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

Abstract

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.

Mold Allergen, Pen c 13, Induces IL-8 Expression in Human Airway Epithelial Cells by Activating Protease-Activated Receptor 1 and 2

“…Although the precise role of Pen c 13 in allergic disease is unclear, fungal proteases have been linked to allergic asthma, and many of the Ags frequently implicated in disease are proteases. In previous studies, the house dust mite allergens, Der p 1, Der p 3, and Der p 9, were found to activate PAR-2 and stimulate the release of the proinflammatory cytokines, GM-CSF, eotaxin, IL-6, and IL-8, by human lung epithelial cells (2,3). Cockroach extract also increases IL-8 expression in human bronchial epithelial cells via PAR-2 activation (4 -6).…”

“…Furthermore, an extract of German cockroach (Blattella germanica) contains proteolytic activity that causes an increase in TNF-␣-induced IL-8 expression in human bronchial epithelial cells (4). The effects of these aeroallergen proteases, acting via protease-activated receptors (PARs), 3 have been implicated in the regulation of the release of proinflammatory cytokines, such as eotaxin, GM-CSF, IL-6, and IL-8, by epithelial cells in a nonallergic inflammatory response (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). PARs appear to play a proinflammatory role by activating proinflammatory cytokines (11,12).…”

“…House dust mite cysteine and serine proteolytic allergens induce the release of proinflammatory cytokines from epithelial cells (1)(2)(3)10). Proteases from A. alternata, C. herbarum, and A. fumigatus induce the production of IL-8 and IL-6 by A549 cells and cause cell detachment (8,9).…”

How epithelial cells detect danger: aiding the immune response

“…The best studied protagonist is PAR-2, as this receptor has been associated with activation by allergens. Activation of PAR-2 by proteases is associated with house dust mite allergen (82), cockroach allergen (83), trypsin (84) or PAR-2 activating peptides (74) and has been reported for many different epithelial cell lines or primary cells (81,85). The extend of these responses nor their similarity is not fully clear, as in many studies only a limited set of outcome parameters (mostly IL-6 and IL-8) are reported.…”

The contribution of PARs to inflammation and immunity to fungi

“…Further studies in HEK293 cells exposed to Candida supernatant or SAP2 revealed that neither stimuli affected par mRNA expression. With Aspergillu s, despite the ability to inhibit trypsin signaling, culture supernatant did not affect either PAR expression while, similar to other protease allergens, 26 Aspf18 downregulated the expression of par1 ( Supplementary Figure S1 online). Thus, Candida activates PAR 1 and PAR 2 by a TLR2-dependent mechanism, an effect that is not mediated by fungal proteases, while Aspergillus proteases may contribute to the downregulation of trypsin-induced activation of PAR 2 , which occurs by a TLR4-dependent mechanism.…”