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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
SPECIFIC AIMS
Our aim was to investigate whether tryptase and proteases released from mast cells and other effector cells in the course of allergic inflammation may cleave allergens and thus regulate allergen-induced effector cell activation. Using a panel of purified respiratory allergen molecules, we analyzed whether mast cell-derived tryptase cleaves allergens at physiological concentrations. Tryptase-exposed allergens were studied by mass spectroscopy to map cleavage sites and were further analyzed to determine whether tryptase treatment had reduced their IgE-binding capacity as well as allergenic activity. The biological relevance of allergen cleavage through tryptase was evaluated by investigating whether lysates, from activated human mast cells that contain tryptase levels as they occur in vivo, cleave allergens. Additionally, protamine, an inhibitor of heparin-dependent proteases, augmented allergen-induced release of mediators from effector cells. Protease-mediated allergen cleavage is discussed as an important mechanism for terminating allergen-induced effector cell activation.
PRINCIPAL FINDINGS
1. ßbeta;-tryptase treated allergens exhibit reduced IgE antibody reactivity and reduced allergenic activity
We compared IgE reactivity of tryptase-treated vs. untreated purified allergens. Tryptase-treated and untreated 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 is in accordance with the fact that tryptase treatment had led to almost complete degradation of the allergen and had generated small fragments as determined by mass spectroscopy. 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.
In case of the major birch pollen allergen Bet v 1, tryptase treatment caused also a reduction of IgE reactivity for each of the tested sera. Likewise, IgE reactivity to Bet v 2 was considerably reduced after tryptase treatment, albeit to a lower extent as for the other allergens.
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-digested vs. nondigested 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. 1
). For each allergen a representative experiment is shown. Mean values ± SE from triplicate determinations are shown (Fig, 1A-E
). The differences of the results between digested and nondigested allergens were significant for almost all concentrations. In agreement with the results from the IgE reactivity assays, 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. 1A, B
: 0.001 µg/ml Phl p 1; Fig. 1C
: 0.01 µg/ml Phl p 2). Furthermore, a shift of the dose-response curves was observed. The reduction of allergenic activity was less for Phl p 5 and Bet v 1, which had retained a considerable proportion of IgE reactivity despite tryptase treatment (Fig. 1D, E
). Nevertheless, we found also here that histamine release was markedly reduced at the lowest allergen concentration causing relevant histamine release (Fig 1D, E
: 0.0001 µg/ml).
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CONCLUSION AND SIGNIFICANCE
Our studies suggest a hitherto unsuspected role of proteases in the regulation of allergen-induced mast cell activation, the key event of the immediate allergic inflammation. The protease 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. In fact, tryptase is released in large amounts in the course of allergic reactions, which permits its use as a serum marker for mast cell activation. Here, we show that tryptase can cleave a panel of important respiratory allergens and thus considerably reduces their allergenic activity. Based on our experiments, we propose that proteases such as tryptase, which are released after allergen-induced effector cell degranulation, cleave allergens and thus may terminate allergen-induced degranulation (Fig. 2
).
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The in vitro experiments supporting this assumption were done with purified allergens and tryptase under conditions that should mimic physiological conditions. We take into consideration that 
500,000 IgE molecules corresponding to 29 pg IgE are present on a basophil, perhaps less on a mast cell. Also, assuming that 8–12 pg tryptase is present in one lung mast cell, we think that the molar ratio of 40:1 (allergen:tryptase) used in the in vitro experiments rather underestimates the abundance of tryptase over allergen in the context of allergen-induced mast cell activation. 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.
We have demonstrated the cleavage of allergens also with lysates from purifed human mast cells containing defined amounts of tryptase. The only partial inhibition of allergen cleavage with mast cell lysates by protamine indicates that also other mast cell-derived proteases may contribute to allergen degradation. To demonstrate that 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 showed 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 protease activity would enhance the allergic effector reaction. In this context, it had been reported earlier in another context that protamine that inhibits heparin, a cofactor of effector cell proteases, caused an enhancement of IgE- and allergen-dependent but not of IgE-independent histamine release from human basophils. Using RBL cells loaded with allergen-specific IgE, we could indeed demonstrate that protamine lead to the augmentation of RBL degranulation, whereas protamine itself did not cause degranulation.
Based on our results, we propose that protease-mediated allergen cleavage may regulate allergen-induced mast cell and effector cell activation. On the one hand, it is possible that tryptase released in the course of allergic reactions degrades allergens into less allergenic fragments and thus down-regulates allergic inflammation or even terminates 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.
Protease-mediated allergen cleavage may, therefore, also explain the frequent observation that the levels of allergen-specific IgE and the biological activity of allergens are badly correlated. Furthermore, it is possible that protease-mediated allergen cleavage plays a role in the termination of allergen-induced effector cell activation. In fact, the allergen-induced effector cell activation ceases spontaneously and does not exaggerate in an unlimited degranulation process, which would lead to a life-threatening condition. Several other mechanisms may be considered for the termination of allergen-induced effector cell activation. They include the potential down-regulation of effector cell activation by Fc
RI-Fc
-receptor co-crosslinking but it is not clear whether 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. It is also possible that endocytosis of IgE may contribute to the termination of effector cell degranulation, but evidence for endocytosis has so far only been provided for rat basophils. 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.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3999fje
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