Biomarkers in the diagnosis of mast cell activation

Abstract

Purpose of review

Mast cell activation is defined by activation of mast cells by varying stimuli with release of chemical mediators either through degranulation or release of de novo synthesized proteins or lipid mediators. Currently, tryptase measurement increase during symptomatic episodes is the most accepted biomarker measurement for mast cell activation. However, newer diagnostic tools including clinically available urinary mast cell mediators are noninvasive and can be more readily obtained compared to serum tryptase levels. This review will highlight biomarker measurement in the diagnosis of mast cell activation.

Recent findings

This review will highlight biomarker measurement in mast cell activation including serum tryptase and urinary mast cell mediators including N-methylhistamine, leukotriene E4, and 2,3-dinor-11beta-prostaglandin F2 alpha.

Summary

Urine mast cell mediators obtained at baseline and during symptom exacerbation are emerging biomarkers in the diagnosis of mast cell activation. Tryptase measurement and urinary mast cell mediator measurement are currently the most accepted biomarkers for mast cell activation. Further research is needed to establish new biomarkers for mast cell activation.

INTRODUCTION

Mast cells are an important immune cell of the myeloid lineage and are located within connective tissues throughout the body [1,2]. Mast cells play a key role in many different physiological responses including vasodilation, vascular homeostasis, innate and adaptive immune responses, angiogenesis, and anaphylaxis [1]. These physiological responses are mediated by many different mediators that are contained within the mast cell [1,2]. Clinically, the disease processes in which mast cells are most implicated include immunoglobulin E (IgE)-mediated hypersensitivity reactions, chronic urticaria, and mast cell related disorders [3]. In these disease processes, the mast cell is activated by varying stimuli and then release different mediators that are either contained in preformed granules within the mast cell or synthesized at the time of activation [3]. The release of these mediators by the mast cell then have physiological downstream effects. These mast cell mediators that are released can be measured as a marker of mast cell activation. Mast cell mediators can be detected in serum as well as in urine [2–5]. In this review, biomarkers of mast cell activation will be discussed including tryptase, chymase, carboxypeptidase A3, histamine, heparin, leukotriene C4, platelet activating factor, prostaglandin D2, cytokines, and chemokines. Tryptase and urinary mast cell mediators will be highlighted in this review as they are currently the most clinically available and studied biomarkers of mast cell activation. 

Box 1.

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MAST CELL ACTIVATION PRODUCTS

Mast cells are an important effector immune cell responsible for many different physiological events. These physiological responses are mediated by different mediators released by the mast cell upon activation. Mast cell activation occurs via several different receptors including FcεR1, G-protein-coupled receptors [mas-related G-protein-coupled receptor-X2 (MRGPRX2) and chemokine/complement receptors], cytokine receptors [KIT and interleukin (IL)-3 receptor], MyD88-dependent receptors (IL-33 receptor and toll-like receptors), and pattern recognition receptors [3,6,7]. Activation of mast cells by these receptors then trigger release of preformed and/or de-novo produced mast cell mediators [2–7]. The preformed mast cell mediators that are stored within mast cell granules include chymase, carboxypeptidase A3, histamine, heparin, and tryptase [3,8]. Figure 1. De novo synthesized mast cell mediators that are produced at the time of mast cell activation are either de novo synthesized proteins including chemokines and cytokines [IL-6, IL-31, tumor necrosis factor (TNF)-α] or de novo synthesized lipid mediators including cysteinyl leukotrienes [leukotriene C4 (LTC4)], platelet activating factor (PAF), and prostaglandin D2 (PGD2) [2,3,6–8] (Fig. 1). While there are many different mast cell activation products, only a few of these mediators have been clinically validated and commercially available for measurement of mast cell activation [2,3,8]. The current clinically validated and commercially available markers of mast cell activation include serum tryptase and urinary mast cell mediators. Of the urinary mast cell mediators, histamine, LTC4, and PGD2 are the current commercially available and clinically relevant mast cell mediators for the measurement of mast cell activation [2–5,8–11,12^▪▪,13,4]. Tryptase and urinary mast cell mediators including histamine, LTC4, and PGD2 will be highlighted in this review.

FIGURE 1.

Biomarkers of mast cell activation.

TRYPTASE

Tryptase is a serine protease that is produced by mast cells as an inactive monomer, stored within mast cell granules in a proteolytically active form (mature β-tryptase tetramer or α/β heterotetramer), and then released following mast cell activation [3,15,16]. Compared to β-tryptase that is released by the mast cell during mast cell activation, α-tryptase cannot be proteolytically activated and is secreted by the mast cell constitutively, therefore representing a marker of mast cell burden [3,17]. The primary functions of tryptase have not been fully elucidated, but are believed to include physiological processes of inflammation, chemotaxis, fibroblast proliferation, neutralizations of venoms, alteration of epithelial barrier integrity, and endothelial permeability [3,18,19]. Tryptase is predominantly found within mast cells, although basophils can also produce tryptase but at low relative levels approximately 0.4% compared to mast cell production of tryptase [3,20]. Tryptase can be measured in serum, and serum tryptase measurements include both α and β tryptase. When obtaining serum tryptase measurements, basal serum tryptase measurements are used as a measure of mast cell burden [3,10,17]. Elevated basal serum tryptase measurements can be detected in mast cell related disorders including hereditary alpha tryptasemia (HAT), cutaneous mastocytosis, and systemic mastocytosis but can also be detected in myeloid malignancies, chronic helminth infection, and chronic renal failure [3,17,21–30]. Symptomatic serum tryptase measurement during anaphylaxis or suspected symptoms of mast cell activation can be an objective biomarker measurement in mast cell mediated anaphylaxis and mast cell activation. An acute tryptase/basal serum tryptase level ratio of ≥1.685 is both a specific and sensitive marker to confirm mast cell mediated anaphylaxis [31]. Currently, the most accepted biomarker measurement and gold standard for diagnosing mast cell activation is an increase in symptomatic serum tryptase concentration of 20% plus 2 ng/ml from the individual's baseline serum tryptase measurement [2–5,9,10,17,23,32] (Table 1). Symptomatic serum tryptase measurement during mast cell activation has a short serum half-life and has to be obtained within 1–4 h of a suspected mast cell activation event [23]. While symptomatic serum tryptase is currently the most accepted biomarker measurement of mast cell activation, it has important limitations. One limitation is that the currently available serum tryptase assays are not able to distinguish between constitutively secreted tryptase and mast cell activated secretion of tryptase [3,16,27,33,34]. Interpretation of serum tryptase assays can be challenging from the lack of specificity between total versus α and β tryptases in commercially available clinical assays [3,16,27,33,34]. Other limitations of symptomatic measurement of serum tryptase is that it is an invasive biomarker measurement requiring venipuncture and must be obtained in a specific time window [2,3,23]. Furthermore, variability of basal serum tryptase in an individual may affect the calculation accuracy of a symptomatic/basal serum tryptase measurement [31]. Due to symptomatic serum tryptase being an invasive diagnostic procedure and the short time window of collection during suspected mast cell activation, urinary mast cell mediators including histamine, LTC4, and PGD2 are emerging biomarkers for measurement in mast cell activation.

Table 1.

Commercially available and clinically studied markers for mast cell activation

Test name	Collection type	Collection timing	Result indicative of mast cell activation
Serum tryptase	Venipuncture	1-4 h after suspected mast cell event	Symptomatic tryptase with a minimum increase of 20% plus 2 ng/ml from baseline
Urinary LTE4	24 h or random urine collection	Within 5 h of suspected mast cell event	Acute/baseline measurement ≥1.36
Urinary N-methylhistamine	24 h or random urine collection	Within 5 h of suspected mast cell event	Acute/baseline measurement ≥1.29
Urinary 2,3-dinor 11β-PGF2α	24 h or random urine collection	Within 5 h of suspected mast cell event	Acute/baseline measurement ≥1.31

LTE4, leukotriene E4; 2,3-dinor 11β-PGF2α, 2,3-dinor 11 beta prostaglandin F2 alpha.

URINARY MAST CELL MEDIATORS

While there are many different mast cell mediators, histamine, LTC4, and PGD2 are important mast cell mediators that are the most clinically validated and have direct physiologic effects [2,3,11,12^▪▪,13,14].

HISTAMINE

Histamine is an endogenous amine with many physiologic effects including increase in vascular permeability, vasodilation, increase in gastric acid production, excitatory effects on neurons, increased heart rate, increased cardiac contraction, increased glandular secretion, bronchial smooth muscle contraction, and increased airway mucus production [11,35]. Histamine is produced by many different cell types including mast cells, basophils, gastric enterochromaffin-like cells, histaminergic neurons, platelets, dendritic cells, and lymphocytes. While many cell types can produce histamine, the only cell types that are known to store large quantities of histamine in secretory granules include mast cells and basophils. In response to immunologic and nonimmunologic stimuli, mast cells and basophils release histamine through degranulation [2,13,36,37]. After histamine is released, it is rapidly metabolized with a half-life of 1–2 min, making plasma and urinary detection of histamine following suspected mast cell activation very challenging [2,3,38,39]. Urinary histamine levels can be increased in certain inflammatory conditions including interstitial cystitis, thereby making plasma and urinary histamine unreliable biomarkers for measurement of mast cell activation [2,14,40]. However, histamine metabolites including N-methylhistamine and N-methylimidazole acetic acid are more appropriate biomarkers for the measurement of mast cell activation given their longer half-lives [41–47]. N-Methylimidazole acetic acid currently does not have a commercially available assay for measurement. N-methylhistamine which is produced via conversion of histamine by N-methyl transferase, is a commercially available urinary test that can be collected over a 24 h urine collection or a random, spot urinary sample [2]. Normal urinary excretion of N-methylhistamine is age dependent and normal reference ranges varies on age as follows: 0–5 years: 120–510 mcg/g creatinine; 6–16 years: 70–330 mcg/g creatinine; >16 years: 30–200 mcg/g creatinine (source: Mayo Clinic Laboratories). Initially, an elevated urinary N-methylhistamine >30% above the upper end of the normal reference range (200 mcg/g creatinine) was considered consistent with a mast cell activation episode [4]. A recent study found that an acute/baseline value of >1.29 of urinary N-methylhistamine correlated with a clinically significant serum tryptase increase of 20% plus 2 ng/ml [12^▪▪] (Table 1). In this study, urinary mast cell mediators including N-methylhistamine, leukotriene E4 (LTE4), and 2,3-dinor 11 beta prostaglandin F2 alpha (2,3-dinor 11β-PGF2α) were measured with serum tryptase at baseline and during suspected mast cell activation episodes. The lowest acute/baseline value of N-methylhistamine that correlated with significant tryptase elevation was 1.29. Overall, the average acute/baseline urinary N-methylhistamine was 3.2 [12^▪▪]. This study suggests that acute/baseline measurement of urinary N-methylhistamine has utility in the diagnosis of mast cell activation in patients with mast cell activation syndrome [12^▪▪]. Urinary N-methylhistamine does have limitations as a biomarker for mast cell activation in that it can be produced by other cell types and can be elevated at baseline in atopic disorders [3]. However, urinary N-methylhistamine is a noninvasive measurement and timing of collection is slightly greater than symptomatic tryptase measurement (can be obtained within 5 h of suspected mast cell activation) [2,12^▪▪].

CYSTEINYL LEUKOTRIENES

Cysteinyl leukotrienes including leukotriene A4, C4, D4, and E4, are important inflammatory lipid mediators that are derived from arachidonic acid [48]. Cysteinyl leukotrienes have many different physiologic effects including increase in vascular permeability, bronchoconstriction, bronchial smooth muscle contraction, increased airway mucus production, mucus hypersecretion, and bronchial hyperreactivity [11]. Leukotriene C4 is produced by conversion of leukotriene A4 and is a bioactive molecule that is important in inflammation and secreted by cells including mast cells, basophils, eosinophils, dendritic cells, monocytes, and macrophages [2,14,49]. After secretion from activated cells, LTC4 is rapidly metabolized into leukotriene D4, which is then further metabolized to leukotriene E4 [14]. Leukotriene E4 is a biomarker that can be measured in plasma or urine to evaluate for increases in cysteinyl leukotriene production [50–52]. Plasma leukotriene E4 is only available in the research setting and is not a commercially available assay. Urinary leukotriene E4 is a commercially available test that can be collected via a 24 h or random, spot urine collection [2,12^▪▪,14]. Commercially available reference values for urinary LTE4 have been established for patients 18 years of age or older and normal excretion is <104 pg/mg Cr (source: Mayo Clinic Laboratories). Reference ranges for commercially available urinary LTE4 values have not yet been established in the pediatric population. 5-Lipoxygenase inhibitors including zileuton can decrease urinary LTE4 measurement, and patients should be off of 5-lipoxygenase inhibitors for accurate detection. Urinary LTE4 has been shown to be increased in anaphylaxis [53,54]. In a recent study, urinary LTE4 was found to be significantly elevated during mast cell activation episodes in patients with mast cell activation syndrome [12^▪▪] (Table 1). In this study, urinary LTE4 was significantly elevated with mast cell activation with the average acute/baseline LTE4 of 35.98 [12^▪▪]. The lowest acute/baseline urinary LTE4 value that accompanied a significant symptomatic serum tryptase increase was 1.36 [12^▪▪]. This study suggests that acute/baseline measurement of urinary LTE4 is an important diagnostic consideration for evaluating mast cell activation [12^▪▪]. Similar to N-methylhistamine, urinary LTE4 has been shown to be increased in atopic disorders and has been reported to be elevated in patients with AERD [3]. However, an acute/baseline assessment of urinary LTE4 over an elevated baseline LTE4 alone is more indicative of mast cell activation and can be used as diagnostic tool in the evaluation of mast cell activation.

PROSTAGLANDIN D2

Prostaglandin D2 (PGD2) is another de novo synthesized lipid mediator from mast cells upon activation similar to LTC4 and platelet activating factor [3]. Like LTE4, PGD2 is synthesized from arachidonic acid but via a different sequential pathway involving the enzyme cyclooxgenase [2,13]. First, cyclooxygenase 1 or 2 converts arachidonic acid into prostaglandin H2. Prostaglandin H2 is then converted to prostaglandin D2 by either hematopoietic or lipocalin type of prostaglandin D2 synthase. Lipocalin-prostaglandin D2 synthase is expressed in the central nervous system and cardiac tissue. Hematopoietic-prostaglandin D2 synthase is expressed by mast cells, megakaryocytes, platelets, monocytes, dendritic cells, TH2 lymphocytes, and lung epithelial cells. Basophils do not express hematopoietic-prostaglandin D2 synthase [2,14,55,56]. As basophils do not express hematopoietic-prostaglandin D2 synthase, mast cells are the predominant source of PGD2 [13]. Upon mast cell activation, mast cells rapidly synthesize and release large amounts of PGD2 [14]. PGD2 has important physiologic effects including increasing vascular permeability, increasing intestinal permeability, increased venular permeability leading to flushing, bronchoconstriction, peripheral vasodilation, coronary and pulmonary artery vasoconstriction, enhancing histamine release from basophils, mucus hypersecretion, and inhibition of platelet aggregation [11].

PGD2 is rapidly degraded into D-, F-, and J-ring metabolites, which are then excreted into urine as more stable urinary metabolites [13]. 2,3-dinor 11 beta prostaglandin F2 alpha (2,3-dinor 11β-PGF2α) is the commercially available urinary assay for measurement of PGD2. Urinary 2,3-dinor 11β-PGF2α can be collected as a 24 h or random, spot urinary sample. The normal reference value for both random and 24 h urine collection are <1802 pg/mg Cr (source: Mayo Clinic Laboratories). Cyclooxygenase-1 inhibitors including aspirin and other NSAIDs, can decrease urinary 2,3-dinor 11β-PGF2α via decreased prostaglandin synthesis, and for accurate measurement, patients should be off these medications prior to collection.

Urinary 2,3-dinor 11β-PGF2α has shown clinical utility in the diagnosis of mast cell activation. A recent study evaluating acute/baseline levels of 2,3-dinor 11β-PGF2α with a corresponding significant acute increase in symptomatic serum tryptase in patients with suspected mast cell activation episodes revealed that the acute/baseline ratio of 2,3-dinor 11β-PGF2α was elevated (Table 1). The average acute/baseline urinary 2,3-dinor 11β-PGF2α level was 7.28. The lowest acute/baseline 2,3-dinor 11β-PGF2α that was associated with a significant in increase in symptomatic serum tryptase was 1.31 [12^▪▪]. This study suggests that urinary PGD2 metabolites are increased in mast cell activation and that an elevated acute/baseline 2,3-dinor 11β-PGF2α ≥ 1.31 is consistent with mast cell activation. Similar to other urinary mast cell mediators, 2,3-dinor 11β-PGF2α can be elevated in other conditions, and urinary metabolites of PGD2 have been noted to be elevated in patients with aspirin exacerbated respiratory disease [57]. Urinary 2,3-dinor 11β-PGF2α should be collected simultaneously with the other urinary mast cell mediators (LTE4 and N-methylhistamine) when evaluating for mast cell activation [2,12^▪▪].

CONCLUSION

Mast cell activation is defined by activation of mast cells by varying stimuli with release of chemical mediators either through degranulation or release of de novo synthesized proteins or lipid mediators. While the mast cell releases several different mediators, the current mediators that are commercially available and studied specifically for mast cell activation include serum tryptase and urinary mast cell mediators of LTE4, N-methylhistamine, and 2,3-dinor 11β-PGF2α. Acute/baseline tryptase measurement is the currently accepted gold standard for evaluation of mast cell activation. Emerging evidence suggests that acute/baseline measurement of urinary mast cell mediators is also a valuable tool for mast cell activation while being noninvasive and more accessible to patients than tryptase measurement alone. While tryptase and urinary mast cell mediators are important tools in the evaluation of mast cell activation, further research is needed to find additional biomarkers for the evaluation of mast cell activation.

Acknowledgements

None.

Financial support and sponsorship

None.

Conflicts of interest

Dr Pongdee receives research funding from Blueprint Medicines. Dr Voelker has no conflicts of interest to disclose.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

• ▪ of special interest

• ▪▪ of outstanding interest