Allergen-dependent oxidant formation requires purinoceptor activation of ADAM 10 and prothrombin.

To the Editor:

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. 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. These events are preventable by Allergen Delivery Inhibitors or antagonists of PAR1 and PAR4 G-protein–coupled receptors. Intracellular ROS formation by any allergen is noteworthy because asthma is associated with deficits in antioxidant defences 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. This convergence opens pannexons, releasing ATP, which is essential for allergen and viral RNA-dependent ROS production. 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.

Fig E1

Generation of ROS by HDM allergens in airway epithelial cells is dependent on EGFR receptor tyrosine kinase signaling. A, Concentration-dependent inhibition by AG1478 of ROS production by HDM allergens (triangles) or BzATP (circles) (†P < .001 vs control HDM, ‡P < .001 vs control BzATP). B, EGFR-directed siRNA reduces the response to HDM allergens (*P < .001 vs vehicle [veh], †P < .001 vs HDM allergens in cells with or without control [con] transfection). C, Reduction in ROS generation activated by BzATP or UTP in cells treated with EGFR-directed siRNA (*P < .001 vs veh, †P < .001 vs BzATP or UTP with or without con transfection). RFU, Relative fluorescence units.

Fig E2

Effects of metalloprotease inhibitors on ROS generation in human airway epithelial cells stimulated by either HDM allergens or BzATP. A, Marimastat has no effect on baseline ROS production (left panel) but inhibits the response to HDM allergens (right panel) (*P < .001 vs vehicle [veh] or marimastat control, †P < .001 vs cells activated with HDM in the absence of marimastat). B, Concentration-dependent inhibition of HDM responses by TAPI-1 (*P < .001 vs veh, †P < .05, ‡P < .001 vs HDM 1). C, Inhibition of BzATP by TAPI-1 (*P < .001 vs veh, †P < .001 vs BzATP). D, Inhibition of BzATP by marimastat (*P < .001 vs veh or marimastat in unstimulated cells, †P < .001 vs BzATP control). E, Inhibition of the response to HDM allergens by TAPI-2. The control response to HDM allergens is displayed in the left section as a bar graph with mean ± SE shown, and as a dotted line in the concentration-inhibition curve in the right section of this panel (*P < .001 vs veh or TAPI-2 control, †P < .001 vs HDM 1). F, Lack of effect of TAPI-2 on the response to BzATP (*P < .001 vs veh or TAPI-2 control). RFU, Relative fluorescence units.

Fig E3

Effects of metalloprotease inhibitors on ROS generation activated by HDM allergens. A, Modulation of baseline ROS production in airway epithelial cells by WAY 170523 (*P < .001, **P < .01 vs vehicle [veh]). B, Inhibition of HDM allergen- or BzATP-dependent ROS production by WAY 170523 (*†P < .001 vs veh, **P < .05-.001 vs HDM, ‡P < .001 vs BzATP). C, Effect of CL 82198 on baseline ROS production and lack of action against HDM allergens (*P < .001 vs veh, **P < .05 vs veh, †P < .001 vs veh). D, Effect of ARP 100 on baseline ROS production and failure to inhibit the response to HDM allergens (*P < .001 vs veh, †P = .01 and ‡P < .001 vs HDM allergen). RFU, Relative fluorescence units.

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 1

Inhibition by GI 254023X suggests that ADAM 10 is a mediator of intracellular ROS production by (A) HDM allergens (*P < .001 vs vehicle [veh], †P < .05-.001 vs HDM 1), (B) BzATP (*P < .001 vs veh, ‡P < .001 vs BzATP, **P < .05 vs veh), (C) UTP (*P < .001 vs veh, †P < .001 vs UTP). D-F, ADAM 10 gene silencing also reduces these responses (*P < .001 vs veh, †P < .001 vs HDM 1, BzATP, or UTP with or without control transfection, ‡P < .05 vs BzATP). RFU, Relative fluorescence units.

Fig 2

Recombinant 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.

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, 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.

Fig E4

Thrombin dependency of intracellular ROS generation in airway epithelial cells. A, Knockdown of prothrombin (PT) expression by siRNA blunts the response to stimulation by mixed HDM allergens (*P < .001 vs vehicle [veh], †P < .05 vs HDM stimulation in cells with control [con] transfection, ‡P < .001 vs HDM stimulation). B, Concentration-dependent generation of ROS by the snake venom protease, ecarin. Dashed line shows ROS production rate in unstimulated cells. C, CL 82198 inhibits ROS generation by exogenous thrombin (*P < .001 vs veh, †P < .001 vs thrombin, ‡P < .05 vs veh). D, GI 254023X inhibits ROS generation by exogenous thrombin (*P < .001 vs veh, †P < .001 vs thrombin). RFU, Relative fluorescence units.

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. In addition, as the principal sheddase of the adherens junction protein, E-cadherin, 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.

These findings expand the growing pleiotropic role of ADAM 10 in allergy. Illustratively, ADAM 10 drives TH2 bias and promotes IgE synthesis by being a CD23 sheddase, an effect incidentally ascribed to Der p 1 itself. In airway epithelial cells, ADAM 10 liberates CCL20 (which recruits dendritic cells and TH17 cells and promotes mucus hyperplasia), CCL2 (chemoattractant for dendritic cells), CCL5 (eosinophil chemokine), CXCL8 (neutrophil chemokine), and CXCL16 (T-cell chemoattractant).6, 7 It is also involved in stem cell factor-dependent mast cell migration. ADAM 10 expression is upregulated in a model of asthma and on B cells in patients with allergy and in TH2-prone mice.8, 9 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.

Intriguingly, ADAM 10 is also the cellular receptor for Staphylococcus aureus α-hemolysin toxin, 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).