Protease Allergens Research Articles

Lung Natural Helper Cells Are a Critical Source of Th2 Cell-Type Cytokines in Protease Allergen-Induced Airway Inflammation

Overproduction of cytokines by T helper 2 (Th2) cells in the lung is thought to be a cause of asthma. Here we report that innate lymphocytes termed lung natural helper (LNH) cells are a T cell-independent source of Th2 cell-type cytokines in protease allergen-treated lungs. LNH (Lin(-)Sca-1(+)c-kit(+/lo)CD25(+)CD127(+)) cells, when stimulated by IL-33 plus IL-2, IL-7, or thymic stroma lymphopoietin (TSLP), produced large amounts of IL-5 and IL-13. Intranasal administration of protease allergen papain induced eosinophil infiltration and mucus hyperproduction in the lung of wild-type and Rag1(-/-) mice, but not in Rag2(-/-)Il2rg(-/-) mice that lack LNH cells. LNH cell depletion inhibited papain-induced airway inflammation in Rag1(-/-) mice whereas adoptive transfer of LNH cells enabled Rag2(-/-)Il2rg(-/-) mice to respond to papain. Treatment of lung explants with papain induced IL-33 and TSLP production by stroma cells and IL-5 and IL-13 production by LNH cells. Thus, LNH cells are critical for protease allergen-induced airway inflammation.

Human blood basophils do not act as antigen‐presenting cells for the major birch pollen allergen Bet v 1

Several studies in mice have recently shown that basophils can act as antigen-presenting cells (APC) inducing Th2-mediated immune responses against parasites or protease allergens. The aim of this study was to investigate whether human basophils function as APC for the major birch pollen allergen Bet v 1. Fluorescently labeled Bet v 1 was used to assess surface binding and internalization of allergen by basophils and different types of APC from birch pollen-allergic and nonallergic individuals. Sorted basophils were analyzed in terms of up-regulation of MHC class II and co-stimulatory molecules in the absence and presence of IL-3 and IFN-γ by flow cytometry. Expression of proteins crucial for antigen presentation, namely cathepsin S and invariant chain, was determined. Basophils were used as APC in co-culture experiments with Bet v 1-specific T-cell clones (TCCs). Basophils from birch pollen-allergic donors very efficiently bound Bet v 1 through IgE/FcεRI complexes on their surface. In contrast to professional APC, basophils did not internalize allergen and expressed marginal levels of cathepsin S and invariant chain. HLA-DP, HLA-DQ, CD80/CD86, and CD40 were absent from purified basophils even when stimulated with IL-3 plus IFN-γ. IL-3/IFN-γ marginally up-regulated HLA-DR. Bet v 1-pulsed basophils failed to induce proliferative and cytokine responses in Bet v 1-specific, HLA-DR-restricted TCCs. Human basophils neither internalize, process nor present Bet v 1. Because Bet v 1 is a highly relevant allergen, we conclude that basophils play no role as APC in IgE-mediated allergy in humans.

The Protease Allergen Pen c 13 Induces Allergic Airway Inflammation and Changes in Epithelial Barrier Integrity and Function in a Murine Model

Fungal allergens are associated with the development of asthma, and some have been characterized as proteases. Here, we established an animal model of allergic airway inflammation in response to continuous exposure to proteolytically active Pen c 13, a major allergen secreted by Penicillium citrinum. In functional analyses, Pen c 13 exposure led to increased airway hyperresponsiveness, significant inflammatory cell infiltration, mucus overproduction, and collagen deposition in the lung, dramatically elevated serum levels of total IgE and Pen c 13-specific IgE and IgG1, and increased production of the Th2 cytokines IL-4, IL-5, and IL-13 by splenocytes stimulated in vitro with Pen c 13. To examine the mechanisms involved in the regulation of allergenicity by Pen c 13, we performed two-dimensional fluorescence difference gel electrophoresis analysis combined with nano-LC-MS/MS, followed by bioinformatics analysis to identify potential targets that associated with allergic inflammation, which suggested that galectin-3 and laminin might be involved in novel pathogenic mechanisms. Finally, we focused on junctional proteins between cells, because, in addition to opening of the epithelial barrier by environmental proteases possibly being the initial step in the development of asthma, these proteins are also associated with actin rearrangement. Taken together, our findings indicate that Pen c 13 exposure causes junctional structure alterations and actin cytoskeletal rearrangements, resulting in increased permeability and airway structural changes. These effects probably change the lung microenvironment and foster the development of allergic sensitization.

Naïve T cells sense the cysteine protease allergen papain and propel basophil-mediated T helper type 2 immunity (149.13)

Abstract Sensitization to protease allergens, such as papain, or helminth infection is associated with basophil recruitment to draining lymph nodes. Basophils have the capacity to present antigen to naïve T cells and promote TH2 differentiation. How papain induces basophil migration is unknown. Here, we show that papain immunization induces production of chemokines including CCL2, CCL17 and CCL22 in lymph nodes preceding basophil accumulation. Papain-evoked basophil trafficking and TH2 differentiation were diminished by the absence of CCR2 or CCR4. Papain binds and activates antigen-inexperienced T cells directly to produce CCL17 and CCL22, and T cells appear to be the major source of chemokine following papain immunization. Papain-elicited accumulation of basophils and production of CCL17 and CCL22 in lymph nodes were abolished by T cell deficiency. These results support a previously unanticipated innate function for naïve T cells in the recognition of a cysteine protease allergen, which lies upstream of basophil-evoked TH2 effector cell development.

Lung natural helper cells are an important source of innate TH2 cytokines (158.2)

Abstract Recently, novel innate TH2 cytokine producing lymphocytes termed nuocytes or natural helper cells have been found in the gut-associated mucosa tissues. Similar TH2 cytokine producing innate cells have also been detected in other organs, including the lung, but their functional significance is not well understood. We have identified TH2 cytokine producing innate lymphocytes, termed lung natural helper (NH) cells in B6 mouse lungs and elucidated their role in lung eosinophilia and asthma. Lung NH cells are Lineage-negative but express of Sca-1, c-Kit, CD127 and CD25. Lung NH cell gene expression microarrays indicate that they likely constitute a novel lymphoid lineage. When purified and stimulated in vitro, they produce large amounts of IL3, 4, 5, 13, and IL17A. They also rapidly produce IL5 and IL13 in lung explant cultures stimulated with IL25, an alarmin released by stressed lung epithelial cells. Lung NH cells are present in Rag-/- mice that lack T and B cells but absent in Rag-/-cγ-/- mice. Intranasal administration of protease allergen papain rapidly induces eosinophil infiltration and mucus hypersecretion, which are hallmark asthma symptoms, in the lung of B6 and Rag-/- mice but not in Rag-/-cγ-/- mice. Papain treatment of lung explants also induces IL13 production by lung NH cells. Thus, lung NH cells are unique lymphocytes of the innate immune system and an important source of T cell-independent TH2 cytokines. They likely play an important role in initiating asthma.

Interactions of airway epithelium with protease allergens in the allergic response

Among the apparently innocuous environmental proteins routinely inhaled by human subjects, only a small proportion of these antigens triggers allergy in susceptible individuals. Although the molecular basis of the allergenicity of these airborne proteins remains to be fully characterized, numerous studies suggest that the ability of such proteins to promote allergic responses is at least due to their proteolytic activity. This review will summarize insights into the interactions of protease allergens with the respiratory epithelium. In addition to their capacity to facilitate their antigen presentation through epithelial barrier degradation, protease allergens can directly activate airway mucosal surfaces to recruit inflammatory cells and to initiate the airway remodelling process. A greater understanding of the effects of protease allergens in the airways inflammation as well as on the relevant targets could define novel therapeutic strategies for the treatment allergic asthma.

Aspergillus fumigatus allergen expression is coordinately regulated in response to hydrogen peroxide and cyclic AMP

BackgroundA. fumigatus has been associated with a wide spectrum of allergic disorders such as ABPA or SAFS. It is poorly understood what allergens in particular are being expressed during fungal invasion and which are responsible for stimulation of immune responses. Study of the dynamics of allergen production by fungi may lead to insights into how allergens are presented to the immune system.MethodsExpression of 17 A. fumigatus allergen genes was examined in response to various culture conditions and stimuli as well as in the presence of macrophages in order to mimic conditions encountered in the lung.ResultsExpression of 14/17 allergen genes was strongly induced by oxidative stress caused by hydrogen peroxide (Asp f 1, -2, -4, -5, -6, -7, -8, -10, -13, -17 and -18, all >10-fold and Asp f 11, -12, and -22, 5-10-fold) and 16/17 allergen genes were repressed in the presence of cAMP. The 4 protease allergen genes (Asp f -5, -10, -13 and -18) were expressed at very low levels compared to the comparator (β-tubulin) under all other conditions examined. Mild heat shock, anoxia, lipid and presence of macrophages did not result in coordinated changes in allergen gene expression. Growth on lipid as sole carbon source contributed to the moderate induction of most of the allergen genes. Heat shock (37°C > 42°C) caused moderate repression in 11/17 genes (Asp f 1, -2, -4, -5, -6, -9, -10, -13, -17, -18 and -23) (2- to 9-fold), which was mostly evident for Asp f 1 and -9 (~9-fold). Anaerobic stress led to moderate induction of 13/17 genes (1.1 to 4-fold) with one, Asp f 8 induced over 10-fold when grown under mineral oil. Complex changes were seen in gene expression during co-culture of A. fumigatus with macrophages.ConclusionsRemarkable coordination of allergen gene expression in response to a specific condition (oxidative stress or the presence of cAMP) has been observed, implying that a single biological stimulus may play a role in allergen gene regulation. Interdiction of a putative allergen expression induction signalling pathway might provide a novel therapy for treatment of fungal allergy.

Basophils as Th2-inducing antigen-presenting cells

Recent studies have revealed a role for basophils as antigen-presenting cells that preferentially induce T(h)2 cells in response to complexes of antigen plus antigen-specific IgE, to protease allergens or to helminthic parasites in vitro and in vivo through the production of 'early IL-4' and the presentation to CD4(+) T cells of complexes of peptide plus MHC class II. These findings have revealed previously unknown functions of basophils and will potentially aid the design of novel therapeutic strategies for T(h)2-IgE-mediated diseases such as bronchial asthma.

Aspergillus 단백분해효소 알러젠에 의해 유도된 Th2 관련 기도염증반응에서 protease activated receptor 2 (PAR2)의 역할

Most allergens have protease activities, suggesting that proteases may be a key link between Th2-type immune reactions in allergic responses. Protease activated receptor (PAR) 2 is activated via the proteolytic cleavage of its N-terminal domain by proteinases. To know the role of PAR2 in Aspergillus protease allergen activated Th2 immune responses in airway epithelial cells, we investigated and compared immune cell recruitment and level of chemokines and cytokines between PAR2 knock out (KO) mice and wild type (WT) mice. There were evident immune cell infiltrations into the bronchial alveolar lavage fluid (BALF) of WT mice, but the infiltrations in PAR2 KO mice were significantly lowered than those of WT mice. The IL-25, TSLP, and eotaxin gene expressions were profoundly increased after Aspergillus protease, but their expression was significantly lowered in PAR2 KO mice in this study. Compared to PAR2 KO mice, OVA specific IgE concentrations in serum of WT mice were quite increased; moreover, the IgE level of PAR2 KO mice was lower than in WT mice. The IL-25 expression by Aspergillus protease stimulation was significantly reduced by p38 specific inhibitor treatment. In this study, we determined that Th2 response was initiated with IL-25 and TSLP mRNA up-regulation in lung epithelial cells via PAR2 after Aspergillus protease allergen treatment.

TH9: the latest addition to the expanding repertoire of IL‐25 targets

Interleukin (IL)-9 was first described as a TH2-associated cytokine and, although it is clear that it is involved in ‘TH2-like’ inflammation and pathology, it has recently become apparent that its regulation and biology are unlike that of the other type 2 cytokines, IL-4, IL-5 and IL-13. In vitro, IL-9-producing T cells appear to be a subset distinct from those that produce the other type 2 cytokines.1 These IL-9-secreting T cells are dependent upon transforming growth factor (TGF-β) signalling and have been recognized as an independent T helper subset, termed TH9 (Figure 1). A new report from the lab of Chen Dong has identified a role for the IL-17 cytokine family member IL-25 in the regulation of TH9 cells and has highlighted how IL-25 signalling might affect IL-9-mediated inflammatory responses in a mouse model of allergic airways disease.2

Molecular Cloning and Immunochemical Characterization of a Novel Major Japanese Cedar Pollen Allergen Belonging to the Aspartic Protease Family

Background:Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis in Japan. Protease activity in the pollen grains may trigger pro-allergic responses but no such proteases have yet been identified as pollen allergens. Objectives:We report the molecular cloning and immunochemical characterization of a novel C. japonica pollen allergen belonging to the aspartic protease family. Methods:We focused on the C. japonica pollen allergen spot No. 63 (CPA63, 47.5% IgE binding frequency) on our 2-dimensional IgE immunoblot map. The internal amino acid sequences were determined using time-of-flight mass spectrometry. Full-length cpa63 cDNA was cloned by rapid amplification of cDNA ends (RACE)-PCR. Recombinant CPA63 (r-CPA63) was expressed using the baculovirus-insect cell culture system and its IgE binding capacity was analyzed by enzyme-linked immunosorbent assay (ELISA). The proteolytic activity of r-CPA63 was also assessed using a putative mature enzyme produced upon autolysis. Results: cpa63 cDNA encoded a 472 amino acid polypeptide showing about 40% sequence identity to members of the plant atypical aspartic protease family. ELISA showed that r-CPA63 was recognized by IgE antibodies in the serum of 58% (18/31) of Japanese cedar pollinosis patients. We also demonstrated an aspartic protease-like enzyme activity of the putative mature r-CPA63. Conclusions:We have identified the first plant aspartic protease allergen from Japanese cedar pollen. The availability of the CPA63 sequence and its recombinant allergen production system are useful not only for pharmaceutical applications but also for further examination of the role of protease activity in the pathogenesis of cedar pollinosis.

Basophils as antigen presenting cells

Recent reports demonstrate that basophils act as antigen presenting cells to drive Th2 and IgE responses against protease and protein allergens and helminth parasites. Through MHC class II-dependent cognate interactions with CD4(+) T cells in the context of co-stimulatory molecules, and through secretion of IL-4, IL-13 and thymic stromal lymphopoietin, basophils drive antigen-specific Th2 responses. These results have uncovered previously unknown functions of basophils, and should aid in designing novel therapeutic strategies for asthma and allergic conditions.

Protease Allergens Induce the Expression of IL-25 via Erk and p38 MAPK Pathway

Allergic diseases, including asthma, are characterized by T helper type 2 (Th2) cell-mediated inflammations, coupled with tissue infiltration by eosinophils. In this study, we demonstrate that multiple protease allergens, including papain and DerP1, efficiently induce interleukin (IL)-25 and thymic stromal lymphopoietin (TSLP) gene expression, and this phenomenon is dependent on the protease activities of these allergens. The IL-25 cytokine level in bronchial alveolar lavage (BAL) was also profoundly and significantly increased after treatment with papain. Additionally, the levels of Th2 cytokines were significantly increased, as compared to those in the OVA-only treatment group. The various protease allergens triggered the expression of IL-25 and TSLP mRNA in mouse lung epithelial cells (MLE12) and primary mouse lung epithelial cells; these effects were inhibited by the deactivation of the protease activity of papain. The allergen papain activates the ErK and p38 MAP pathways; the inhibition of these pathways, but not the NFκB or PI-3 kinase pathways, impairs the induction of IL-25 and TSLP expression by proteases. In this study, we demonstrate that the protease allergens induce IL-25 and TSLP via the MAP kinase signal pathways, and their protease activities are essential to this pathway.

Molecular Cloning and Immunochemical Characterization of a New Japanese Cedar Pollen Allergen Homologous to Plant Subtilisin-Like Serine Protease

Protease activities in allergen sources are thought to be involved in triggering allergic inflammation through the disruption of epithelial barrier or the induction of proinflammatory cytokines. Protease allergens may also work as type 2 helper T cell (TH2) adjuvants through the cleavage of cell surface receptors. Here, we report molecular cloning and immunochemical characterization of a new Japanese cedar (Cryptomeria japonica) pollen allergen (CPA9) homologous to serine protease, which is initially found as a high IgE-binding spot on our two-dimensional (2-D) IgE immunoblotting map. The cpa9 cDNA encoded a 757 amino acid polypeptide showing a significant sequence identity with plant subtilisin-like serine protease family members including melon major allergen Cuc m 1. We found that native CPA9 purified from C. japonica pollen showed a high IgE-binding frequency and IgE cross-reactivity with melon extract.

Three-dimensional model of the honeybee venom allergen Api m 7: structural and functional insights

Api m 7 is one of the major protease allergens of the honeybee venom. It consists of a serine protease-like (SPL) and a CUB domain. The knowledge about the structure and function of Api m 7 is limited mainly to its amino acid sequence. Three-dimensional models of the two structural domains were constructed using their amino acid sequences and the crystallographic coordinates of prophenoloxidase-activating factor (PPAF-II) as a template for the SPL domain and the coordinates of porcine spermadhesin PSP-II for the CUB domain. The structural organization of Api m 7 suggests that the CUB domain is involved in interactions with natural substrates while the SPL domain probably activates zymogens. IgE epitopes and antigenic sites were predicted. Api m 7 shows structural and functional similarity to the members of the PPAF-II family. Possible substrates, function and evolution of the enzyme are discussed in the paper.

Molds, mycotoxins, and sick building syndrome

The following is a review of some of the work we have done since 2004 regarding the importance of molds and their mycotoxins in the phenomenon of sick building syndrome (SBS). In these studies we showed that the macrocyclic trichothecene mycotoxins (MTM) of Stachybotrys chartarum (SC) are easily dissociated from the surface of the organism as it grows and could therefore be consequently spread in buildings as the fungus experiences additional water events. We then showed that SC and Penicillium chrysogenum (PC) colonies remain viable long after a water source has been removed, and the MTM produced by SC remain toxic over extended periods of time. We next showed that PC when inhaled, can release in vivo, a protease allergen that can cause a significant allergic inflammatory reaction in the lungs of mice. We then showed, in a laboratory study, that the MTM of SC can become airborne attached to spores or SC particulates smaller than spores. Following that study, we next showed that the same phenomenon actually occurred in SC infested buildings where people were complaining of health problems potentially associated with SBS. Finally, we were able to demonstrate the presence of MTM in the sera of individuals who had been exposed to SC in indoor environments. This last study was done with enough mold exposed individuals to allow for the statistical significance of SC exposure to be evaluated.

Basophils function as antigen-presenting cells for an allergen-induced T helper type 2 response

TH2 mediated immune responses are induced upon infection with multicellular parasites and can be triggered by a variety of allergens. Mechanisms of induction and the antigen-presenting cells involved in activation of TH2 responses remain poorly defined and the innate immune sensing pathways activated by parasites and allergens are largely unknown. Basophils are required for the in vivo induction of TH2 responses by protease allergens. Here we show that basophils also function as antigen presenting cells. We show that, while dendritic cells were dispensable, antigen presentation by basophils was necessary and sufficient for allergen-induced activation of TH2 responses in vitro and in vivo. Thus, basophils function as antigen presenting cells for TH2 differentiation in response to protease allergens.

Serine protease activity of Per a 10 augments allergen‐induced airway inflammation in a mouse model

We recently reported an immunodominant serine protease allergen (Per a 10) from Periplaneta americana. This study investigates the role of its proteolytic activity in driving the immune responses towards self and other allergens. Groups of Balb/c mice were sensitized intraperitoneally and subcutaneously with proteolytically active Per a 10 or inactivated Per a 10 (using aminoethyl benzenesulfonyl fluoride hydrochloride) or whole body P. americana extract and subsequently challenged intranasally with the respective antigens. Mice were also sensitized and challenged with ovalbumin (OVA) alone or co-administered with active or inactive Per a 10. The immune-inflammatory responses were measured by airway hyperresponsiveness (AHR) and cellular infiltration of lungs i.e. eosinophil counts, eosinophil peroxidase (EPO) activity, myeloperoxidase activity (MPO), lung histopathology, serum levels of specific-antibodies and levels of Th1/Th2 interleukins in bronchoalveolar lavage fluid (BALF) and in spleen cells culture supernatant. Mice challenged with active Per a 10/P. americana extract showed a significant airway inflammation demonstrated by enhanced AHR and increased cellular infiltration of lungs as evidenced by high eosinophil counts, EPO activity, IL-4 and IL-5 in BALF. Active Per a 10 also induced a significant proliferation of spleen cells, increased secretion of IL-4 and IL-5 in the spleen cells culture supernatant and systemic production of specific-IgE and IgG1. However, exposure with inactive Per a 10 elicited a low cellular infiltration and systemic antibody production. Exposure to OVA with active Per a 10 demonstrated a significantly high cellular infiltration and production of OVA-specific IgE and IgG1, than exposure to OVA alone or with inactive Per a 10. Proteolytic activity of Per a 10 plays an important role in driving the allergic immune response by providing an adjuvant effect, towards self and other potential allergens present in the same microenvironment.

Divergent functions for airway epithelial matrix metalloproteinase 7 and retinoic acid in experimental asthma

The innate immune response of airway epithelial cells to aeroallergen initiates the development of T cell responses that are central to allergic inflammation. Although proteinase allergens induce the expression of interleukin 25 we show that epithelial matrix metalloproteinase 7 (MMP7) was expressed in asthma and was required for maximal activity of IL-25 in promoting T helper type 2 cell differentiation. Allergen-challenged Mmp7−/− mice showed reduced airway hyperreactivity, allergic inflammatory cytokine production and increased expression of retinal dehydrogenase (RALDH)-1. Inhibition of RALDH-1 restored the asthma phenotype in Mmp7−/− mice and inhibited lung T regulatory cell responses while exogenous administration of retinoic acid attenuated the asthma phenotype. Thus, MMP7 coordinates allergic lung inflammation by activating IL-25 while simultaneously inhibiting retinoid-dependent T regulatory cell development.

Allergen-driven suppression of thiol production by human dendritic cells and the effect of thiols on T cell function

Dendritic cells are a major source of extracellular thiols needed for T cell activation, a process in which CD40-mediated stimulation plays a pivotal role. The Dermatophagoides pteronyssinus group 1 mite allergen (Der p 1) has previously been shown to cleave CD40 from the surface of human dendritic cells, thereby suggesting that Der p 1 might compromise the ability of these cells to sustain thiol production during T cell activation. This has therefore prompted us to examine the effect of the mite protease allergen Der p 1 on thiol production by human dendritic cells. Monocyte-derived dendritic cells were treated with either proteolytically active or inactive Der p 1 and then stimulated through CD40 for extracellular thiol detection. The effect of thiol ( N-acetyl- l-cysteine) and thiol inhibitors on naïve T cell responses, including CD25 and FOXP3 expressions, cell proliferation and cytokine production, was determined. Here, we show that Der p 1-mediated cleavage of CD40 from the surface of dendritic cells suppresses the ability of these cells to produce extracellular thiols, and that reducing thiols are needed for the generation of the T helper type 1 (Th1), T cytotoxic type 1 (Tc1) and T regulatory (Treg) cell phenotypes. We conclude that Der p 1-driven suppression of thiol production by dendritic cells may disrupt Th1/Tc1 and Treg cell development, and in doing so could lead to Th2/Tc2 cell responses and allergy.