Fabien Schmidlin

Protease-Activated Receptor 2 Mediates Eosinophil Infiltration and Hyperreactivity in Allergic Inflammation of the Airway

Trypsin and mast cell tryptase can signal to epithelial cells, myocytes, and nerve fibers of the respiratory tract by cleaving proteinase-activated receptor 2 (PAR2). Since tryptase inhibitors are under development to treat asthma, a precise understanding of the contribution of PAR2 to airway inflammation is required. We examined the role of PAR2 in allergic inflammation of the airway by comparing OVA-sensitized and -challenged mice lacking or overexpressing PAR2. In wild-type mice, immunoreactive PAR2 was detected in airway epithelial cells and myocytes, and intranasal administration of a PAR2 agonist stimulated macrophage infiltration into bronchoalveolar lavage fluid. OVA challenge of immunized wild-type mice stimulated infiltration of leukocytes into bronchoalveolar lavage and induced airway hyperreactivity to inhaled methacholine. Compared with wild-type animals, eosinophil infiltration was inhibited by 73% in mice lacking PAR2 and increased by 88% in mice overexpressing PAR2. Similarly, compared with wild-type animals, airway hyperreactivity to inhaled methacholine (40 μg/ml) was diminished 38% in mice lacking PAR2 and increased by 52% in mice overexpressing PAR2. PAR2 deletion also reduced IgE levels to OVA sensitization by 4-fold compared with those of wild-type animals. Thus, PAR2 contributes to the development of immunity and to allergic inflammation of the airway. Our results support the proposal that tryptase inhibitors and PAR2 antagonists may be useful therapies for inflammatory airway disease.

c-Cbl mediates ubiquitination, degradation, and down-regulation of human protease-activated receptor 2

Mechanisms that arrest G-protein-coupled receptor (GPCR) signaling prevent uncontrolled stimulation that could cause disease. Although uncoupling from heterotrimeric G-proteins, which transiently arrests signaling, is well described, little is known about the mechanisms that permanently arrest signaling. Here we reported on the mechanisms that terminate signaling by protease-activated receptor 2 (PAR2), which mediated the proinflammatory and nociceptive actions of proteases. Given its irreversible mechanism of proteolytic activation, PAR2 is a model to study the permanent arrest of GPCR signaling. By immunoprecipitation and immunoblotting, we observed that activated PAR2 was mono-ubiquitinated. Immunofluorescence indicated that activated PAR2 translocated from the plasma membrane to early endosomes and lysosomes where it was degraded, as determined by immunoblotting. Mutant PAR2 lacking intracellular lysine residues (PAR2Δ14K/R) was expressed at the plasma membrane and signaled normally but was not ubiquitinated. Activated PAR2 Δ14K/R internalized but was retained in early endosomes and avoided lysosomal degradation. Activation of wild type PAR2 stimulated tyrosine phosphorylation of the ubiquitin-protein isopeptide ligase c-Cbl and promoted its interaction with PAR2 at the plasma membrane and in endosomes in an Src-dependent manner. Dominant negative c-Cbl lacking the ring finger domain inhibited PAR2 ubiquitination and induced retention in early endosomes, thereby impeding lysosomal degradation. Although wild type PAR2 was degraded, and recovery of agonist responses required synthesis of new receptors, lysine mutation and dominant negative c-Cbl impeded receptor ubiquitination and degradation and allowed PAR2 to recycle and continue to signal. Thus, c-Cbl mediated ubiquitination and lysosomal degradation of PAR2 to irrevocably terminate signaling by this and perhaps other GPCRs.

Involvement of MMP-12 and phosphodiesterase type 4 in cigarette smoke-induced inflammation in mice

The aim of the present study was to characterise a mouse model of airways inflammation induced by cigarette smoke and to compare it with a lipopolysaccharide (LPS) model with regards to the efficacy of a PDE4 inhibitor (cilomilast), a corticosteroid (dexamethasone) and macrophage metalloelastase (MMP)-12 gene deletion. Cigarette smoke exposure for 3 days induced a time-dependent airway neutrophilia associated with an increased level of keratinocyte-derived chemokine (KC), macrophage inflammatory protein (MIP)-2, MIP-1α and MMP-9 in the bronchoalveolar lavage (BAL). LPS exposure also induced an increase in the number of neutrophils in BAL. Studies in MMP-12-/- mice showed that in contrast to the smoking model, MMP-12 did not have a critical role in LPS-induced inflammation. Both cilomilast and dexamethasone blocked LPS-induced neutrophilia in a dose-dependent manner. Cilomilast inhibited cigarette smoke-induced neutrophilia and MIP-1α, but only 10 mg·kg−1 of dexamethasone was effective. Both anti-inflammatory treatments had no effect on the levels of KC and MIP-2 in the BAL. Although the inflammatory response was very similar in the smoking model and LPS, the pharmacological modulation and the MMP-12 gene deletion highlighted the differences in the mechanisms involved. Furthermore, the cigarette smoke model seemed to better represent the situation described in chronic obstructive pulmonary disease patients. In conclusion, these differences underline the importance of using an acute smoke-exposure model to investigate potential new treatments for chronic obstructive pulmonary disease.

Dynamin and Rab5a-dependent Trafficking and Signaling of the Neurokinin 1 Receptor

Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca2+ mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP.

Heterologous regulation of trafficking and signaling of G protein-coupled receptors: β-Arrestin-dependent interactions between neurokinin receptors

Cells express multiple G protein-coupled receptors that are simultaneously or sequentially activated by agonists. The consequences of activating one receptor on signaling and trafficking of another receptor are unknown. We examined the effects of selective activation of the neurokinin 1 receptor (NK1R) on signaling and trafficking of the NK3R and vice versa. Selective agonists of NK1R and NK3R induced membrane translocation of β-arrestins (β-ARRs). Dominant negative β-ARR319–418 inhibited endocytosis of NK1R and NK3R. Whereas an NK1R agonist caused sequestration of NK1R with β-ARR in the same endosomes, thereby depleting them from the cytosol, β-ARRs did not prominently sequester with the activated NK3R and rapidly returned to the cytosol. In cells coexpressing both receptors, prior activation of the NK1R inhibited endocytosis and homologous desensitization of the NK3R, which was dose-dependently reversed by overexpression of β-ARR1. Similar results were obtained in enteric neurons that naturally coexpress the NK1R and NK3R. In contrast, activation of the NK3R did not affect NK1R endocytosis or desensitization. Thus, the high-affinity and prolonged interaction of the NK1R with β-ARRs depletes β-ARRs from the cytosol and limits their role in desensitization and endocytosis of the NK3R. Because β-ARRs are critical for desensitization, endocytosis, and mitogenic signaling of many receptors, this sequestration is likely to have important and widespread implications.

PLA2 phosphorylation and cyclooxygenase-2 induction, through p38 MAP kinase pathway, is involved in the IL-1β-induced bradykinin B2 receptor gene transcription

Abstract. We hypothesized that inflammatory mediators such as interleukin-1β (IL-1β) might be responsible for the hyperreactivity to bradykinin observed in asthmatic patients. We reported previously that Il-1β induced a prostanoid-dependent increase in the density of bradykinin B2 receptors in cultured human bronchial smooth muscle cells. Our experiments demonstrate that the rapid prostaglandin E2 (PGE2) synthesis induced by IL-1β is abolished by cycloheximide, suggesting the involvement of protein synthesis. The formation of PGE2 is preceded by the phosphorylation of cPLA2 and the expression of cyclooxygenase-2 (Cox-2). The inhibition of p38 MAP kinase inhibited PGE2 synthesis, cPLA2 phosphorylation and abolished Cox-2 expression. The inhibition of Cox-2 expression correlated with a decrease of bradykinin B2 receptor expression. These data demonstrate that the activation of p38 MAP kinase elicited by IL-1β leads to the phosphorylation of cPLA2 and Cox-2 overexpression, allowing rapid synthesis of PGE2 as a prerequisite for bradykinin B2 gene expression in human bronchial smooth muscle cells which could explain the hyperresponsiveness of asthmatic patients to bradykinin.

Glucocorticoids inhibit the bradykinin B2 receptor increase induced by interleukin-1β in human bronchial smooth muscle cells

We studied the effect of the glucocorticoids, dexamethasone and budesonide, on the interleukin-1beta-induced increase of bradykinin B2 receptors in cultured human bronchial smooth muscle cells, a cellular model of bronchial hyperreactivity. Both compounds prevented the increase of the bradykinin B2 mRNA and the bradykinin-induced inositol phosphate accumulation. These results demonstrate a direct effect of glucocorticoids on airway smooth muscle hyperresponsiveness mediated through inhibition of the over-expression of receptors for contractile mediators induced by inflammatory mediators.

Evidence for in vitro expression of B1 receptor in the mouse trachea and urinary bladder

Motor responses to des‐Arg9‐bradykinin and bradykinin were studied in the isolated mouse trachea (precontracted with carbachol, 10 μM) and the urinary bladder of either Swiss, C57Bl/6J or bradykinin B2 receptor knockout (Bk2r−/−) mice after 1–6 h in vitro. The expression of mRNA for the mouse B1 receptor in tracheal and urinary bladder tissues was also studied by using Northern blot analysis. In isolated tracheae, des‐Arg9‐bradykinin produced a relaxant response that increased over time: no response was observed after 1 h of incubation, whereas after 6 h the maximum response (1 μM) was 68–84% of the relaxation produced by isoproterenol (1 μM) in the three mouse strains. The relaxant response to bradykinin (1 μM) observed at 1 h (38–51% of isoproterenol) was increased (62–65% of isoproterenol) after 6 h in Swiss and C57Bl/6J mice, but was absent in Bk2r−/− mice. In the presence of cycloheximide, des‐Arg9‐bradykinin did not cause any response at 6 h. Similar findings were obtained in the urinary bladder: at 1 h des‐Arg9‐bradykinin (1 μM) did not cause any motor effect, whereas at 6 h it caused a contraction that was 28–59% of that produced by carbachol (1 μM) in the three mouse strains. Cycloheximide blocked the response to des‐Arg9‐bradykinin. Bradykinin (1 μM) contracted urinary bladders at 1 h (34–35% of carbachol), as well as at 6 h (66–77% of carbachol) in Swiss and C57Bl/6J strains, but was without effect in Bk2r−/− mice. Northern blot hybridization with a specific cDNA probe against mouse B1 receptor mRNA using total RNA extracted from tracheae and urinary bladders freshly removed from Swiss and Bk2r−/− mice revealed minimal expression. However, marked hybridization was detected 150 min after in vitro exposure in both tissues. Evidence is provided that in vitro exposure of mouse trachea and urinary bladder causes a time‐dependent induction of B1 receptors that cause relaxation and contraction, respectively.

Glucocorticoids increase bradykinin B2 receptor gene transcription in cultured guinea-pig tracheal smooth muscle cells.

We investigated the effect of the glucocorticoid methylprednisolone on the modulation of the expression of the bradykinin B2 receptors in cultured, guinea-pig, tracheal, smooth muscle cells. These receptors are implicated in the pathogenesis of human asthma. Untreated cells expressed a single population of binding sites for [3H]bradykinin with a dissociation constant, Kd, of 87.7±12.0 pM and a maximum binding site density, Bmax, of 245.4±71 fmol/mg protein. Treatment of the cultured guinea-pig tracheal smooth muscle cells with methylprednisolone 10–5 M for 6 h increased the number of bradykinin receptors; this response reached a maximum of 78% and returned to the basal value after 12 h. Bradykinin (10–12 M) elicited a sixfold higher calcium level in treated cells than in control cells. To investigate bradykinin B2 receptor mRNA expression in guinea-pig cells, we used the reverse transcription polymerase chain reaction (RT-PCR) technique to synthesize a specific bradykinin B2 cDNA probe of 296 bp corresponding to nucleotides 456–751 of the human sequence. This guinea-pig cDNA had 88%, 86% and 83% homology with the corresponding human, mouse and rat sequences, respectively, but no homology with any other known sequences. Following methylprednisolone treatment, Northern blot hybridization indicated that mRNA increased fourfold after 3 h compared with control cells, and returned to basal level within 7 h. The rate of gene transcription, assessed by nuclear run-on assays, increased fourfold after 3 h treatment with 10–5 M methylprednisolone. These results indicate that glucocorticoids induce early up-regulation of bradykinin B2 receptors in cultured guinea-pig tracheal smooth muscle cells by increasing the rate of transcription of the bradykinin B2 receptor gene.

Bradykinin B2 receptors mediate contraction in the normal and inflamed human gallbladder in vitro

BACKGROUND & AIMS The components of the kinin system, including kinongens, kininogenases, and B(2) and B(1) receptors, are expressed and activated during inflammation. Here, we investigated the expression of the kinin B(2) receptor messenger RNA, kininogen and kallikrein immunoreactivity, and the ability of kinins to contract control and inflamed gallbladders in vitro. METHODS Human gallbladders, obtained from patients undergoing cholecystectomy either for acute cholecystitis secondary to gallstone disease or during elective gastro-entero-pancreatic surgery (controls), were processed for reverse-transcription polymerase chain reaction analysis, kallikrein and kininogen immunohistochemistry, binding studies, and in vitro contractility studies. RESULTS Tissue expression of B(2) receptor messenger RNA and specific binding of [(3)H]-bradykinin increased significantly in acute cholecystitis compared to controls. Kallikrein immunoreactivity was detected in the epithelium and infiltrating leukocytes, whereas kininogen immunoreactivity in the lumen of blood vessels and interstitial space. Bradykinin contracted isolated strips of control and acute cholecystitis gallbladders. In acute cholecystitis tissue, efficacy of bradykinin was higher than that of control gallbladders and similar to that of cholecystokinin. The contraction induced by bradykinin was significantly attenuated by B(2) receptor antagonism but not by cyclooxygenase inhibition and B(1), muscarinic, or tachykinin receptor antagonism. CONCLUSIONS All the components of the kinin system are expressed in the human gallbladder. Bradykinin is a powerful spasmogen via B(2) receptor activation in the normal and, especially, in the inflamed human gallbladder.