The role of exogen proteases in Th 2 cell differentiation and in the pathogenesis of asthma

Transcription

1 Ph.D Thesis The role of exogen proteases in Th 2 cell differentiation and in the pathogenesis of asthma By Attila Kiss M.D. Semmelweis University Doctoral School Semmelweis University Faculty of Pulmonology Supervisor: Prof. Dr. Magyar Pál Budapest 2004

2 Introduction Asthma is a chronic disorder of the airways that is characterized by reversible airflow obstruction, eosinophyl airway inflammation, persistent airway hyperreactivity and airway remodeling. Over the past 25 years, both the prevalence and the mortality have risen substantially and 6-12% of the adult populations in industrialized country carry the asthma diagnosis. In Hungary the notified asthmatic cases shows 8 times increase in the last twenty years and the registered asthmatic number reached the 1.5% of the population. Defining the immune pathologic basis of allergic inflammation is paramount to revealing mechanisms of allergic disease and developing improved therapies or establishing preventive strategies for diseases like asthma. Central to these efforts is to understand the unique properties of respiratory allergens which initiate and sustain the allergic inflammation what underlies pathologic processes such as airway obstruction or mucus overproduction. Using the analogy of the role of pattern recognition receptors, PRRs in initiating Th1 type immune defenses against intracellular pathogens, might brings us closer to understand the molecular mechanism which underlies the Th2 cell differentiation and initiation of allergic inflammation. As the innate immune system recognizes conserved molecular patterns by pattern recognition receptors to identify the type of invaders and launch the proper adaptive defense mechanism against intracellular pathogen, there might be other still unknown molecular patterns in allergens, which can be recognized by the host immunity. In order to this we scrutinized different allergens, which have been used in experimental asthma model and those, which play pathologic roles in human allergy and asthma. Two distinct class of Ag has been identified and used in experimental respiratory allergic models. The first class, we termed as type I antigen, is exemplified by chicken egg albumin (OVA) which, is incapable of inducing a broad spectrum of lung allergic changes which include airway hyperresponsiveness, goblet cell metaplasia, mucus hypersecretion and eosinophil inflammation of the bronchial mucosa, if given strictly by inhalation. However, type I Ag is capable of inducing all of these allergic changes if it is given remote from the lung (intraperitonealy or subcutaneously) in a series of priming doses, typically 2

3 with aluminum-based adjuvant before intrapulmonary challenges. In contrast, the other major class of allergen, in this study termed type II, is derived from the fungus Aspergillus fumigatus, readily induces allergic lung inflammation when given only through the airway, without the need for additional adjuvant. The distinction between allergen classes is further illustrated by comparing their immune properties. OVA is given only through the airway induces a relative tolerogenic state termed IgE tolerance in which murine B cells are induced to secrete only the Ig isotypes IgG1 and IgG2a and Th effector responses are not observed. In contrast to OVA, immunity to type II allergens derived from A. fumigatus is distinctly allergic, dominated by the presence of lung parenchymal and airway eosinophils and Th 2, elevated serum titers of IgE, and the physiologic changes of the airways caused by the allergic inflammation. Therefore, these two types of respiratory allergens differ immunologically in terms of their intrinsic ability to bypass induction of airway tolerance. Because human allergic lung disease is likely induced by inhaled allergen, it is critical to understand the adjuvant properties of type II allergens, which enable them to bypass normal tolerogenic mechanisms, inducing Th2 type immune response and establishing allergic disease. Pathogenic roles have previously been suggested for proteases and amylases and other exogenous molecules common to human allergens. However, it has not been shown that exposure by inhalation to these microbial products is necessary to overcome airway tolerogenic mechanisms, enable Th2 priming, and establish allergic lung inflammation. Aims of the study 1. In the study we were focusing on antigens like pollen and different fungal antigens, which often contribute to human allergy and asthma. Using an experimental model of asthma, we tried to identify various type II allergens and compare their allergenic property to type I and previously tested type II allergens. Comparing these type II allergens we intend to define common biochemical properties, which potentially can underlie their intrinsic allergenicity. 3

4 2. We id entified three distinct type II allergens, all of which contained active protease and amylase activities. Our goal was to dissect weather the active amylase or the protease activity play critical role, or are responsible for bypassing the state of anergy and eliciting allergic immune reaction. 3. If the presence of active protease is responsible for allergenicity of type II allergens, our goal was to show, whether the type I, protease-free antigen can be converted to a type II allergen with high allergen potential by co-administering it intranasally with a single highly purified fungal protease. We would like to prove that antigens together with exogenous proteases are both necessary and sufficient to bypass tolerogenic responses to inhaled Ag and initiate Th2-dependent allergic lung inflammation. Therefore, as allergenic adjuvants, proteases are implicated as important virulence factors in diverse allergic reactions. Materials and Methods Mice Female C57BL/6 mice were used between 4 and 8 wk of age and bred at the American Association for the Accreditation of Laboratory Animal Care-accredited Baylor College of Medicine (Houston, TX) vivarium. Mice were maintained under specific pathogen-free conditions and used according to institutional guidelines. Antigens A. fumigatus Ag was prepared from live cultures. Whole ragweed pollen extract allergen was prepared by pulverizing 1 g of ragweed pollen (Sigma-Aldrich, St. Louis, MO) in 8 ml of PBS, centrifuging at 10,000 rpm for 20 min and sterile filtering the supernatant. Aspergillus oryzae allergen was obtained commercially (Sigma-Aldrich) and 4

5 reconstituted to 10 mg/ml using sterile PBS. Chicken egg OVA (grade V; Sigma-Aldrich) was reconstituted in sterile endotoxin tested PBS and was either given alone or was added in identical amount (25 µg/dose) to protease-containing preparations immediately before administration. OVA fragments were prepared by incubating OVA (1 mg/ml) with A. oryzae allergen for 90 min at 37 C followed by centrifugation through a molecula r-size exclusion column (Centricon 10). Fragment isolation and exclusion of protease were confirmed by SDS-PAGE followed by silver staining and determination of total protease activity (none detected in fragments). Mice were then administered 25 µg protein/dose intranasally. Total protein content of allergens was determined using a bicinchoninic acid - based protein assay according to the manufacturer s instructions (Pierce, Rockford, IL). The purified 33-kDa serine protease from A. fumigatus was prepared by PE Kolattukudy and inhibited >95% by repeated addition of PMSF (1 mm; Roche, Basel, Switzerland) for 2 h at room temperature followed by overnight dialysis against PBS at 4 C. Protease activity of A. oryzae allergen was inhibited >95% by repeated addition of phosphoramidon (500 µm; Roche) for 2 h at room temperature followed by overnight dialysis against PBS. Induction and analysis of the asthma phenotype The doses of Ag preparations used for these studies were based on protease activity we empirically determined to yield equivalent induction of the asthma phenotype following intranasal challenge. Doses of inhibited protease allergens were adjusted based on equivalent total protein content relative to protease-uninhibited allergen. For intranasal Ag challenge, mice were anesthetized with metofane vapor (Vedco, St. Joseph, MO) and allowed to inhale 50 µl of allergen following application to the nares with a pipette every 4th days for five total challenges. Data were collected 24 h following the final Ag challenge and were expressed as means ± SEM. Airway hyperresponsiveness by PC200, bronchoalveolar lavage (BAL) cytology, serum antibody isotypes, lung histopathology and lung cytokine profiles by ELISPOT assay were determined. Briefly, mice were ventilated using 100% oxygen under conditions that 5

6 maintained physiologic ph, arterial PCO2 and body temperature. After establishing a stable baseline for respiratory system resistance as determined by continuously quantitating Pt/V (where Pt = change in tracheal pressure and V = air flow) at 70% tidal volume, acetylcholine chloride was administered i.v. over 1 s in escalating doses. The provocative concentration of acetylcholine in microgram per gram of body weight that caused a 200% increase in respiratory system resistance, designated PC200, was calculated by linear interpolation of appropriate dose-response curves. BAL cells were collected by instilling and withdrawing 2 times 1-ml aliquots of PBS (ph 7.2) from the tracheal cannula. The cells were centrifuged onto glass slides, using Shandon Cytospin 3 cyto-centrifuge, the slides were stained after air dry using modified Giemsa stain (Protocol Hema 3?, Fisher Diagnostics, Middletown, VA), and used to determine the absolute numbers of eosinophils. Suspensions of whole lung cells were prepared by removing the lungs and dissecting away lymph nodes and thymic tissue. Lungs were finely minced and the fragments were pressed through a 40-µm nylon mesh filter. The cells were spin at 350 RCF for 5min and re-suspend in equal 1000? l of RPMI 1640 with 10% FBS and antibiotics. For ELISPOT assays, duplicate cell samples were distributed to 96-well microtiter plates that had been precoated with mab 11B11 anti-murine-il-4 Ab, serial 2-fold dilutions of the cells were conducted, and the plates were incubated undisturbed for 18 h at 37 C. After washing away the cells, biotinylated secondary Ab against IL-4, BVD6-24G.2 (BD PharMingen, San Diego, CA), was added. Captured IL-4 was revealed using streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA) and developed using 5-bromo-4-chloro-indolyl-phosphate (Sigma- Aldrich) in 0.1 M 2-amino-2-methyl-1-propanol buffer (Sigma-Aldrich) suspended in 0.6% low melting agarose. Individual blue spots were counted after solidification of the agar using inverted microscopy and the IL-4 producing sell number were calculated per lungs. Serum was prepared from whole blood collected at the time of death for determination of total IgE and OVA- specific IgG1 and IgG2a. For total IgE, serum diluted 1/5 and 1/50 was added to plates precoated with anti-ige Ab R35-72 (BD PharMingen) and developed with biotinylated Ab R (BD PharMingen) followed by streptavidin- 6

7 conjugated alkaline phosphatase and nitrophenylphosphate (NPP; Sigma-Aldrich) substrate and the results compared with a standard curve prepared with monoclonal murine IgE (Sigma-Aldrich). For determination of OVA specific IgG1 and IgG2a the ELISA plates were coated with OVA (100 µg/ml in PBS), serum diluted 1/500, and 1/5000 was added, followed by biotinylated isotype-specific Abs (IgG1: LO-MG1 (Caltag Laboratories, Burlingame, CA); IgG2a: LO-MG2a (Caltag Laboratories)). Plates were washed, developed with streptavidin-conjugated alkaline phosphatase and NPP substrate, read in 405 nm wavelength, using standard spectrophotometer and relative concentrations were expressed as OD405. Secreted airway glycoprotein concentration, which reveals airway mucin concentration was quantitated using the glycoprotein-binding lectin, jacalin. A total of 40 µl of BAL diluted 1/100 and 1/1000 were added to individual wells of microtiter ELISA plates and incubated 2 h at 37 C. Plates were washed and blocked with 3% I-block (Applied Biosystems, Foster City, CA) followed by addition of 0.002% biotinylated jacalin (Calbiochem, La Jolla, CA). After incubation for 1 h at 37 C, plates were washed and developed with streptavidin-conjugated alkaline phosphatase and NPP. Results were quantitated by comparison with a mucin (Sigma-Aldrich) standard curve. Statistics All data are representative of at least three independent experiments with four to five mice in each in vivo experiment and are expressed as means ± SEM. Significant differences (p?0.05) were determined using Student s t test (log PC200) or Mann-Whitney U test. 7

8 Results Allergens prepared from A. fumigatus, A. oryzae and the pollen of Ambrosia artemisiifolia (Ragweed) were compared with chicken egg albumin (OVA) for their ability to induce asthma phenotype in mice when given intranasally. The intranasal antigen challenge was also compared to the widely used OVA based allergic murin model, where the sensitization takes place distant from the lung, either intracutan or intraperitoneally, followed by series of intranasal ovalbumin challenges. Compared with a previously standardized dose of the A. fumigatus allergen, the other fungal- and pollen-derived allergens yielded comparable induction of airway hyperresponsiveness, BAL eosinophilia, comparable degrees of peribronchovascular inflammation and airway goblet cell metaplasia. Similar results were obtained with OVA administered intradermaly or intraperitonealy, followed by intranasal challenges, whereas OVA introduced strictly intranasaly, in the absence of prior extra-pulmonary sensitization, was incapable to induce any allergic inflammation and asthmatic phenotype. These results indicate that those diverse antigen preparations, which elicit allergic response strictly intranasal administration, are potent type II allergens and all have a special property, which overcomes the innate resistance of the airway to immune activation in response to antigen challenge. Therefore, we try to identify a factor common to these allergens that is capable of overcoming induction of tolerance and inducing strong lung allergic reactions in resistant mice. Further analyses confirmed in all three allergens the presence of protease and amylase activities, which have both been linked to allergens and allergic phenomena. To explore the allergenic potential of amylases and proteases, we performed additional experiments with the A. oryzae allergen prepared with OVA (AOO) and administered intranasally in doses of 10-fold increasing concentration based on protease content. This protocol induced progressively severe allergic lung disease as assessed by airway eosinophilia, airway hyperresponsiveness and secreted airway mucin equivalent at the highest dose to that elicited by a previously standardized dose of the A. fumigatus allergen. In contrast, no induction of these allergic parameters was observed in mice challenge with 8

9 saline or OVA alone, whereas an intermediate dose of A. oryzae allergen without OVA showed no significant alteration in allergenic activity. Thus, the quantity of active protease, not OVA, determined the degree of expression of allergic disease induced by the A. oryzae allergen. In order to understand weather the present of the active protease or amylase enzymes in the A. oryzae/ova allergen were responsible eliciting allergic lung disease, the protease activity of A. oryzae allergen was inhibited more than 95% compare to the initial activity using phosphoramidon, a specific metalo-protease inhibitor. These small inhibitor molecules reacting with the active center of the enzyme, inhibits the protease activity, without altering the enzyme protein conformation and antigenic property. Using this protocol our data showed that the inhibition of the protease in the allergen, regardless of the presence of active amylase, resulted almost complete abrogation of all aspects of the asthma phenotype, including airway-hyperresponsiveness, mucin secretion, and airway eosinophilia. Inhibition of the protease activity abrogated the robust peribronchovascular inflammation and airway goblet cell metaplasia induc ed by the A. oryzae allergen. Even though all the antigen mixture was freshly prepared right before the intranasal challenge, we could not exclude that the potent proteases we were using, did not cleave the ovalbumin molecule, providing small OVA fragments available for the immune system. To confirm that protease activity, and its act on an undefined endogenous target and not the digestion product of the exogenously delivered antigen is allergenic, we compared reactivity of A. oryzae allergen alone to an antigen containing only A.oryzae protease-hydrolyzed OVA fragments (OVA peptides). As expected, A. oryzae allergen reconstituted airway hyperresponsiveness and airway eosinophilia while the OVA peptides showed no potential to induce the asthma phenotype or elicit lung inflammation. These data demonstrate that the present of the active protease in the A. oryzae allergen and not the amylase activity, was required for the development of allergic lung disease, furthermore we showed that the small OVA protein fragments, potentially cleaved by the active protease were not responsible in initiating allergic immune response. 9

10 It has been showed that the Ag-induced asthma phenotype is dependent on the generation of Th2 cells and their produced cytokines. We detected Th2 cells following challenge with A. oryzae/ova allergen. B cells under control of Th1 or Th2 cells producing distinct isotype of antigen specific antibodies. OVA added to allergenic preparations permitted detection of Ag-specific IgG1, which in the mouse is largely Th2- and IL-4-dependent and IgG2a, an IFN-? and Th1-dependent isotype. Similar to indices of allergic lung disease, relative quantities of total IgE and OVA-specific IgG1 titrated according to the strength of A. oryzae/ova allergen administered and were comparable at the highest dose to those achieved with A. fumigatus/ova allergen. IgE and, in contrast to prior studies, IgG1, were largely absent from sera of mice receiving protease-inactive A. oryzae/ova allergen or OVA alone, consistent with induction of Ag-specific tolerance. Furthermore, IL-4-producing cells, consisting of predominantly Th2 cells and eosinophils, were only detected in lungs of mice receiving active protease. Together, these studies demonstrate that, in addition to allergic lung disease, A. oryzae/ova allergen induced an Ag-specific Th2 response dependent only on the presence of active protease. To demonstrate that these findings are applicable to other proteases and distinct allergens, we combined OVA with the secreted serine protease purified from the extracellular fluid of A. fumigatus cultures. When administered intranasally to mice according to the same protocol as used with previous allergens, A. fumigatus serine protease/ova induced airway hyperresponsiveness identical with that elicited by the more complex A. fumigatus allergen and significant eosinophilia. We further documented recruitment of IL-4-producing cells to the lungs of A. fumigatus serine protease/ovachallenged mice in numbers consistent with results obtained with other allergens. In contrast, no significant induction of these allergic parameters was observed following challenge with saline, OVA alone, protease alone, or OVA and protease inactivated >95% by PMSF, a serine class protease inhibitor. These studies demonstrated that an active protease distinct from that present in the A. oryzae allergen is sufficient for induction of allergic lung disease. Furthermore, our findings demonstrate that active protease in combination with Ag is both necessary and sufficient to overcome the innate resistance of the airway to Th2 activation and allergic lung disease. 10

11 Conclusions We were investigating several common and potent allergens, which were strongly linked to human atopic and allergic diseases like asthma. We applied these antigens to murin experimental model and compared them to other widely used antigens, which caused tolerance by simply inhalation and they were incapable to induce allergic inflammation without prior extrapulmonary sensitization. These antigens had a special features as they were able to bypass the natural defend mechanism of lung immune environment, the tolerance. Our studies demonstrate that type II allergens, which do not require additional adjuvants or immune priming remote from the lung to elicit allergic lung inflammation, are not unique to A. fumigatus, but rather are easily derived from diverse species relevant to human atopy. We have further shown that active protease accounts for the intrinsic allergenic activity of the A. oryzae allergen and that a highly purified protease derived from A. fumigatus is sufficient to convert the type I allergen OVA to a type II when combined with protease. Therefore, regarding the type II allergens explored in this study, active proteases are the essential adjuvant factor responsible for allergic potency. Although absolute protease activity varied considerably in different preparations having equivalent allergenic activity, only protease activity correlated with disease induction, complete inactivation of protease in the A. oryzae allergen abrogated both inflammation and Th2 activation. Neither OVA (a type I allergen), a purified active protease (A. fumigatus serine protease), nor a protease-inactive allergen complex (A. oryzae allergen) was sufficient to induce the asthma phenotype if given intranasally. Remarkably however, a combination of only two molecules, a protease and type I allergen, was sufficient to induce allergic lung disease in a manner dependent only on the dose of active protease. Therefore, active protease, in combination with a type I allergen, is sufficient to bypass airway tolerance induction, stabilize Th2 effector commitment to the type I allergen, and sustain allergic lung inflammation. 11

12 Although these studies alone cannot exclude the existence of protease-inactive allergenic adjuvants, our data more importantly demonstrate the possibility that Th2 commitment and allergic phenomena arising from airway allergen exposure are ultimately driven by a protease-based mechanism. Publications Kheradmand F, Kiss A, Xu J, Lee SH, Kolattukudy PE, Corry DB A protease-activated pathway underlying Th cell type 2 activation and allergic lung disease. J Immunol Nov 15;169(10): Lee SH, Kiss A, Xu J, Qian Y, Bashoura L, Kheradmand F, Corry DB. Airway glycoprotein secretion parallels production and predicts airway obstruction in pulmonary allergy. J Allergy Clin Immunol Jan;113(1):72-8. Corry DB, Rishi K, Kanellis J, Kiss A, Song Lz LZ, Xu J, Feng L, Werb Z, Kheradmand F. Decreased allergic lung inflammatory cell egression and increased susceptibility to asphyxiation in MMP2-deficiency. Nat Immunol Apr;3(4): Epub 2002 Mar11 Lectures A Kiss, D Corry. The role of exogen proteases in Th2 cell differenciation and in the pathogenesis of allergic diseases. Annual Meeting of Hungarian Society of Allergology and Clinical Immunology, Keszthely, 2004.May. A Kiss, D Corry. The role of exogen proteases in Th2 cell differenciation and in the pathogenesis of allergic diseases. The Hungarian Thoracic Society 53th Meeting, Debrecen, 2004.June