Geoffrey A. Stewart

Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions

House dust mite (HDM) allergens are important factors in the increasing prevalence of asthma. The lung epithelium forms a barrier that allergens must cross before they can cause sensitization. However, the mechanisms involved are unknown. Here we show that the cysteine proteinase allergen Der p 1 from fecal pellets of the HDM Dermatophagoides pteronyssinus causes disruption of intercellular tight junctions (TJs), which are the principal components of the epithelial paracellular permeability barrier. In confluent airway epithelial cells, Der p 1 led to cleavage of the TJ adhesion protein occludin. Cleavage was attenuated by antipain, but not by inhibitors of serine, aspartic, or matrix metalloproteinases. Putative Der p 1 cleavage sites were found in peptides from an extracellular domain of occludin and in the TJ adhesion protein claudin-1. TJ breakdown nonspecifically increased epithelial permeability, allowing Der p 1 to cross the epithelial barrier. Thus, transepithelial movement of Der p 1 to dendritic antigen-presenting cells via the paracellular pathway may be promoted by the allergens own proteolytic activity. These results suggest that opening of TJs by environmental proteinases may be the initial step in the development of asthma to a variety of allergens.

Activation of Protease-Activated Receptor (PAR)-1, PAR-2, and PAR-4 Stimulates IL-6, IL-8, and Prostaglandin E2 Release from Human Respiratory Epithelial Cells

Epithelia from many tissues express protease-activated receptors (PARs) that play a major role in several different physiological processes. In this study, we examined their capacity to modulate IL-6, IL-8, and PGE2 production in both the A459 and BEAS-2B cell lines and primary human bronchial epithelial cells (HBECs). All three cell types expressed PAR-1, PAR-2, PAR-3, and PAR-4, as judged by RT-PCR and immunocytochemistry. Agonist peptides corresponding to the nascent N termini of PAR-1, PAR-2, and PAR-4 induced the release of cytokines from A549, BEAS-2B, and HBECs with a rank order of potency of PAR-2 > PAR-4 > PAR-1 at 400 μM. PAR-1, PAR-2, and PAR-4 also caused the release of PGE2 from A549 and HBECs. The PAR-3 agonist peptide was inactive in all systems tested. PAR-1, PAR-2, or PAR-4, in combination, caused additive IL-6 release, but only the PAR-1 and PAR-2 combination resulted in an additive IL-8 response. PAR peptide-induced responses were accompanied by changes in intracellular calcium ion concentrations. However, Ca2+ ion shutoff was ∼2-fold slower with PAR-4 than with PAR-1 or PAR-2, suggesting differential G protein coupling. Combined, these data suggest an important role for PAR in the modulation of inflammation in the lung.

House Dust Mite Allergens Induce Proinflammatory Cytokines from Respiratory Epithelial Cells: The Cysteine Protease Allergen, Der p 1, Activates Protease-Activated Receptor (PAR)-2 and Inactivates PAR-1

In previous studies, we demonstrated that allergenic house dust mite proteases are potent inducers of proinflammatory cytokines from the respiratory epithelium, although the precise mechanisms involved were unclear. In this study, we investigated whether this was achieved through activation of protease-activated receptor (PAR)-1 or -2. Pretreatment of A549 respiratory epithelial cells with the clinically important cysteine protease allergen, Der p 1, ablated subsequent PAR-1, but not PAR-2 agonist peptide-induced IL-6 and IL-8 release. HeLa cells transfected with the plasmid coding for PAR-2, in contrast to PAR-1, released significant concentration of IL-6 after exposure to Der p 1. Exposure of HeLa cells transfected with either PAR-1/enhanced yellow fusion protein or PAR-2/enhanced yellow fusion protein to Der p 1 caused receptor internalization in the latter cells only, as judged by confocal microscopy with re-expression of the receptor within 120-min postenzyme exposure. Der p 1-induced cytokine release from both A549 and transfected HeLa cells was accompanied by changes in intracellular Ca2+ concentrations. Desensitization studies showed that Der p 1 pretreatment of the A549 cells resulted in the abolition of both trypsin- and PAR-2 agonist peptide-induced Ca2+ release, but not that induced by subsequent exposure to either thrombin or PAR-1 agonist peptide. These data indicate for the first time that the house dust mite allergen Der p 1-induced cytokine release from respiratory epithelial cells is, in part, mediated by activation of PAR-2, but not PAR-1.

The transmembrane protein occludin of epithelial tight junctions is a functional target for serine peptidases from faecal pellets of Dermatophagoides pteronyssinus

There have been only a few studies of how allergens cross the airway epithelium to cause allergic sensitization. House dust mite fecal pellets (HDMFP) contain several proteolytic enzymes. Group 1 allergens are cysteine peptidases, whilst those of groups 3, 6 and 9 have catalytic sites indicative of enzymes that mechanistically behave as serine peptidases. We have previously shown that the group 1 allergen Der p 1 leads to cleavage of tight junctions (TJs), allowing allergen delivery to antigen presenting cells.

The association between Dermatophagoides mites and the increasing prevalence of asthma in village communities within the Papua New Guinea highlands

The prevalence of asthma among adults but not children living in eight South Fore villages of the Eastern Highlands of Papua New Guinea has risen dramatically over the past decade and now is 6 per 1000 in the age group less than 20 yr and 73 per 1000 in the age group more than 20 yr. Allergy to house dust mites appears to be a significant feature in the disease pathogenesis, and it is likely that this is associated with modifications to traditional lifestyles by the recent introduction of blankets and changes in sleeping habits that promote a more fertile environment for growth and multiplication of mites. Asthma is now provoked by a wide range of factors such as exertion, stress, and antecedent respiratory tract infections, which have always been common features of the South Fore way of life. This suggests that allergic reactivity may be the precursor of bronchial hyperreactivity that, once manifested, can be triggered by a variety of stimuli, not all of which are immunologic in origin.

Quantitative structural and biochemical analyses of tight junction dynamics following exposure of epithelial cells to house dust mite allergen Der p 1.

House dust mite allergen Der p 1 is a cysteine peptidase. Previously, we have suggested that the proteolytic activity of this allergen may contribute to asthma by damaging the barrier formed by the airways epithelium.

The biochemistry of common aeroallergens

In recent years, our knowledge ofthe structure of many of the important aeroallergens has increased greatly due to the application of sophisticated molecular biological and protein chemistry techniques. We now know the complete amino acid sequences of more than 50 allergens, and the number of clinically significant allergens remaining to be sequenced is rapidly growing smaller. The data obtained from such studies have been extensively reviewed [1] and they clearly indicate that, from a structural standpoint, allergens are indistinguishable from conventional antigens, and that factors known to contribute to antigenicity per se will apply to allergens entering the respiratory tract. However, it has become apparent too that many allergens demonstrate a range of biochemical activities suggesting these properties may also contribute to immunogenicity [2]. In this article, the available information on the physicochemical characteristics and biochemical properties of many of the individual protein allergens from common aeroallergenic sources such as plants, fungi and bacteria (Tables 1-6), dust mites and insects (Table 7, 8), animals (Tables 6, 9) and birds (Table 10) have been collated.

The isolation and characterization of a novel collagenolytic serine protease allergen (Der p 9) from the dust mite Dermatophagoides pteronyssinus

BACKGROUND Dust mites have been shown to contain a serine protease distinct from the previously reported trypsin and chymotrypsin. The latter enzymes have been shown to be allergens, but the allergenic importance of the former is unknown. OBJECTIVE This study was performed to isolate and characterize the novel mite serine protease and determine its allergenicity. METHODS The mite serine protease was isolated from feces-enriched extracts of Dermatophagoides pteronyssinus by ion-exchange chromatography and affinity chromatography, and its physicochemical properties were determined. The allergenicity of the protease was assessed by using the RAST. RESULTS The protease was enzymatically similar to chymotrypsin and cathepsin G-like enzymes from a variety of sources and was shown to cleave collagen. It had a molecular mass of 23,780 d. N-terminal sequence analysis (18 residues) indicated homology with the mite tryptic allergen, Der p 3, and the chymotryptic allergen, Der p 6. RAST analyses showed that the frequencies of reactivity to the novel allergen and to Der p 1, Der p 2, Der p 3, and Der p 6 were 92%, 97%, 100%, 97%, and 65%, respectively (n = 35). RAST inhibition studies showed some cross-reactivity between the protease and Der p 3 but not Der p 6. CONCLUSIONS A novel mite serine protease was isolated from D. pteronyssinus and found to be a major allergen. This allergen has been tentatively designated Der p 9.

House dust mite-derived amylase: Allergenicity and physicochemical characterization

Amylase activity was found in extracts of both Dermatophagoides pteronyssinus whole mite (0.16 U/mg) and spent growth medium (0.01 U/mg) but not in unused growth medium. It was also detected in all extracts of house dust obtained from mattresses (n = 20; geometric mean, 1.95 U/gm) and in 18 extracts of dust obtained from lounge room carpets (n = 20; geometric mean, 0.54 U/gm). Although the origins of amylase in dust are unclear, enzyme activity correlated with mite counts (n = 40; r = 0.35; p less than 0.05) and Der p I concentrations (r = 0.41; p less than 0.01). Mite amylase was purified from spent growth medium by affinity chromatography, gel filtration, and chromatofocusing. It was physicochemically similar to mammalian amylase with regard to molecular weight (60,000), charge heterogeneity (isoelectric point, 5 to 7) and the capacity to bind to an organomercurial affinity matrix. The optimum pH for enzymatic activity was revealed to be 6.4. IgE immunoblot studies demonstrated that the enzyme was allergenic and that its expression was dependent on the integrity of intrachain disulfide bonds. Sera from 25% of mite-allergic children and 46% of mite-allergic adults contained specific IgE to mite amylase. IgE to amylase was associated (p less than 0.01) with increased concentrations of total mite-specific IgE determined with a direct RAST assay.

Class specific inhibition of house dust mite proteinases which cleave cell adhesion, induce cell death and which increase the permeability of lung epithelium

1 House dust mite (HDM) allergens with cysteine and serine proteinase activity are risk factors for allergic sensitization and asthma. A simple method to fractionate proteinase activity from HDM faecal pellets into cysteine and serine class activity is described. 2 Both proteinase fractions increased the permeability of epithelial cell monolayers. The effects of the serine proteinase fraction were inhibited by 4‐(2‐aminoethyl)‐benzenesulphonyl fluoride hydrochloride (AEBSF) and soybean trypsin inhibitor (SBTI). The effects of the cysteine proteinase fraction could be inhibited by E‐64. No reciprocity of action was found. 3 Treatment of epithelial monolayers with either proteinase fraction caused breakdown of tight junctions (TJs). AEBSF inhibited TJ breakdown caused by the serine proteinase fraction, whereas E‐64 inhibited the cysteine proteinase fraction. 4 Agarose gel electrophoresis revealed that the proteinases induced DNA cleavage which was inhibited by the matrix metalloproteinase inhibitor BB‐250. Compound E‐64 inhibited DNA fragmentation caused by the cysteine proteinase fraction, but was without effect on the serine proteinase fraction. Staining of proteinase‐treated cells with annexin V (AV) and propidium iodide (PI) revealed a diversity of cellular responses. Some cells stained only with AV indicating early apoptosis, whilst others were dead and stained with both AV and PI. 5 HDM proteinases exert profound effects on epithelial cells which will promote allergic sensitization; namely disruption of intercellular adhesion, increased paracellular permeability and initiation of cell death. Attenuation of these actions by proteinase inhibitors leads to the conclusion that compounds designed to be selective for the HDM enzymes may represent a novel therapy for asthma.