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

Abstract

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 e ects of the serine proteinase fraction were inhibited by 4-(2-aminoethyl)-benzenesulphonyl¯uoride hydrochloride (AEBSF) and soybean trypsin inhibitor (SBTI). The e ects 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 e ect 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 e ects 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.

Tight junction properties of the immortalized human bronchial epithelial cell lines Calu‐3 and 16HBE14o‐

“…Previous work has demonstrated that Calu-3 cells can express tight junctions, secretory component, secretory leukocyte proteinase inhibitor, lysozyme, lactoferrin, and the MUC-2 mucin gene, demonstrate constitutive and phorbol 12-myristate 13-acetate-inducible gelatinase B activity, have an active cystic fibrosis transmembrane conductance regulator and display functional cyclic adenosine monophosphate-dependent chloride secretion [18, 36±38]. 16HBE14o-cells possess tight junctions, cystic fibrosis transmembrane conductance regulator and functional chloride conductances [19], they express functional b-adrenoceptors [39], and the cytokines interleukin-2 [40], and interleukin-8 [39], utilize the transcription factor nuclear factor-kB [41] and contain type-III phosphodiesterase [42]. Taken together with the evidence from other investigations [25], these data collectively suggest that the 16HBE14o-and Calu-3 epithelial cell lines may prove to be useful paradigms of the permeability functions of human airway epithelium, especially in studies of tight junction assembly and disassembly.…”

“…Measurements of [ 14 C]-mannitol clearance and permeability were performed exactly as described previously [19]. Experiments were conducted in confluent monolayers of epithelial cells which had been propagated on ultrathin undercoats of ungelled Matrigel in Costar Transwells (Corning-Costar Ltd., High Wycombe, Buckinghamshire, UK).…”

Allergic Rhinitis and its Impact on Asthma (ARIA) 2008*

“…However, it is now also appreciated that allergens, on account of their enzymatic proteolytic activity, may directly activate epithelial cells (318,772,773) and eventually lead to a Th2-immune response, inducing cytokine and chemokine release (774), thus having the potential to induce airway inflammation independent of IgE (775). Moreover, Der p 1 is able to alter the epithelial tight junctions (316), thereby increasing epithelial permeability (776). The relative importance of non-IgE-to IgE-mediated mechanisms is undetermined.…”

The Skin Barrier Function and the Development of Dermatitis.

“…Studies have shown that these proteins degrade adhesion proteins and enhance the permeability of the lung epithelium (Winton et al, 1998). It can then be suggested that the same effect may occurs at the skin barrier level.…”