triggered MC activation contributes to leukocyte recruitment and (nonhistaminergic) itch in the context of contact dermatitis⁷⁴. On the other end of the spectrum is work focusing on the beneficial role of MRGPRX2, for example, in bacterial clearance^83,84.

Other studies aim to discern differences across ligands, a strategy which has led to the identification of G-protein biased (eg, Icatibant) versus balanced (ie, G-protein and β-arrestin activating, such as c48/80) ligands of MRGPRX2⁸⁸, and, associated with this aspect, the elucidation of MRGPRX2’s destiny following ligand binding, whereby internalization was detected following cathelicidin and c48/80, but not Icatibant or AG-30/5C^82,88. An earlier report found that, on comparison with C3AR, phosphorylation and desensitization of MRGPRX2 was slow using cathelicidin as stimulus⁶⁰. Future experiments will have to reveal the intracellular events elicited by different ligands and their impact on MRGPRX2 recycling versus degradation.

Moreover, comparisons across MC subsets are being carried out to ascertain where in the body MRGPRX2 may be preferentially activated. In a recent study MCs derived from human skin, lung, synovial, and heart tissue were tested for responsiveness toward MRGPRX2 ligands. MCs from skin showed robust stimulability, while synovial MCs responded weakly, and lung and heart MCs were unresponsive—despite the latter being typically classified as MC_TC-type MCs¹¹². Although further confirmation, for example, from immunohistochemistry of the intact heart is required, the study implies that chymase and MRGPRX2 expression may be dissociated, even though they constitute 2 highly selective markers of MC_TC cells. In a micromilieu with high RA or chronic IL-33 exposure, we observed that both MC_TC features were coordinately down-regulated (at least at mRNA level), so that some overlap likely exists in the prerequisites driving chymase and MRGPRX2 transcription^102,106,107.

Deciphering the various aspects of MRGPRX2 biology will contribute to a deeper understanding of many pathologies to which MCs supposedly contribute and help uncover how MCs act in disorders not primarily associated with type-I allergy. Because MRGPRX2, like FcεRI, triggers acute MC activation and anaphylactic shock, comprehensive knowledge of its regulation and signaling is mandatory and the receptor has reasonably become a premier topic of MC research.

Together, MRGPRX2 is vital to understand MC biology, explain MC reactivity patterns and open avenues for therapeutic interventions alike. Though still in its infancy compared with its much better-defined “cousin” FcεRI, research into the MRGPRX2 network is currently gaining momentum and should soon identify differences across ligands, reveal the response patterns of clinically relevant MCs to MRGPRX2 triggering, unveil the involvement of MRGPRX2 in a number of processes in vivo, and disclose factors that predispose individuals to react vigorously via the alternative route in order to identify patients at risk for “pseudo-allergic anaphylaxis.”

Source of funding

The author’s research on MRGPRX2 is funded by the Deutsche Forschungsgemeinschaft DFG (BA-3769/4-1).

Conflict of interest disclosures

The author declares that there is no financial conflict of interest with regard to the content of this report.