Nithiananthan Asokananthan

Activation of Protease-Activated Receptor (PAR)-1, PAR-2, and PAR-4 Stimulates IL-6, IL-8, and Prostaglandin E2 Release from Human Respiratory Epithelial Cells

Epithelia from many tissues express protease-activated receptors (PARs) that play a major role in several different physiological processes. In this study, we examined their capacity to modulate IL-6, IL-8, and PGE2 production in both the A459 and BEAS-2B cell lines and primary human bronchial epithelial cells (HBECs). All three cell types expressed PAR-1, PAR-2, PAR-3, and PAR-4, as judged by RT-PCR and immunocytochemistry. Agonist peptides corresponding to the nascent N termini of PAR-1, PAR-2, and PAR-4 induced the release of cytokines from A549, BEAS-2B, and HBECs with a rank order of potency of PAR-2 > PAR-4 > PAR-1 at 400 μM. PAR-1, PAR-2, and PAR-4 also caused the release of PGE2 from A549 and HBECs. The PAR-3 agonist peptide was inactive in all systems tested. PAR-1, PAR-2, or PAR-4, in combination, caused additive IL-6 release, but only the PAR-1 and PAR-2 combination resulted in an additive IL-8 response. PAR peptide-induced responses were accompanied by changes in intracellular calcium ion concentrations. However, Ca2+ ion shutoff was ∼2-fold slower with PAR-4 than with PAR-1 or PAR-2, suggesting differential G protein coupling. Combined, these data suggest an important role for PAR in the modulation of inflammation in the lung.

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

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.

Direct effects of corticotropin-releasing hormone and thyrotropin-releasing hormone on fetal lung explants

Fetal lung produces corticotropin-releasing hormone (CRH) without known direct effects. We tested the hypothesis that CRH can directly regulate lung development. In baboon fetal lung explants, CRH strongly induces surfactant phospholipid synthesis and SP-C immunostaining, plus [(3)H]thymidine incorporation. CRH receptor mRNA was detected in lung from multiple baboons at e125. Testing thyrotropin (TRH) as a specificity control, we did demonstrate different direct effects with only modest stimulation of surfactant phospholipid synthesis and strong induction of cytidylyltransferase gene expression. Therefore, CRH, similar to ACTH and glucocorticoids, is a potent inducer of cell differentiation in fetal lung.

Oncostatin M synergises with house dust mite proteases to induce the production of PGE2 from cultured lung epithelial cells

The release of PGE2 and nitric oxide (NO) from the respiratory epithelium may act to dampen inflammation. In other tissues, oncostatin M (OSM), a potent inducer of epithelial antiproteases, has also been shown to interact with IL‐1β to stimulate PGE2 release. However, whether OSM interacts with pro‐inflammatory cytokines and proteases in the production of anti‐inflammatory eicosanoids and NO from airway epithelium is unknown. The effect of OSM and the related cytokine leukaemia inhibitory factor (LIF) on PGE2 and NO production by the respiratory epithelial cell line, A549 in response to pro‐inflammatory cytokines as well as protease‐rich house dust mite (HDM) fractions and a protease‐deficient rye grass pollen extract was examined by immunohistochemistry, cell culture, ELISA and enzyme‐immunoassay. Cells treated with a mixture of IL‐1β, IFNγ and LPS for 48 h produced a 9 fold increase in PGE2 and a 3 fold increase in NO levels (both P<0.05). Both OSM and LIF were without effect. However, OSM added together with the cytokine mixture synergistically enhanced PGE2 production (22 fold, P<0.05). OSM also synergistically enhanced PGE2 production in response to a cysteine protease‐enriched, but not serine protease‐enriched HDM fraction (P<0.05). Rye grass extract, neither alone nor in combination with OSM, induced PGE2 or NO production, although it did induce the release of GM‐CSF. These observations suggest that OSM is an important co‐factor in the release of PGE2 and NO from respiratory epithelial cells and may play a role in defense against exogenous proteases such as those derived from HDM.

Activation of protease‐activated receptors (PARs)‐1 and ‐2 promotes alpha‐smooth muscle actin expression and release of cytokines from human lung fibroblasts

Previous studies have shown that protease‐activated receptors (PARs) play an important role in various physiological processes. In the present investigation, we determined the expression of PARs on human lung fibroblasts (HLF‐1) and whether they were involved in cellular differentiation and pro‐inflammatory cytokine and prostaglandin (PGE2) secretion. PAR‐1, PAR‐2, PAR‐3, and PAR‐4 were detected in fibroblasts using RT‐PCR, immunocytochemistry, and flow cytometry. Increased expression of PAR‐4, but not other PARs, was observed in fibroblasts stimulated with phorbol myristate acetate. The archetypical activators of PARs, namely, thrombin and trypsin, as well as PAR‐1 and PAR‐2 agonist peptides, stimulated transient increases in intracellular Ca2+, and promoted increased α‐smooth muscle actin expression. The proteolytic and peptidic PAR activators also stimulated the release of IL‐6 and IL‐8, as well as PGE2, with a rank order of potency of PAR‐1 > PAR‐2. The combined stimulation of PAR‐1 and PAR‐2 resulted in an additive release of both IL‐6 and IL‐8. In contrast, PAR‐3 and PAR‐4 agonist peptides, as well as all the PAR control peptides examined, were inactive. These results suggest an important role for PARs associated with fibroblasts in the modulation of inflammation and remodeling in the airway.

Delayed and persistent suppression of bronchoconstriction by trypsin in the airway lumen

Mucosal trypsin, a protease-activated receptor (PAR) stimulant, may have an endogenous bronchoprotective role on airway smooth muscle. To test this possibility the effects of lumenal trypsin on airway tone in segments of pig bronchus were tested. Bronchial segments from pigs were mounted in an organ chamber containing Krebs solution. Contractions were assessed from isovolumetric lumen pressure induced by acetylcholine (ACh) or carbachol added to the adventitia. Trypsin, added to the airway lumen (300 µg·mL−1), had no immediate effect on smooth muscle tone but suppressed ACh-induced contractions after 60 min, for at least 3 h. Synthetic activating peptides (AP) for PAR1, PAR2 or PAR3 were without effect, but PAR4 AP caused rapid, weak suppression of contractions. Lumenal thrombin was without effect and did not prevent the effects of trypsin. Effects of trypsin were reduced by Nω-nitro-l-arginine methyl ester but not indomethacin. Trypsin, thrombin and PAR4 AP released prostaglandin E2. Adventitially, trypsin, thrombin and PAR4 AP (but not PAR2 AP) relaxed carbachol-toned airways after <3 min. The findings of this study show that trypsin causes delayed and persistent bronchoprotection by interacting with airway cells accessible from the lumen. The signalling mechanism may involve nitric oxide synthase but not prostanoids or protease-activated receptors.

Immunomodulatory effects of functionalised chalcones on pro-inflammatory cytokine release from lung epithelial cells

Chalcones, prepared in high yield in polyethylene glycol, Mw = 300 (PEG300), were screened for inhibition of the thrombin-mediated release of pro-inflammatory interleukin-6 (IL-6) and interleukin-8 (IL-8) from the A549 lung epithelial cell line. Several show significant down-regulation of these pro-inflammatory mediators in a dose-dependant manner with one exhibiting pro-inflammatory properties. Computational studies on selected chalcones revealed a detailed understanding of the structure, electron density and biological activity relationship.