Allergen-dependent oxidant formation requires purinoceptor activation of ADAM 10 and prothrombin
Authors: Jie Chen, Jihui Zhang, Theresa Tachie-Menson, Neha Shukla, David R. Garrod, Clive Robinson
Journal: Journal of Allergy and Clinical Immunology (2017)

Group 1 allergens, exemplified by Der p 1, are the most significant triggers within the allergenic repertoire of house dust mite (HDM) proteins capable of eliciting the intracellular generation of reactive oxidant species (ROS) by airway epithelial cells.1Zhang J. Chen J. Allen-Philbey K. Perera Baruhupolage C. Tachie-Menson T. Mangat S.C. et al.Innate generation of thrombin and intracellular oxidants in airway epithelium by allergen Der p 1.J Allergy Clin Immunol. 2016; 138: 1224-1227Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar This is because Der p 1, a cysteine peptidase, behaves as a prothrombinase, thereby triggering canonical activation of protease-activated receptor (PAR) 1 and 4 by thrombin.1Zhang J. Chen J. Allen-Philbey K. Perera Baruhupolage C. Tachie-Menson T. Mangat S.C. et al.Innate generation of thrombin and intracellular oxidants in airway epithelium by allergen Der p 1.J Allergy Clin Immunol. 2016; 138: 1224-1227Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar These events are preventable by Allergen Delivery Inhibitors or antagonists of PAR1 and PAR4 G-protein–coupled receptors.1Zhang J. Chen J. Allen-Philbey K. Perera Baruhupolage C. Tachie-Menson T. Mangat S.C. et al.Innate generation of thrombin and intracellular oxidants in airway epithelium by allergen Der p 1.J Allergy Clin Immunol. 2016; 138: 1224-1227Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar Intracellular ROS formation by any allergen is noteworthy because asthma is associated with deficits in antioxidant defences1Zhang J. Chen J. Allen-Philbey K. Perera Baruhupolage C. Tachie-Menson T. Mangat S.C. et al.Innate generation of thrombin and intracellular oxidants in airway epithelium by allergen Der p 1.J Allergy Clin Immunol. 2016; 138: 1224-1227Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar and ROS promote inflammation through transcription factor regulation, histone modifications, and the direct activation of signal transduction. The partially delineated pathway that leads to ROS production by HDM allergens converges with signaling from the ligation of Toll-like receptor 3 or melanoma differentiation–associated protein-5, which are key in host responses to respiratory viruses associated with asthma exacerbations.1Zhang J. Chen J. Allen-Philbey K. Perera Baruhupolage C. Tachie-Menson T. Mangat S.C. et al.Innate generation of thrombin and intracellular oxidants in airway epithelium by allergen Der p 1.J Allergy Clin Immunol. 2016; 138: 1224-1227Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar This convergence opens pannexons, releasing ATP, which is essential for allergen and viral RNA-dependent ROS production.1Zhang J. Chen J. Allen-Philbey K. Perera Baruhupolage C. Tachie-Menson T. Mangat S.C. et al.Innate generation of thrombin and intracellular oxidants in airway epithelium by allergen Der p 1.J Allergy Clin Immunol. 2016; 138: 1224-1227Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar Other pertinent effects of ATP include stimulation of IL-33 release, TH2 bias in dendritic antigen presenting cells, mast cell activation, and dyspnea. "Sheddase"-dependent activation of epidermal growth factor receptor is implicated in G-protein–coupled receptor crosstalk, so we explored whether HDM allergen-dependent ROS generation requires the participation of sheddase metalloenzymes, especially those of the a disintegrin and metalloprotease (ADAM) family. To investigate the production of intracellular ROS, we loaded human airway epithelial cells with dihydrorhodamine 123 and exposed them to a natural mixture of Dermatophagoides pteronyssinus allergens or 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) and uridine 5′-triphosphate (UTP) (to mimic the activation of P2X7 and P2Y purinoceptors by endogenously-released ATP) (see the Methods section in this article's Online Repository at www.jacionline.org). Exploration of metalloenzymes capable of ectodomain cleavage or regulated intracellular proteolysis was prompted by the finding that epidermal growth factor receptor signaling is crucial for ROS generation in cells stimulated by HDM allergens, BzATP or UTP (see Fig E1, A-C, in this article's Online Repository at www.jacionline.org). The metalloenzyme inhibitors marimastat and TAPI-1 (N-[(2R)-2-[2-(hydroxyamino)-2-oxoethyl]-4-methyl-1-oxopentyl]-3-(2-napthalenyl)-1-alanyl-N-(2-aminoethyl)-1-alaninamide acetate) blunted ROS production by either HDM allergens or BzATP (see Fig E2, A-D, in this article's Online Repository at www.jacionline.org). Surprisingly, TAPI-2 (N-(R)-(2-(hydroxyaminocarbonyl)methyl)-4-methylpentanoyl-L-t-butyl-glycine-L-alanine 2-aminoethyl amide acetate) (which has greater selectivity than TAPI-1 for the "classical" sheddase ADAM 17) did not affect responses to BzATP, although it was an effective inhibitor of mixed HDM allergens (see Fig E2, E and F). From these results, and consistent with additional data (see Fig E3, A-D, in this article's Online Repository at www.jacionline.org), we inferred that ROS production involved a metalloprotease component distinct from ADAM 17. Unexpectedly, the potent and selective ADAM 10 inhibitor, GI 254023X, attenuated intracellular generation of ROS by HDM, and was particularly efficacious in cells stimulated by BzATP or UTP (Fig 1, A-C), whereas it lacked effect in quiescent cells. Substantial involvement of ADAM 10 in responses to all 3 stimuli was confirmed by siRNA knockdown (Fig 1, D-F). As further proof, exogenously added recombinant human ADAM 10 elicited concentration-dependent ROS generation, which was inhibited by GI 254023X, thus authenticating its action (Fig 2, A-C). The effect of ADAM 10 was sensitive to AG 1478, confirming a receptor tyrosine-kinase–dependent component of the activation cycle (Fig 2, D).Fig 2Recombinant human (rh) ADAM 10 stimulates intracellular ROS formation in airway epithelial cells. A and B, Progress curves and concentration-response relationship for dihydrorhodamine oxidation following vehicle (veh) or rhADAM 10. All concentrations P < .001 with respect to the dashed line. C-E, Inhibition by GI 254023X, AG 1478, or argatroban, respectively, of responses to ADAM 10. BzATP is shown for reference (*P < .001 vs veh, **P < .001 vs ADAM 10, †P < .01 vs veh, ‡P < .001 vs veh). F, Gene-silencing prothrombin (PT) blunts the response to BzATP (*P < .001 vs veh, **P < .05 vs BzATP, †P < .05 vs BzATP stimulation in control transfection, ‡P < .001 vs BzATP stimulation). G, As in Fig 2, F, but stimulation by UTP (*P < .001 vs veh, **P < .001 vs UTP, †P < .05 vs UTP stimulation in control transfection, ‡P < .001 vs UTP). RFU, Relative fluorescence units; rhADAM 10, recombinant human ADAM 10.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Surprisingly, argatroban inhibited responses to rhADAM 10, implying the formation of thrombin (Fig 2, E). We have previously shown that Der p 1 is a prothrombinase,1Zhang J. Chen J. Allen-Philbey K. Perera Baruhupolage C. Tachie-Menson T. Mangat S.C. et al.Innate generation of thrombin and intracellular oxidants in airway epithelium by allergen Der p 1.J Allergy Clin Immunol. 2016; 138: 1224-1227Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar confirmed here by demonstrating that siRNA knockdown of prothrombin attenuated the response to mixed HDM allergens (see Fig E4, A, in this article's Online Repository at www.jacionline.org). Moreover, we have now found that prothrombin knockdown blunted the responses to BzATP or UTP (Fig 2, F and G). This is consistent with ADAM 10 activation, which we show to be downstream from purinoceptor stimulation, operating a pathway to enhance thrombin formation. The principle of metalloprotease-initiated thrombin formation and ROS production was further exemplified using the snake venom protease, ecarin, whose ability to activate prothrombin by proteolytic cleavage is well established from its use as a clinical diagnostic in the ecarin clotting test. Like ADAM 10, ecarin is a member of the M12B protease subfamily and comprises metalloprotease, disintegrin, and cysteine-rich domains. Like ADAM 10, ecarin is a potent generator of intracellular ROS (Fig E4, B). Detailed biochemical studies investigating the activation of prothrombin by ADAM 10 are underway and will be reported separately. Our data implicate purinoceptor-dependent activation of ADAM 10 as a downstream effector of ROS production in an innate response to HDM allergens. Significantly, ADAM 10 establishes a signaling cycle capable of sustaining prothrombin activation after its initiation by group 1 HDM allergens.1Zhang J. Chen J. Allen-Philbey K. Perera Baruhupolage C. Tachie-Menson T. Mangat S.C. et al.Innate generation of thrombin and intracellular oxidants in airway epithelium by allergen Der p 1.J Allergy Clin Immunol. 2016; 138: 1224-1227Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar In addition, as the principal sheddase of the adherens junction protein, E-cadherin,2Inoshima I. Inoshima N. Wilke G.A. Powers M.E. Frank K.M. Wang Y. et al.A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice.Nat Med. 2011; 17: 1310-1314Crossref PubMed Scopus (297) Google Scholar activation of ADAM 10 has the potential to augment any dysregulation of the epithelial barrier arising from targeted cleavage of tight junctions by group 1 HDM allergens.3Robinson C. Zhang J. Newton G.K. Perrior T.R. 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ADAM 10 expression is upregulated in a model of asthma and on B cells in patients with allergy and in TH2-prone mice.8Di Valentin E. Crahay C. Garbacki N. Hennuy B. Gueders M. Noel A. et al.New asthma biomarkers: lessons from murine models of acute and chronic asthma.Am J Physiol Lung Cell Mol Physiol. 2009; 296: L185-L197Crossref PubMed Scopus (96) Google Scholar, 9Cooley L.F. Martin R.K. Zellner H.B. Irani A.M. Uram-Tuculescu C. El Shikh M.E. et al.Increased B cell ADAM10 in allergic patients and Th2 prone mice.PLoS One. 2015; 10: e0124331Crossref PubMed Scopus (11) Google Scholar The combination of high ADAM 10 expression on B cells within a TH2 cytokine environment causes mimicry of disease pathophysiology, namely, mucus cell hyperplasia, airway constriction, inflammation, and IgE production, whereas development of these is attenuated in mice deficient in ADAM 10.9Cooley L.F. Martin R.K. Zellner H.B. Irani A.M. Uram-Tuculescu C. El Shikh M.E. et al.Increased B cell ADAM10 in allergic patients and Th2 prone mice.PLoS One. 2015; 10: e0124331Crossref PubMed Scopus (11) Google Scholar Intriguingly, ADAM 10 is also the cellular receptor for Staphylococcus aureus α-hemolysin toxin,2Inoshima I. Inoshima N. Wilke G.A. Powers M.E. Frank K.M. Wang Y. et al.A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice.Nat Med. 2011; 17: 1310-1314Crossref PubMed Scopus (297) Google Scholar suggesting that ADAM 10–dependent responses to allergens and infections, both viral and bacterial, may represent a signaling nexus in chronic severe disease exacerbations, which merits further examination in the clinic. Additional information is available (see this article's Methods, Results, and References section in the Online Repository at www.jacionline.org). 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Redox control of asthma: molecular mechanisms and therapeutic opportunities.Antioxid Redox Signal. 2010; 12: 93-124Crossref PubMed Scopus (179) Google Scholar In the experiments described here, we investigated the role of endogenous epithelial proteases in the signal transduction pathway leading to ROS production by Der p 1. Our specific focus was the role of zinc metalloproteases, especially ADAM enzymes. This supplementary information provides background data supporting the disclosure made in the accompanying "Letter to the Editor." ARP 100 (2-[((1,1′-biphenyl)-4-ylsulfonyl)-(1-methylethoxy)amino]-N-hydroxyacetamide), CL 82198 (N-[4-(4-morpholinyl)butyl]-2-benzofurancarboxamide hydrochloride), GI 254023X ((2R)-N-[(1S)-2,2-dimethyl-1-[(methylamino)carbonyl]propyl]-2-[(1S)-1-(N-hydroxyformamido)ethyl]-5-phenylpentanamide), WAY 170523 (N-[2-[4-[[[2-[(hydroxyamino)carbonyl]-4,6-dimethylphenyl](phenylmethyl)amino]sulfonyl]phenoxy]ethyl]-2-benzofurancarboxamide), and AG1478 (N-(3-chlorophenyl)-6,7-dimethoxy-4-quinazolinanine hydrochloride) were obtained from Tocris (Avonmouth, Bristol, United Kingdom [UK]). TAPI-1 acetate (N-[(2R)-2-[2-(hydroxyamino)-2-oxoethyl]-4-methyl-1-oxopentyl]-3-(2-naphthalenyl)-l-alanyl-N-(2-aminoethyl)-l-alaninamide acetate salt), TAPI-2 acetate (N-(R)-(2-(hydroxyaminocarbonyl)methyl)-4-methylpentanoyl-l-t-butyl-glycine-l-alanine 2-aminoethyl amide),argatroban monohydrate ((2R,4R)-1-[(2S)-5-[(aminoiminomethyl)amino]-1-oxo-2-[[(1,2,3,4-tetrahydro-3-methyl-8-quinolinyl)sulfonyl]amino]pentyl]-4-methyl-2-piperidinecarboxylic acid), marimastat ((2S,3R)-N4-[(1S)-2,2-dimethyl-1-[(methylamino)carbonyl]propyl]-N1,2-dihydroxy-3-(2-methylpropyl)butanediamide), BzATP (2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt), UTP (uridine 5′-triphosphate trisodium salt dehydrate), LPSs from Escherichia coli 0111:B4, and human thrombin were from Sigma-Aldrich (Poole, Dorset, UK). Dihydrorhodamine-123 was obtained from Life Technologies (Paisley, Renfrewshire, UK). Cell culture media and reagents were obtained from Life Technologies, Sigma-Aldrich, and GE Healthcare (Little Chalfont, Buckinghamshire, UK). Transfection reagents and siRNA duplexes (typically mixtures of 3 target-specific 19-25 nt siRNAs or scrambled controls against no known targets) were obtained from Santa Cruz Biotechnology (Dallas, Tex). Baculovirus-derived, rhADAM 10 (Thr214 – Glu672) expressed in sf21 cells was obtained from R&D Systems (Abingdon, Oxfordshire, UK). Ecarin from Echis carinatus venom and thrombin from human plasma were supplied by Sigma-Aldrich. Mixed, native HDM allergens in their natural proportions were prepared from laboratory cultures of D pteronyssinus according to our standard procedures. Der p 1 content of the allergen extracts was determined by ELISA (Indoor Biotechnologies, Cardiff, UK), whereas functional catalytic activity of Der p 1 was determined fluorimetrically using a Der p 1–selective substrate as described elsewhere.E4Newton G.K. Perrior T.R. Jenkins K. Major M.R. Key R.E. Stewart M.R. et al.The discovery of potent, selective, and reversible inhibitors of the house dust mite peptidase allergen Der p 1: an innovative approach to the treatment of allergic asthma.J Med Chem. 2014; 57: 9447-9462Crossref PubMed Scopus (21) Google Scholar, E5Zhang J. Chen J. Allen-Philbey K. Perera Baruhupolage C. Tachie-Menson T. Mangat S.C. et al.Innate generation of thrombin and intracellular oxidants in airway epithelium by allergen Der p 1.J Allergy Clin Immunol. 2016; 138: 1224-1227Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar HDM mixtures were normalized by reference to Der p 1 content expressed as μg/mL. Thus, HDM 1 refers to mixed HDM whose Der p 1 concentration is 1 μg/mL. Normalization was necessary because the extracts contain multiple components and batch-to-batch variation in activity is seen. Der p 1 is the most important normalization denominator in these studies because it is the principal active component responsible for intracellular ROS generation.E4Newton G.K. Perrior T.R. Jenkins K. Major M.R. Key R.E. Stewart M.R. et al.The discovery of potent, selective, and reversible inhibitors of the house dust mite peptidase allergen Der p 1: an innovative approach to the treatment of allergic asthma.J Med Chem. 2014; 57: 9447-9462Crossref PubMed Scopus (21) Google Scholar For consistent batch-to-batch proteolytic activity of Der p 1 in allergen preparations, experiments were conducted in the presence of 5 mM l-cysteine. Endotoxin content in allergen preparations was determined by kinetic chromogenic limulus amebocyte assay (Endochrome-K, Charles River Laboratories International, Inc, Wilmington, Mass) according to manufacturer instructions. Calu-3 cells, which are both well validated and a relevant cellular model for these investigations, were cultured according to our standard procedures as described elsewhere.E23Winton H.L. Wan H. Cannell M.B. Gruenert D.C. Thompson P.J. Garrod D.R. et al.Cell lines of pulmonary and non-pulmonary origin as tools to study the effects of house dust mite proteinases on the regulation of epithelial permeability.Clin Exp Allergy. 1998; 28: 1273-1285Crossref PubMed Scopus (98) Google Scholar, E24Wan H. Winton H.L. Soeller C. Tovey E.R. Gruenert D.C. Thompson P.J. et al.Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions.J Clin Invest. 1999; 104: 123-133Crossref PubMed Scopus (601) Google Scholar, E25Wan H. Winton H.L. Soeller C. Stewart G.A. Thompson P.J. 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