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* Division of Immunopathology, Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Austria;
Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Austria;
Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Austria; and
ZAUM–Center for Allergy and Environment, Division of Environmental Dermatology and Allergy GSF/TUM, Department of Dermatology and Allergy, Technical University of Munich, Germany
1Correspondence: Division of Immunopathology, Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, General Hospital Vienna, 3Q, Waehringer Guertel 18–20, A-1090 Vienna, Austria. E-mail: rudolf.valenta{at}meduniwien.ac.at
ABSTRACT
The key event of allergic inflammation, allergen-induced crosslinking of mast cell-bound IgE antibodies, is accompanied by release of inflammatory mediators, cytokines, and proteases, in particular ß-tryptase. We provide evidence that protease-mediated cleavage of allergens represents a mechanism that regulates allergen-induced mast cell activation. When used in molar ratios as they occur in vivo, purified ß-tryptase cleaved major grass and birch pollen allergens, resulting in defined peptide fragments as mapped by mass spectrometry. Tryptase-cleaved allergens showed reduced IgE reactivity and allergenic activity. The biological relevance is demonstrated by the fact that lysates from activated human mast cells containing tryptase levels as they occur in vivo cleaved allergens. Additionally, protamine, an inhibitor of heparin-dependent effector cell proteases, augmented allergen-induced release of mediators from effector cells. Protease-mediated allergen cleavage may represent an important mechanism for terminating allergen-induced effector cell activation.—Rauter, I., Krauth, M.-T., Flicker, S., Gieras, A., Westritschnig, K., Vrtala, S., Balic, N., Spitzauer, S., Huss-Marp, J., Brockow, K., Darsow, U., Ring, J., Behrendt, H., Semper, H., Valent, P., Valenta, R. Allergen cleavage by effector cell-derived proteases regulates allergic inflammation.
Key Words: allergy • mast cells • tryptase • allergen
MORE THAN 25% of the population suffers from IgE-mediated allergies, including allergic rhinoconjunctivitis; bronchial asthma; food allergy; allergic dermatitis; and, in severe cases, life-threatening anaphylactic shock (1
, 2)
. The key immunological mechanism underlying the most common manifestations of allergy, i.e., the mucosal forms of allergy, is the induction of mast cell and effector cell (e.g., basophil) degranulation by allergens. It is well established that the crosslinking of Fc
RI- (FcRI) bound IgE antibodies by allergens leads to mast cell activation and the release of preformed inflammatory mediators, such as histamine as well as de novo synthesis of lipid mediators, cytokines, and chemokines (3
, 4)
. Activated mast cells release large amounts of proteases, in particular tryptase, which calculated on a wt basis, accounts for 
20% of their total cellular protein (5)
. Tryptase is a neutral protease consisting of four subunits with a MW of 134 kDa. In contrast to histamine release, which peaks within the first 30 min after mast cell activation, maximum amounts of soluble tryptase are found as late as 30–60 min after allergen challenge in extracellular fluids (6)
. This delayed release reflects a slower diffusion of tryptase when it is complexed with negatively charged proteogycan (e.g., heparin) (5
, 6)
. The enzymatic activity and three-dimensional structure of tryptase have been studied in detail, but the biological function of allergen-induced tryptase release remains enigmatic (5
6
7
8)
.
In this study we investigated whether tryptase and effector cell-derived proteases may play a role in the regulation of allergen-induced effector cell activation by proteolytic cleavage of allergenic molecules.
For this purpose, we exposed a panel of important respiratory allergens to tryptase using molar ratios of the components as they may occur in vivo (9
, 10)
and studied the effects of tryptase treatment on IgE reactivity and allergenic activity of the allergen molecules. Furthermore, we analyzed mast cell lysates with known tryptase contents regarding their capacity to degrade allergens and showed that protamine, an inhibitor of heparin-dependent proteases, augments effector cell degranulation.
MATERIALS AND METHODS
Allergens, recombinant human tryptase, patients’ sera, and antibodies
Recombinant major timothy grass pollen (rPhl p 1, rPhl p 2, rPhl p 5, and rPhl p 6) and birch pollen allergens (rBet v 1, rBet v 2) were expressed in E. coli and purified as biologically active allergens equivalent to the corresponding natural allergens (Biomay, Vienna, Austria; 11
12
13
14
15
16
). With the exception of rPhl p 1, each of the allergen molecules used in this study represented folded proteins, as determined by circular dichroism (14
, 17
18
19
20
21)
. Recombinant human ß-tryptase was expressed in Pichia pastoris as biologically active protein and is supplied in a storage solution containing 0.5 mg/ml heparin (Promega, Madison, WI). Sera were obtained from allergic patients with grass and birch pollen allergy as well as from nonallergic individuals. The diagnosis of grass and birch pollen allergy was based on case history, skin-prick test results, and serological testing (22)
. A rabbit antiserum with specificity for Phl p 1 was raised against the purified recombinant allergens (17)
. 125I-labeled anti-human IgE antibodies were purchased from Pharmacia, Uppsala, Sweden, and 125I-labeled donkey anti-rabbit antibodies from Amersham, UK.
In vitro tryptase cleavage of allergens
In a typical reaction 10 µg purified allergen was incubated with tryptase in a molar ratio of 1:40 (tryptase:allergen) in 20 µl ddH2O at 37°C for 2 h (9
, 10)
. For control purposes, allergens were incubated for the same time period without addition of tryptase. The reactions were stopped by freezing in liquid nitrogen until analysis. Tryptase-mediated allergen cleavage was investigated by using three different tryptase inhibitors, a mouse monoclonal anti-ß-tryptase antibody (Ab) (Promega), protamine (ICN, Frankfurt, Germany), which blocks the cofactor heparin (23)
, and the serine-protease inhibitor leupeptin (Sigma-Aldrich). Allergen (2 µg) was treated with tryptase in vol of 20 µl with a molar ratio of 1:10 with or without inhibitor (1 µg anti-ß-tryptase moAb, 10 µg leupeptin, 10 IU protamine (1 IU/µl) at 37°C. Each experiment was repeated at least three times. Reactions were stopped after 10 min by adding SDS sample buffer containing ß-mercaptoethanol and heating of the samples at 95°C for 5 min (24)
. Samples were analyzed by 14% SDS-PAGE and Coomassie staining (25)
and, after blotting onto nitrocellulose (Schleicher and Schüll, Dassel, Germany) (26)
, were detected with specific IgE antibodies (Figs. 3B
, 6A-C
) from patients or allergen-specific rabbit antibodies (Fig. 3A
) (13)
.
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Mass spectroscopical analysis of tryptase-cleaved allergen fragments
For the two major grass pollen allergens Phl p 1 and Phl p 5, a mass-spectroscopical analysis of proteolytic fragments was performed. Reaction products obtained after tryptase treatment as well as the untreated allergens were fractionated by HPLC using Nucleosil 100, RP-18, 150 x 4 mM, 5 µm columns, and a gradient of 10 to 90% v/v acetonitrile (0.1% v/v TFA). Maldi-TOF analysis of the fractions was performed with an Axima CFR (Kratos, Manchester, UK) in positive linear mode, using alpha-cyano-4-hydroxycinnamic acid (CHCA) as a matrix as described (27)
.
Allergenic activity of tryptase-treated allergens
The allergenic activity of tryptase-treated allergens was compared with untreated allergens by exposing various concentrations of the reaction products to basophils from allergic patients. Peripheral blood polymorphonuclear leukocytes (PMNs) enriched for basophils were prepared from heparinized blood samples by Dextran sedimentation (28)
. Cells were incubated with increasing concentrations of reactants in histamine release buffer (25 mmol/L Tris-HCl, pH =7.6, 5 mmol/L KCl, 130 mmol/L NaCl, 0.6 mmol/L Ca2+, 1 mmol/L Mg2+, 0.33 mg/ml human serum albumin) at 37°C for 30 min. Histamine released into the culture supernatants was determined by RIA (Immunotech, Marseille, France) and is expressed as percentage of total histamine measured in cell lysates (28)
. The determinations were done in triplicates and are displayed as mean values ± SE Statistical significance of the differences of results obtained with and without tryptase treatment was determined by the Student’s t-test. Differences were considered statistically significant for P < 0.05.
Cleavage of allergens by mast cell lysates
Cord blood-derived mast cells were generated as described previously (29
30
31)
. In brief, cord blood mononuclear cells (n =2) were cultured in the presence of SCF and interleukin (IL)-6 for 3–4 wk, with a resulting purity of cultured mast cells of 60–90%. Cord blood mast cells (2 x 105) were washed with protein-free Pipes buffer (Pipes 25 mmol/L, NaCl 110 mmol/L, KCl 5 mmol/L, CaCl2 2 mmol/L, glucose 1g/L, pH =7.35). Cell lysates were obtained by sonication of unstimulated cells. Tryptase levels were measured in lysates by RIA (Pharmacia, Uppsala, Sweden).
The effects of cell lysates on allergens were determined by incubating 1–2 µg purified allergens with 30 µl lysate at 37°C for 1 h. In certain experiments, 10 IU protamine (1 IU/µl; ICN) was added to inhibit heparin-dependent proteases (23)
. For control purposes, an equal vol of water (i.e., 10 µl) was added. Reactions were stopped by freezing the samples in liquid nitrogen until analysis. Thereafter, samples were separated by 14% SDS-PAGE, blotted onto nitrocellulose, and probed with allergen-specific antibodies.
Rat basophil leukemia cell degranulation experiments
The rat basophil leukemia cell subline RBL-2H3 (32)
was cultured in RPMI 1640 medium (Biochrome AG, Berlin, Germany) containing 10% fetal calf serum (PAA, Pasching, Austria). Four x 104 cells/well were plated in 96-well tissue culture plates (Szabo, Vienna, Austria) and incubated overnight at 37°C and 5% CO2. The cells were loaded with a mouse monoclonal IgE Ab specific for Phl p 1 (Gieras & Valenta, unpublished data) for 2 h at 37°C and 5% CO2. The cell layer was washed twice in Tyrode’s buffer (Sigma-Aldrich, Vienna, Austria) (137 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 1.8 mM CaCl2, 0.4 mM NaH2PO4, 5.6 mM D-glucose, 12 mM NaHCO3, 10 mM N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (HEPES), and 0.1% w/v BSA, pH 7.2). Crosslinking of cell-bound IgE antibodies was induced by adding a 100 µl sample of a Phl p 1 dilution (0.3 µg/ml) or this dilution containing different amounts of protamine (1, 2, 5, 10 IU) in Tyrode’s buffer. The cells were incubated for 60 min at 37°C in a humidified atmosphere. A control was performed by adding only the highest amount (i.e., 10 IU) of protamine without allergen to the cells. The supernatants were analyzed for ß-hexosaminidase activity by adding 80 mM 4-methyl-umbelliferyl-N-acetyl-ß-D-glucosamide (Sigma-Aldrich) in citrate buffer (0.1 M, pH =4.5) for 1 h at 37°C (33)
. The reaction was stopped by the addition of 100 µl glycine buffer (0.2 M glycine, 0.2 M NaCl, pH = 10.7). Fluorescence was measured at 
ex:360/em:465 nm using a fluorescence microplate reader (CytofluorTM 2350, Millipore, Billerica, MA). Results are reported as percentage of total ß-hexosaminidase released after addition of 1% Triton X-100. The experiments were repeated three times, and a representative experiment is shown. The determinations were done in triplicates and are displayed as mean values ± SE. Statistical significance of the differences of results obtained without and with protamine was determined by the Student’s t-test. Differences were considered statistically significant for P < 0.05.
RESULTS
ß-tryptase cleaves allergens in molar ratios occurring in vivo
To study whether human ß-tryptase cleaves important environmental allergens, we have exposed purified grass and birch pollen allergens to the recombinant enzyme using allergen excess (9
, 10)
. A molar ratio of 1:40 (enzyme:allergen) was chosen, which is even lower than that calculated for the surface of activated mast cells. In Fig. 1
, we have analyzed samples of purified allergens that were incubated with (lanes +) or without (lanes –) tryptase. Many of the allergens (grass pollen allergens: Phl p 1, Phl p 6; birch pollen: Bet v 2) were almost completely degraded (Fig. 1)
. The major Timothy grass pollen allergen, Phl p 5, and the major birch pollen allergen, Bet v 1, were fragmented and degraded (Fig. 1)
. Only Phl p 2, a major grass pollen allergen showed no visible signs of degradation in the Coomassie-stained gel but, according to mass-spectroscopy, was cleaved at the NH2 terminus (data not shown). The amount of ß-tryptase used to digest the allergens was too low to be detected by Coomassie staining.
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ß-tryptase processing of allergens generates defined allergen fragments
The major Timothy grass pollen allergens Phl p 1 and Phl p 5 belong to the most frequent environmental allergens with known three-dimensional structure and IgE epitopes (17
, 19;
MMDB: 21806, PDB: 1N10). The HPLC profile of the digests showed that the recombinant enzyme had yielded a partial cleavage of the allergens (data not shown). Figures 2
A, B show a mapping by mass spectroscopy of the proteolytic fragments, which were generated by the tryptase treatment of the grass pollen allergens Phl p 1 and Phl p 5. Many of the Phl p 1 proteolytic fragments represent peptides that were smaller than the previously determined minimal IgE-reactive portions (34)
. However, even among the small Phl p 1 fragments, it was obvious that no complete fragmentation of Phl p 1 was achieved because cleavage had not occurred at all predicted sites (e.g., aa 4–17, aa 45–64, aa 166–179, aa 77–117) (Fig. 2A
).
|
Similar results were obtained for Phl p 5. The mass spectroscopical mapping of tryptase-generated fragments of the major grass pollen allergen Phl p 5 showed that the experimental fragments were smaller than the currently known continuous IgE-reactive portions of Phl p 5 (Fig. 2B
) (35
36
37
38
39)
. Most of the Phl p 5 fragments generated by in vitro tryptase cleavage were in the range of 10 amino acids. According to the IgE epitope mapping and three-dimensional structure data for Phl p 1 and Phl p 5, it can be anticipated that a complete tryptase cleavage of the allergens would totally abolish their IgE reactivity and allergenic activity. The specificity of allergen cleavage is not only documented by the mass spectroscopic analysis of fragments but also by inhibition experiments using inhibitors of tryptase activity (Fig. 3
A, B). Exposure of rPhl p 1 to recombinant ß-tryptase generates Phl p 1 fragments of less than 14 kDa, which can be detected with Phl p 1-specific rabbit antibodies (Fig. 3A
: lanes 3 and 4). Addition of the protease inhibitor leupeptin prevents tryptase cleavage of Phl p 1, indicating the specificity of the proteolytic cleavage reaction (Fig. 3A
: lanes 1, 2). The Phl p 1 allergen migrated in its monomeric form at 28 kDa without signs of degradation when leupeptin, but no tryptase, was added (Fig. 3A
: lanes 5, 6) and when incubated alone (Fig. 3A
: lanes 7–8). Figure 3B
shows that a ß-tryptase–specific mouse monoclonal antibody inhibits specifically tryptase-mediated cleavage of Phl p 1 (lane 1) as visualized with patients IgE antibodies.
ß-tryptase treated allergens exhibit reduced IgE Ab reactivity
Next, we compared IgE reactivity of tryptase-treated vs. untreated purified allergens (Fig. 4
A, B). Figure 4A
shows the IgE reactivity of the major grass pollen allergens Phl p 1, Phl p 2, Phl p 5, and Phl p 6. Tryptase-treated (panels +) and untreated (panels –) allergens were blotted onto nitrocellulose and exposed to sera from five allergic patients. The reduction of IgE reactivity obtained by tryptase treatment was strongest for Phl p 1 and Phl p 2 where the sera failed to react or showed strongly reduced IgE reactivity to the cleaved allergens (Fig. 4A
). The reduction of IgE reactivity to Phl p 1 is in accordance with the fact that tryptase treatment had lead to almost complete degradation of the allergen and generated small fragments (Fig. 1
; Fig. 2A
). In the case of Phl p 2, it is possible that tryptase has removed small portions from the termini of the allergen, which may have led to the destruction of its ß-sheet structure without causing a visible change of the MW detectable by SDS-PAGE (Fig. 1)
(40)
. The latter assumption was supported by mass spectroscopical analysis, which showed that tryptase frequently cleaved at the NH2 terminus of the protein (data not shown). A considerable reduction of IgE reactivity of tryptase-treated Phl p 6 was noted for all tested sera and in, case of Phl p 5, we observed the appearance of allergen fragments below 30 kDa in the tryptase-treated samples.
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In case of the major birch pollen allergen, Bet v 1, tryptase treatment caused a dissociation of Bet v 1 dimers at 34 kDa and IgE reactivity to the monomeric form at 17 kDa was reduced for all sera (Fig. 4B
). Likewise, IgE reactivity to Bet v 2 was considerably reduced after tryptase treatment, albeit to a lower extent as for the other allergens (Fig. 4B
).
ß-tryptase-processed allergens exhibit reduced allergenic activity
To analyze whether tryptase treatment reduces also the allergenic activity of allergens, basophils from grass and birch pollen allergic individuals were exposed to the tryptase-exposed vs. nonexposed allergens. Basophils from five allergic patients were incubated with different concentrations of Phl p 1, Phl p 2, Phl p 5, and Bet v 1 (Fig. 5
). For each allergen a representative experiment is shown. Mean values ± SEM from triplicate determinations are shown (Fig, 5A, B
). The differences of the results between digested and nondigested allergens were significant for almost all concentrations (Phl p 1: c =0.00001 µg/ml, P =0.002; c =0.0001 µg/ml, P =0.002; c =0.001 µg/ml, P =0.0001; c =0.01 µg/ml, P =0.003; Phl p 2: c =0.0001 µg/ml, P =0.037; c =0.001 µg/ml, P =0.000;c =0.01 µg/ml, P =0.000; Phl p 5: c =0.0001 µg/ml, P =0.0048; Bet v 1: c =0.00001 µg/ml, P =0.002;c =0.0001 µg/ml, P =0.000; c=0.001 µg/ml, p = 0.027; c =0.01 µg/ml, P =0.024). In agreement with the results from the IgE reactivity assays (Fig. 4)
, we found that the allergenic activity of Phl p 1 and Phl p 2 was strongest reduced. Tryptase treatment caused a clear reduction of histamine release at all tested allergen concentrations. This reduction caused by tryptase treatment was most pronounced at the lowest allergen concentration yielding significant histamine release (Fig. 5A, B
: 0.001 µg/ml Phl p 1; Fig. 5C
: 0.01 µg/ml Phl p 2). Furthermore, a shift of the dose-response curves was observed. The reduction of allergenic activity was less pronounced for Phl p 5 and Bet v 1, which also had retained a considerable proportion of IgE reactivity despite tryptase treatment (Fig. 5D, E
; Fig. 4
). Nevertheless, we found also here that histamine release was markedly reduced at the lowest allergen concentration causing relevant histamine release (Fig 5D, E
: 0.0001 µg/ml).
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Cleavage of Phl p 1 by mast cell lysates and tryptase is partially inhibited by protamine
Next, we investigated whether natural, mast cell lysates containing tryptase can degrade allergens. Human cord blood-derived mast cells were grown to more than 90% purity and homogenated in protein-free buffer. Aliquots of 1–2 µg Phl p 1 were exposed to 30 µl human mast cell lysate (derived from 2x105 cells) containing 70 ng ß-tryptase or to 30 µl PBS. Thereafter, both aliquots were blotted onto nitrocellulose and exposed to serum IgE from an allergic patient (Fig. 6
A). A reduction of IgE reactivity to the Phl p 1-derived band at 28 kDa and the appearance of degradation products were observed after exposure to the mast cell lysate compared to the PBS control. The cell lysate did not react with the Phl p 1-specific IgE antibodies (Fig. 6A
). Figure 6B
shows that protamine, an inhibitor of heparin-dependent proteases (e.g., tryptase, chymase) (23)
, reduced allergen degradation. For example, the degradation product of 16 kDa was not found in samples with protamine (Fig. 6B
: asterisk). Phl p 1 samples without mast cell lysates were not degraded, and samples without Phl p 1 showed no reactions with the Phl p 1-specific antibodies (Fig. 6B
). In addition, Figure 6C
demonstrates that protamine inhibits the allergen cleavage by tryptase. Again, protamine prevented the appearance of a low MW Phl p 1 cleavage product at 14–16 kDa (Fig. 6C
: asterisk). We have also studied whether natural allergen contact induces levels of ß-tryptase in vivo, which are comparable with those used in the in vitro experiments. First, we found by microdialysis experiments that skin-prick testing of an allergic patient with rPhl p 1 induced intradermal concentrations of 30–40 µg/L ß-tryptase (data not shown). Second, we detected in blister fluids collected from a patient’s skin, which had been challenged with rPhl p 1 ß-tryptase levels close to 250 µg/L (data not shown). The in vivo ß-tryptase levels thus corresponded to the tryptase levels measured in mast cell lysates used for the in vitro experiments (41)
.
Protamine, an inhibitor of heparin-dependent proteases, enhances Phl p 1-induced rat basophil leukemia cell degranulation
An earlier study had reported that protamine potentiates IgE-dependent and allergen-induced but not IgE-independent histamine release from human basophils (42)
. We, therefore, investigated whether protamine would enhance mediator release induced by the Phl p 1 allergen. For this purpose we used rat basophil leukemia cells loaded with Phl p 1-specific IgE antibodies and challenged them with Phl p 1 in the presence and absence of protamine (Fig. 7
). The release of hexosaminidase induced by allergen alone (28.2%±1.28) was always increased when protamine was added, reaching a maximal augmentation at a concentration of 50 IU/ml protamine (43.54%±0) (Fig. 7)
. When the highest dose of protamine was added without allergen almost no release was detected (0.23%±2.27) (Fig. 7)
. Statistical analysis revealed significant differences between allergen-induced mediator release obtained without and with various concentrations of protamine (10 IU: P =0.007; 20 IU: P =0.000; 50 IU: P =0.003; 100 IU: P =0.005).
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DISCUSSION
Our studies suggest a hitherto unsuspected role of tryptase and effector cell-derived proteases in the regulation of allergen-induced immediate allergic inflammation (3
, 4)
. Tryptase is a specific marker of mast cells and mast cell subsets and represents the major component of these cells on a wt basis, accounting for almost 20% of the total cellular protein (9)
. In fact, tryptase is released in large amounts in the course of allergic reactions, which permits to use it as a serum marker for mast cell activation (5
, 6)
. Here, we show that tryptase can cleave a panel of important respiratory allergens and thus considerably reduces their allergenic activity. The in vitro experiments were performed with purified allergens and tryptase under conditions that should mimic physiological conditions. Taking into consideration that 500,000 IgE molecules corresponding to 29 pg IgE are present on a basophil, perhaps less on a mast cell, and assuming that 8–12 pg tryptase are present in one lung mast cell, we think that the molar ratio of 40:1 (allergen:tryptase) used in the in vitro experiments, rather underestimate the abundance of tryptase over allergen in the context of allergen-induced mast cell activation (9
, 10)
. Nevertheless, we could show under these conditions that tryptase degrades different respiratory allergens, irrespective of their MW, three-dimensional structure and biological properties, yielding a considerable reduction of their IgE reactivity and their allergenic activity as demonstrated by histamine release experiments. The specificity of the tryptase-mediated allergen cleavage is demonstrated by the fact that two inhibitors of tryptase activity (i.e., a tryptase specific moAb and protamine) inhibited the process. Although we observed a strong reduction of allergenic activity for most of the allergens, especially at concentrations as they occur in vivo, some allergens, (e.g., the major grass pollen allergen Phl p 5 and the major birch pollen allergen Bet v 1) appeared less sensitive to tryptase cleavage. Notably these allergens were found to be extremely potent inducers of allergic reactions in vivo as tested by skin-prick testing and nasal provocation testing in allergic patients (43)
.
We have also demonstrated the cleavage of allergens with lysates from purifed human mast cells containing defined amounts of tryptase. However, the only partial inhibition of allergen cleavage by mast cell lysates with protamine indicates that also other mast cell and effector cell-derived proteases (e.g., chymases) may contribute to allergen degradation (44)
. Nevertheless, it should be emphasized that tryptase is the major mast cell protease in terms of amount (9
, 44)
. To investigate whether the recombinant allergens used for the in vitro experiments induce indeed also in vivo allergic reactions where concentrations of tryptase occur as have been used in vitro, we conducted skin test experiments and analyzed the tissue fluids from the sites of allergic reactions. These experiments confirmed that the concentrations of tryptase occurring at sites of allergic reactions correspond to the concentrations used in the experiments performed with the mast cell lysates.
To demonstrate the biological significance of our findings, we investigated whether inhibition of effector cell-derived proteases would enhance the allergic effector reaction. In this context it had been reported earlier in another context that protamine, which inhibits heparin a cofactor of effector cell-derived proteases (23)
, caused an enhancement of IgE- and allergen-dependent but not of IgE-independent histamine release from human basophils (42)
. Using a defined model system consisting of RBL cells loaded with allergen-specific IgE, we could indeed demonstrate that protamine lead to the augmentation of allergen-induced RBL degranulation, whereas protamine itself did not cause degranulation (Fig. 7)
.
Based on our results we propose that allergen cleavage by effector cell-derived proteases such as tryptase may regulate allergen-induced effector cell activation. On the one hand, it is possible that proteases released in the course of allergic reactions degrade allergens into less allergenic fragments and thus down-regulate allergic inflammation or even terminate effector cell activation. On the other hand, it has been shown for certain allergens, e.g., Phl p 5, that allergen cleavage may even expose highly IgE reactive domains, which can induce even stronger allergic reactions than the intact allergen (39
, 45)
.
Allergen cleavage by effector cell-derived proteases may also explain the frequent observation that the levels of allergen-specific IgE and the biological activity of allergens are badly correlated (43)
. Furthermore, it is possible that allergen cleavage by proteases such as tryptase plays a role in the termination of allergen-induced mast cell activation. In fact, the allergen-induced mast cell activation ceases spontaneously and does not exaggerate in an unlimited degranulation process, which would lead to a life-threatening condition (3
, 4)
. Several other mechanism may be considered for the termination of allergen-induced effector cell activation. They include the potential down-regulation of effector cell activation by FcRI-Fc
-recptor co-crosslinking, but it is not clear whether mast cells and other effector cells express this receptor under physiological conditions in patients and whether sufficient amounts of allergen-specific IgG antibodies of the correct isotype are present in all allergic patients (46)
. It is also possible that endocytosis of IgE may contribute to the termination of mast cell degranulation but evidence for endocytosis has so far only been provided for rat basophils (47)
.
We, therefore, believe that protease-mediated allergen cleavage may represent an important and fundamental mechanism for the regulation of allergen-induced effector cell activation. This process may explain the different sensitivities to different allergens and maybe involved in the control of allergic reactions.
ACKNOWLEDGMENTS
This study was supported by grants F01803, F01809, F01815, and T165-B09 of the Austrian Science Fund.
Received for publication March 17, 2005. Revision received December 15, 2005. REFERENCES
receptors on mast cells: activatory and inhibitory regulation of mediator release. Int. Arch. Allergy Immunol. 133,305-315[CrossRef][Medline]This article has been cited by other articles:
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  U. Baranyi, B. Linhart, N. Pilat, M. Gattringer, J. Bagley, F. Muehlbacher, J. Iacomini, R. Valenta, and T. Wekerle Tolerization of a Type I Allergic Immune Response through Transplantation of Genetically Modified Hematopoietic Stem Cells J. Immunol., June 15, 2008; 180(12): 8168 - 8175. [Abstract] [Full Text] [PDF]
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