Effect of sex and haplotype on plasma tryptase levels in healthy adults

BACKGROUND: The total level of alpha-tryptase and ss-tryptase in serum or plasma is used as a clinical indicator of the mast cell burden. OBJECTIVE: The effect of the tryptase haplotype and of sex on the total tryptase level of healthy individuals was determined. METHODS: A novel hot-stop PCR technique was used to determine the tryptase genotype, and a standard fluoroenzyme immunoassay was used to measure total plasma tryptase levels in 106 healthy subjects. Mx modeling and the QTL association routine of Mendel 5.0 were used to analyze the data. RESULTS: Tryptase haplotypes exhibit a 1 (betaalpha/betaalpha):2 (betabeta/betaalpha):1 (betabeta/betabeta) distribution, monomorphic for ss at 1 position and allelic for ss and alpha at the other position. The betaalpha haplotype has a frequency of 0.49. The betaalpha haplotype increases total tryptase levels by 0.5 ng/mL from the overall mean, whereas female sex increases the level by 0.2 ng/mL from the mean. CONCLUSION: The tryptase haplotype and sex each have a statistically significant effect on the total plasma tryptase level of healthy subjects.     

Tryptase genetics and anaphylaxis

Tryptases secreted by tissue mast cells and basophils can enter the bloodstream. In human subjects tryptases are encoded by several genes and alleles, including alpha, beta, gamma, and delta. Common variations include complete absence of alpha genes. Until recently, alpha tryptase was considered to be the major tryptase secreted at baseline and in mastocytosis. However, lack of alpha tryptase genes has little effect on circulating tryptase levels, which are now thought mainly to consist of inactive pro-beta tryptase secreted constitutively rather than stored in granules with mature tryptases. Pro-beta tryptase levels thus might reflect total body mast cell content. In contrast, mature beta tryptase can increase transiently in severe systemic anaphylaxis and confirm the diagnosis. However, it might fail to increase in food anaphylaxis or might increase nonspecifically in samples acquired after death. Thus pro- and mature beta tryptase measurements are useful but associated with false-negative and false-positive results, which need to be considered in drawing clinical conclusions in cases of suspected anaphylaxis.     

Characterization of mast-cell tryptase-expressing peripheral blood cells as basophils

BACKGROUND: Mast-cell tryptase is a protease with proinflammatory activity, the expression of which by peripheral blood leukocytes (PBLs) has not been fully characterized. OBJECTIVE: We examined tryptase expression in human PBLs to further characterize this tryptase-expressing cell population for lineage and disease association. METHODS: PBLs were fixed, permeabilized, stained with antibodies to tryptase and a panel of mast cell- and basophil-specific markers, and analyzed by means of flow cytometry. RESULTS: Tryptase expression was restricted to a population of cells that stained positive for IgE and negative for the panel of lineage markers (IgE(+), lin(-)). This IgE(+), lin(-) population did not stain for the mast-cell markers Kit or chymase but did stain for the basophil-specific granule proteins recognized by the 2D7 and BB1 mAbs. Per-cell tryptase expression demonstrated a greater than 100-fold range of expression among donors but did not correlate with disease status (asthma or mastocytosis), FEV(1), or serum tryptase concentration. Tryptase was released by purified basophils after anti-IgE activation. CONCLUSIONS: The phenotype of tryptase-expressing PBLs and their lack of increase in patients with mastocytosis demonstrates that these cells are basophils. Per-cell basophil tryptase expression is highly variable between donors, with some donors expressing levels approaching those of mast cells. As such, anti-tryptase antibodies cannot be used to distinguish these 2 cell types from one another by means of flow cytometry. These results demonstrate that tryptase represents an additional mediator through which basophils may contribute to allergic inflammation.     

Tryptase haplotype in mastocytosis: relationship to disease variant and diagnostic utility of total tryptase levels

Serum mast cell tryptase levels are used as a diagnostic criterion and surrogate marker of disease severity in mastocytosis. Approximately 29% of the healthy population lacks alpha tryptase genes; however, it is not known whether lack of alpha tryptase genes leads to variability in tryptase levels or impacts on disease severity in mastocytosis. We have thus analyzed tryptase haplotype in patients with mastocytosis, computing correlations between haplotype and plasma total and mature tryptase levels; and disease category. We found: (1) the distribution of tryptase haplotype in patients with mastocytosis appeared consistent with Hardy-Weinberg equilibrium and the distribution in the general population; (2) the disease severity and plasma tryptase levels were not affected by the number of alpha or beta tryptase alleles in this study; and (3) information about the tryptase haplotype did not provide any prognostic value about the severity of disease. Total and mature tryptase levels positively correlated with disease severity, as well as prothrombin time and partial thromboplastin time, and negatively correlated with the hemoglobin concentration.     

Tryptase release and clinical severity of anaesthetic reactions

Plasma tryptase measurements provide a convenient measure of mast cell degranulation and are being increasingly used in the investigation of drug induced anaphylactoid reactions. A study involving 30 patients exhibiting life threatening response to anaesthetic drugs was carried out to explore the limitations of this diagnostic assay. The patients divided into two roughly equal groups “tryptase releasers” and “non-tryptase releasers”, despite having comparable clinical severity. The tryptase releasers showed the more typical manifestations of the anaphylactoid response, predominantly hypotension, and a relationship was apparent between the measured tryptase level and systolic blood pressure (BP). Tryptase levels of>25 ng/ml were associated with unmeasurable BP. In contrast, the non-releasers exhibited acute bronchospasm, either alone or as the initial and predominant clinical manifestation. However, urinary methylhistamine assays indicated that, despite normal plasma tryptase levels, some of these patients had undergone mast cell or basophil degranulation. The importance of measuring both plasma tryptase and urinary methylhistamine to obtain further discrimination of non-tryptase releasing reactions is illustrated by reference to thein vivo behaviour of a mast cell tumour.     

Human mast cell tryptase in biology and medicine

The most abundant prestored enzyme of human mast cell secretory granules is the serine-protease tryptase. In humans, there are four tryptase isoforms, but only two of them, namely the alpha and beta tryptases, are known as medically important. Low levels of continuous tryptase production as an immature monomer makes up the major part of the baseline serum tryptase levels, while transient release of mature tetrameric tryptase upon mast cell degranulation accounts for the anaphylactic rise of serum tryptase levels. Serum tryptase determination contributes to the diagnosis or monitoring of mast cell disorders including mast cell activation – induced anaphylaxis, mastocytosis and a number of myeloproliferative conditions with mast cell lineage involvement. Baseline serum tryptase levels are predictive of the severity risk in some allergic conditions.     

Possible molecular mechanisms to account for the involvement of tryptase in the pathogenesis of psoriasis

Tryptase has been suggested to take part in the pathophysiology of psoriasis mainly through the production of C3a by cleaving C3. However, studies using tryptase preparations of high purity do not support this notion. Therefore, although tryptase is unanimously believed to be involved in the immunopathogenesis of psoriasis, no convincing mechanism has been proposed for its role. This paper proposes several mechanisms by which this enyme may exert its role in the pathobiology of psoriasis. Tryptase is a mitogen for epithelial cells and stimulates IL-8 production and ICAM-1 expression by these cells. It also induces the expression of mRNA for IL-1beta and IL-8 and stimulates the selective release of IL-8 from endothelial cells and TNF-alpha, IL-1beta, and IL-6 from lymphocytes and monocytes. Besides itself being a chemoattractant for neutrophils, tryptase activates mast cells and generates kinins from kininogen, thereby playing a crucial role in leukocyte infiltration into psoriatic lesions. This enzyme also induces leukocyte infiltration partly through activating endothelial PAR-2, which contributes to leukocyte rolling, adherence and recruitment by inducing the release of endothelial platelet-activating factor. Through activating PAR-2, tryptase could also trigger the development of Langerhans cells which play a crucial role in the pathophysiology of psoriasis. This enzyme is a mitogen for fibroblasts, which are probably involved in the pathophysiology of psoriasis through production of insulin-like growth factor-I (IGF-I). Tryptase is a gelatinase and also activates stromelysin-1 (MMP-3), thereby contributing to the disruption of psoriatic basement membrane and to the joint damage seen in psoriatic arthritis. Increase of tryptase levels following trauma could also provide a mechanism for Koebner phenomenon seen in psoriasis.     

Hereditary alpha-tryptasemia in 101 patients with mast cell activation–related symptomatology including anaphylaxis

BackgroundHereditary alpha-tryptasemia (HαT) is an autosomal dominant genetic trait characterized by multiple copies of the alpha-tryptase gene at the TPSAB1 locus. Previously described symptomatology involves multiple organ systems and anaphylaxis. The spectrum of mast cell activation symptoms is unknown, as is its association with specific genotypes.ObjectiveTo describe clinical, laboratory, and genetic characteristics of patients referred for the evaluation of mast cell activation–related symptoms and genotype-confirmed HαT.MethodsWe retrospectively describe clinical characteristics, baseline tryptase, and tryptase genotype in 101 patients. Patients were referred for mast cell activation–related symptoms and underwent genotyping to confirm diagnosis of HαT.ResultsOf 101 patients, 80% were female with average tryptase of 17.2 ng/mL. Tryptase was less than 11.4 ng/mL in 8.9% and greater than 20 ng/mL in 22.3% (range 6.2-51.3 ng/mL). KIT D816V mutation was negative in all subjects tested. 2α:3β was the most common genotype but did not correlate with tryptase levels. Unprovoked anaphylaxis was noted in 57% of the subjects with heterogeneous genotypes. Most common symptoms include gastrointestinal, cutaneous, psychiatric, pulmonary, cardiovascular, and neurologic. A total of 85% of patients were taking H₁- or H₂-antihistamines with partial symptom relief. Omalizumab was effective at suppressing anaphylaxis or urticaria in 94% of the patients.ConclusionHαT encompasses a broad range of baseline tryptase and should be considered in patients with symptoms of mast cell activation and tryptase levels greater than 6.2 ng/mL. Patients may present with complex symptomatology including cutaneous, gastrointestinal, neurologic, and psychiatric symptoms and anaphylaxis, some of which respond to omalizumab.     

Tryptase in type I hypersensitivity

Tryptase is currently the main mast cell biomarker available in medical practice. Tryptase determination is a quantitative test performed in serum or plasma for the diagnosis, stratification, and follow-up of mast cell–related conditions. The continuous secretion of monomeric α and β protryptases forms the baseline tryptase level. Transient, activation-induced release of tryptase is known as acute tryptase. Because mast cells are tissue-resident cells, the detection of an acute tryptase release in the bloodstream is protracted, with a delay of 15 to 20 minutes after the onset of symptoms and a peak at approximately 1 hour. Constitutive release of tryptase is a marker of mast cell number and activity status, whereas transient release of mature tryptase is a marker of mast cell degranulation. Although consensual as a concept, the application of this statement in clinical practice has only been clarified since 2020. For baseline tryptase to be used as a biomarker, reference values need to be established. In contrast, defining a transient increase using acute tryptase can only be achieved as a function of the baseline status.     

Release of tryptase together with histamine during the immediate cutaneous response to allergen

Better in vivo techniques are needed for objective assessment of mast cell-dependent events. Tryptase, a neutral protease selectively concentrated in human mast cells, appears along with histamine in skin chamber fluid overlying sites of allergen challenge in sensitive human subjects. Maximal amounts of histamine were found 0 minutes to 30 minutes after challenge; maximal amounts of tryptase were found 30 minutes to 60 minutes after challenge. The later appearance of tryptase most likely reflects its slower diffusion through tissue after release of tryptase from cutaneous mast cells as a macromolecular complex with proteoglycan. The mean weight ratio of tryptase (134,000 molecular weight tetramer) to histamine (111 molecular weight) in chamber fluid after allergen challenge during a 1-hour time course was 4:1. Total amounts of tryptase and histamine recovered in the 0.3 ml chamber fluid samples after a 1-hour challenge averaged 95 ng and 26 ng, respectively. Tryptase levels in skin chamber fluid are an accurate indicator of mast cell activation.     

Serum total tryptase levels are increased in patients with active chronic urticaria

Background We have demonstrated previously mast cell histamine release upon incubation with chronic urticaria (CU) sera, presumably by degranulation. Objective To explore total and mature tryptase in order to assess whether any increase in total tryptase levels is due in part to mast cell degranulation or to mast cell burden. We also wanted to explore differences between the autoimmune groups called idiopathic (serum unable to activate basophils), and to correlate total and mature tryptase levels with different urticaria features. Methods We measured total and mature tryptase serum levels in 81 CU patients, 16 atopic donors and 21 healthy control sera. We assessed autoimmunity by measuring the CD63 expression in normal basophil donors upon incubation with CU sera. Results We found significantly higher levels of total tryptase in the sera of CU patients (6.6 ±4.1 μg/L) than in sera from healthy non‐atopic subjects (4.4 ±2.8 μg/L) and from atopic subjects (4.5 ±1.7 μg/L). Mature tryptase levels were undetectable (<1 ng/mL). Total tryptase levels in the autoimmune urticaria group were significantly higher (9.8 ±5.4 μg/L) than the idiopathic urticaria group (4.4 ±2.2 μg/L). A significant difference in total tryptase was found between symptomatic patients (7.3 ±4.1 μg/L) compared with asymptomatic ones (5.7 ±4.1 μg/L) at the time of venesection. No difference was found in mature tryptase levels either. Conclusion Total elevated tryptase levels are not accompanied by an elevated mature tryptase levels, as might be expected if the serum levels reflected mast cell degranulation. Cite this as: M. Ferrer, J. M. Nuñez‐Córdoba, E. Luquin, C. E. Grattan, J. M. De la Borbolla, M. L. Sanz, L. B. Schwartz, Clinical & Experimental Allergy, 2010 (40) 1760–1766.     

Expression of mast cell tryptases in Hodgkin and Reed-Sternberg (HRS) cells

Tryptase is the most abundant protease in human mast cells, and is often used as a marker for the enumeration of mast cells in tissue. Here we report that tumour cells from Hodgkin lymphoma, the so called Hodgkin and Reed-Sternberg cells, can express tryptase. Hodgkin lymphoma cell lines expressed mRNA for both α- and β-tryptase and also produced the protein, although at much lower concentrations than mast cells. However, the frequency of tryptase positive HRS-cells in situ was very low. This report demonstrates that tumour cells of lymphoid origin can express tryptase in vitro and in situ .     

Mast cell tryptase stimulates both human dermal fibroblast proliferation and type I collagen production

BACKGROUND: Mast cell tryptase has been shown to be mitogenic for fibroblasts, however, it still remains unknown whether mast cell tryptase stimulates collagen production by human derrmal fibroblasts. OBJECTIVE: We have investigated the effect of mast cell tryptase on type I collagen production by human dermal fibroblasts as well as the proliferation of the fibroblasts. METHODS: Tryptase isolated from human lung tissue was added to the culture of fibroblasts from normal dermis, and the fibroblast proliferation and the activity of type I collagen synthesis in the supernatants were assayed, respectively. RESULTS: Fibroblast proliferation was increased with tryptase in a concentration-dependent manner, and a significant increase was observed in the presence of tryptase at concentrations from 0.01 to 10 microg/mL. The increase of fibroblast proliferation with 3 microg/mL tryptase was significantly reduced by 15 microg/mL antitryptase IgG antibody, which was demonstrated to inhibit fibrinogenolysis of tryptase. On the other hand, the production of type I collagen by the fibroblasts was significantly increased with tryptase at a concentration of 10 microg/mL. The collagen production in the presence of 10 microg/mL tryptase was significantly inhibited by 50 microg/mL antitryptase IgG antibody. CONCLUSION: Tryptase increases not only the proliferation of human dermal fibroblasts but also type I collagen production.     

The potential clinical utility of serum alpha-protryptase levels.

BACKGROUND: Because biopsy criteria for diagnosing systemic mastocytosis are not precise, the value of serum alpha-protryptase levels in the work-up of suspected systemic mastocytosis should be considered. OBJECTIVE: A retrospective analysis was performed on subjects with total tryptase serum levels that were high (>/=20 ng/mL), while beta-tryptase serum levels were normal (<1 ng/mL) or modestly elevated (1 to 5 ng/mL). METHODS: Over a 3.5-year period, 52 qualifying specimens were identified from 1369 consecutive samples. The corresponding subjects were divided into those with suspected mastocytosis and those with suspected anaphylaxis. Subjects with suspected mastocytosis were subdivided into 3 subgroups on the basis of biopsy results (positive, negative, or not available). Subjects with suspected anaphylaxis were subdivided into living and deceased subgroups. RESULTS: Among the 15 subjects who underwent biopsy, alpha-protryptase serum levels (the difference between directly-measured levels of serum total tryptase and beta-tryptase), when greater than 75 ng/mL (n = 9), were always associated with a positive biopsy result for systemic mastocytosis; levels from 20 to 75 ng/mL (n = 6) were associated with a positive biopsy result in 50% of subjects. alpha-Protryptase serum levels may be a more sensitive screening test than a bone marrow biopsy for this disorder. Also, elevated alpha-protryptase serum levels in some adult patients return to normal over time, suggesting that mast cell hyperplasia resolved in these patients. Finally, a high alpha-protryptase level may reveal anaphylaxis to be a presenting manifestation of systemic mastocytosis or mast cell hyperplasia. CONCLUSION: Levels of serum alpha-protryptase, relative to those of beta-tryptase, appear to be useful in the diagnostic work-up and follow-up of subjects with suspected systemic mastocytosis.