Abstract
We investigated and compared the mechanisms by which two dust mite proteolytic allergens, Der p 1 and Der p 3, and a peptide agonist of proteinase-activated receptor 2 (PAR(2)AP) trigger interleukin (IL)-8 release from human pulmonary epithelial cells (A549). Although all three stimuli tested induced the up-regulation of IL-8 (mRNA and protein), the Der p 1-mediated signaling events did not exactly match those induced by PAR(2)AP and Der p 3. First, Der p 1 was less effective in stimulating IL-8 gene transcriptional activity than PAR(2)AP and Der p 3. Second, Der p 1-mediated IL-8 expression was mainly dependent on NF-kappaB, whereas Der p 3 and PAR(2)AP regulated IL-8 expression through the activation of both NF-kappaB and AP-1. Third, although all three MAP kinases, ERK1/2, p38, and JNK, were activated, Der p 1 induced IL-8 release exclusively via the ERK1/2 signaling pathway, whereas PAR(2)AP and Der p 3 also involved the other kinases. Fourth, in HeLa cells, Der p 1 was able to up-regulate IL-8 secretion independent of PAR(2) expression, and in contrast with PAR(2)AP and Der p 3, Der p 1 was unable to affect calcium signaling via PAR(2) in PAR(2)-expressing KNRK cells. Finally, cleavage by Der p 1 of a synthetic peptide representing the N-terminal activation-cleavage site of PAR(2) did not release a high potency activator of PAR(2) as does Der p 3. We conclude that Der p 1 (but not Der p 3)-induced IL-8 production in A549 epithelial cells is independent of PAR(2) activation.
Highlights
Signaling and Cytokine Induction by Der p 1 and Der p 3 receptor is cleaved to expose a tethered ligand sequence that binds to the extracellular body of the receptor, leading to the G protein-coupled signal transduction (e.g. activation of phospholipase C, generation of inositol 1,4,5-triphosphate, increased intracellular Ca2ϩ, and activation of protein kinase C)
Der p 1 and Der p 3 Induce IL-8 Expression in A549 Cells—To determine whether Der p 1 and Der p 3 could enhance the synthesis of IL-8 protein in bronchoalveolar cells, A549 epithelial cells were cultured with various concentrations of Der p 1 and Der p 3 for 7 h, and IL-8 levels were measured in the supernatants by enzyme-linked immunosorbent assay (ELISA)
We investigated and compared the molecular mechanisms by which the dust mite proteolytic allergens, Der p 1 and Der p 3, trigger the release of the proinflammatory cytokine IL-8 from human pulmonary epithelial cells (A549), with particular attention paid to a potential role for PAR2
Summary
Signaling and Cytokine Induction by Der p 1 and Der p 3 receptor is cleaved to expose a tethered ligand sequence that binds to the extracellular body of the receptor, leading to the G protein-coupled signal transduction (e.g. activation of phospholipase C, generation of inositol 1,4,5-triphosphate, increased intracellular Ca2ϩ, and activation of protein kinase C (reviewed in Ref. 18)). A relatively small region of the 5Ј-flanking region of the IL-8 gene (nucleotides Ϫ1 to Ϫ133) is essential and sufficient for its transcriptional induction by most stimuli [24] This promoter contains an NF-B element that is required for activation in all cell types studied as well as AP-1 and CCAAT enhancer-binding protein (NF-IL6) binding sites. The latter two sites are not essential for induction but are required for maximal gene expression [24]. We have investigated and compared the mechanisms whereby Der p 1, Der p 3 and an agonist peptide-activator of PAR2 (PAR2AP) induce the expression and the production of IL-8 in human airway epithelial cells. All of our data lead us to conclude that, in contrast with Der p 3, Der p 1-induced IL-8 production in human airway epithelial cells appears to be independent of PAR2 activation
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