Claude Bertrand

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.

Activation of the Fas receptor on lung eosinophils leads to apoptosis and the resolution of eosinophilic inflammation of the airways.

While considerable progress has been made in understanding the events by which eosinophils accumulate in various pathophysiological conditions, the mechanisms controlling the resolution of eosinophilic inflammation are poorly understood. In the present study, we demonstrate that lung eosinophils obtained by bronchoalveolar lavage (BAL) after aerosol allergen provocation of immunized mice expressed the Fas receptor. Stimulation of purified eosinophils in vitro with a monoclonal anti-Fas mAb (1 ng-1 microg/ml) induced a dose/time dependent loss of cell viability from 24-72 h. Measurement of DNA fragmentation with propidium iodide confirmed that anti-Fas induced eosinophil death by apoptosis. While incubation with IL-3, IL-5, or GM-CSF prevented spontaneous apoptosis, these factors failed to prevent anti-Fas induced apoptosis. Administration of anti-Fas mAb to the lungs after the induction of a lung eosinophilia increased the number of peroxidase positive macrophages in BAL fluid 4-12 h later which was followed by a marked reduction in the number of eosinophils in the airways. Importantly, Fas-mediated resolution of eosinophilic inflammation occurred in the absence of any overt secondary inflammatory changes in the lungs. We speculate that defects in this pathway may at least in part explain the chronic eosinophilic inflammation often observed in the lungs of asthmatic individuals.

Inducible Nitric Oxide Synthase Inhibitors Suppress Airway Inflammation in Mice Through Down-Regulation of Chemokine Expression

Growing evidence demonstrates that inducible NO synthase (iNOS) is induced in the airways of asthmatic patients. However, the precise role of NO in the lung inflammation is unknown. This study investigated the effect of both selective and nonselective iNOS inhibitors in an allergen-driven murine lung inflammation model. OVA challenge resulted in an accumulation of eosinophils and neutrophils in the airways. Expression of iNOS immunostaining in lung sections together with an increase in calcium-independent NOS activity in lung homogenates was also observed after OVA challenge. Treatment with iNOS inhibitors from the day of challenge to the day of sacrifice resulted in an inhibition of the inflammatory cell influx together with a down-regulation of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 production. In contrast, eosinophilic and neutrophilic inhibition was not observed with treatment during the sensitization. Both treatments induced an increased production of Th2-type cytokines (IL-4 and IL-5) with a concomitant decrease in production of Th1-type cytokine (IFN-γ). In vitro exposure of primary cultures of murine lung fibroblasts to a NO donor, hydroxylamine, induced a dose-dependent release of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1. Our results suggest that lung inflammation after allergen challenge in mice is partially dependent on NO produced mainly by iNOS. NO appears to increase lung chemokine expression and, thereby, to facilitate influx of inflammatory cells into the airways.

Interactive involvement of brain derived neurotrophic factor, nerve growth factor, and calcitonin gene related peptide in colonic hypersensitivity in the rat

Background and aims: Neutrophins are involved in somatic and visceral hypersensitivity. The action of nerve growth factor (NGF) on sensory neurones contributes to the development of referred colonic hypersensitivity induced by trinitrobenzene sulfonic acid (TNBS). Based on data on brain derived neurotrophic factor (BDNF) and calcitonin gene related peptide (CGRP) in pain, the aims of the present study were: (1) to investigate the involvement of BDNF and CGRP in this model of referred colonic hypersensitivity, (2) to test the effect of exogenous BDNF and CGRP on the colonic pain threshold, and (3) to investigate the relationship between BDNF, NGF, and CGRP by testing antineurotrophin antibodies or h-CGRP 8–37 (a CGRP antagonist) on bowel hypersensitivity induced by these peptides. Methods: Colonic sensitivity was assessed using a colonic distension procedure. Results: Anti-BDNF antibody and h-CGRP 8–37 reversed the induced decrease in colonic threshold (33.4 (2.1) and 40.3 (4.1) mm Hg, respectively, compared with a vehicle score of approximately 18 mm Hg; p<0.001). BDNF (1–100 ng/rat intraperitoneally) induced a significant dose dependent decrease in colonic reaction threshold in healthy rats. This effect was reversed by an anti-BDNF antibody and an anti-NGF antibody (33.4 (0.6) v 18.7 (0.7) mm Hg (p<0.001), anti-NGF v vehicle). NGF induced colonic hypersensitivity was reversed by h-CGRP 8–37 but not by the anti-BDNF antibody. Finally, antineurotrophin antibody could not reverse CGRP induced colonic hypersensitivity (at a dose of 1 µg/kg intraperitoneally). Conclusion: Systemic BDNF, NGF, and CGRP can induce visceral hypersensitivity alone and interactively. This cascade might be involved in TNBS induced referred colonic hypersensitivity in which each of these peptides is involved.

Tachykinin and kinin receptor antagonists: therapeutic perspectives in allergic airway disease

The morbidity of allergic airway disease and the number of deaths resulting from it have not declined in the past ten years. The multiplicity of mediators released in the acute allergic reaction and our limited knowledge of the basic mechanisms that drive chronic inflammation have hampered the design of effective therapeutic regimens for this type of disease. In this article, Claude Bertrand and Pierangelo Geppetti summarize recent studies in which new, potent and selective tachykinin and kinin receptor antagonists demonstrate the involvement of tachykinins and kinins in airway anaphylaxis, and review how these antagonists might be of use in treating allergic asthma and rhinitis.

IL‐5 deficiency abolishes aspects of airway remodelling in a murine model of lung inflammation

Background and objectives Lung remodelling is a recognized feature of chronic asthma. In the present study, we have used IL‐5‐deficient mice to evaluate the role of this cytokine and eosinophilic inflammation in the initial stages of the structural changes occurring in the lung after antigen challenge.

Role of kinins in anaphylactic-induced bronchoconstriction mediated by tachykinins in guinea-pigs.

1 In the present study, we have investigated the role of kinins in allergen‐induced bronchoconstriction. 2 Anaesthetized guinea‐pigs were sensitized to ovalbumin, ventilated artificially, pretreated with atropine (1.4 μmol kg−1, i.v.) and total pulmonary resistance (RL) measured. In preliminary studies in the presence of the neutral endopeptidase inhibitor, phosphoramidon (4.5 μmol kg−1, i.v.), the bradykinin B2 receptor antagonist Hoe 140 (0.1 μmol kg−1, i.v.) completely abolished the increase in RL following aerosolized bradykinin (1 mm, 40 breaths), but had no effect on the increase in RL following aerosolized neurokinin A (NKA, 10μm, 40 breaths). On the other hand, a combination of the NK1 (CP‐96,345, 2 μmol kg‐1, i.v.) and NK2 (SR 48968, 0.3 μmol kg−1, i.v.) tachykinin receptor antagonists abolished completely the increase in RL produced by NKA and partially inhibited the increase in RL produced by bradykinin. These results confirm previous studies that suggest that bradykinin induces the release of tachykinins from sensory nerves in guinea‐pig airways. 3 Aerosolized ovalbumin (0.5%, 5 breaths) increased RL in sensitized guinea‐pigs pretreated with atropine (1.4mmol kg−1, i.v.), an effect that began within 2 min and reached a maximum within 5 min; RL remained above baseline at 20 min. Pretreatment with the bradykinin B2 receptor antagonist, Hoe 140, decreased the bronchoconstrictor effect of ovalbumin markedly at 10 to 20 min. In the presence of phosphoramidon (4.5 μmol kg−1, i.v.) the inhibition induced by Hoe 140 was apparent earlier and remained over the 20 min period of study. 4 Pretreatment with a combination of NK1 (CP‐96,345) and NK2 (SR 48968) tachykinin receptor antagonists also markedly inhibited ovalbumin‐induced bronchoconstriction; addition of the bradykinin B2 receptor antagonist to the NK1 and NK2 tachykinin receptor antagonists had no additional inhibitory effect on antigen‐induced bronchoconstriction. 5 These findings confirm that activation of sensory nerves to release tachykinins in guinea‐pig airways contribute to antigen‐induced bronchoconstriction, and provide evidence that tachykinin release is due to kinins generated during the allergic response.

Evidence that tachykinins relax the guinea-pig trachea via nitric oxide release and by stimulation of a septide-insensitive NK1 receptor

1 This study investigated the possibility that tachykinins relax the guinea‐pig isolated trachea by releasing nitric oxide (NO) from the epithelium. The types of tachykinin receptor mediating both relaxation and contraction of the trachea were also studied. Isometric tension was recorded in isolated tracheal tube preparations precontracted with acetylcholine (10 μm) in which compounds were administered intraluminally in the presence of phosphoramidon and indomethacin (both 1 μm) and the tachykinin NK2 receptor antagonist, SR 48,968 ((S)‐N‐methyl‐N[4‐(4‐acetyl amino‐4‐phenylpiperidino)‐2‐(3,4‐dichlorophenyl)butyl]benzamide), 0.1 μm). 2 In the presence of the inactive enantiomer of an NO‐synthase inhibitor, NG‐monomethyl‐D‐arginine (D‐NMMA, 100 μm), substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and the selective NK1 receptor agonist, [Sar9, Met(O2)11]‐SP, (0.1–10 nM) relaxed tracheal tube preparations. This relaxation was changed into a contraction by pretreatment with the NO‐synthase inhibitor, NG‐monomethyl‐L‐arginine (L‐NMMA, 100 μm). The effect of L‐NMMA on SP‐ and [Sar9, Met(02)11]‐SP‐induced responses was reversed by L‐arginine (L‐Arg, 1 mM), but not by D‐Arg (1 mM). After removal of the epithelium SP, NKA and NKB and [Sar9, Met(O2)11]‐SP (0.1–10 nM) evoked contractile responses in the presence of either L‐NMMA (100 μm) or D‐NMMA (100 μm). The effects of SP and [Sar9, Met(O2)11]‐SP obtained in the presence of another NO‐synthase inhibitor, NG‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μm) or its inactive enantiomer, NG‐nitro‐D‐arginine methyl ester (D‐NAME, 100 μm) were similar to those observed with L‐NMMA or D‐NMMA, respectively. 3 The selective NK1 receptor agonist, [pGlu6, Pro9]‐SP(6–11) (septide, 0.1–10 nM) evoked contractile responses of tracheal tube preparations in the presence of either d‐NMMA (100 μm) or L‐NMMA (100 μm). The log concentration‐response curve to septide obtained in the presence of L‐NMMA was similar to that obtained in the presence of d‐NMMA. [Sar9, Met(O2)11]‐SP (0.1–10 nM) relaxed tracheal tube preparations precontracted with septide (1 μm), whereas septide (0.1 nM‐1 μm) further contracted tracheal tube preparations precontracted with [Sar9, Met(O2)11]‐SP (1 μm). 4 Relaxant and contractile responses evoked by SP, NKA, NKB and by [Sar9, Met(O2)11]‐SP (0.1–10 nM) were not affected by a combination of the histamine H1 (pyrilamine, 1 μm) and H2 (cimetidine, 1 μm) receptor antagonists, but were abolished by the tachykinin NK1 receptor antagonist, CP‐99,994 ((2S,3S)‐3‐(2‐methoxybenzylamino)‐2‐phenylpiperidine, 1 μm), though not by its inactive enantiomer CP‐100,263 (1 μm). Contractile responses evoked by septide (10 nM and 1 μm) were also abolished by CP‐99,994 (1 μm) but not by CP‐100,263 (1 &M). 5 These results demonstrate that tachykinins relax guinea‐pig tracheal tube preparations by releasing NO via the stimulation of epithelial NK1 receptors by a mechanism independent of histamine release. The NK2 receptor type involved is sensitive to SP, NKA, NKB and [Sar9, Met(O2)11]‐SP but not to septide, and is pharmacologically distinct from the NK1 receptor that mediates contraction, which is stimulated by all the agonists, including septide.

NK1 receptor antagonist CP-99,994 inhibits cigarette smoke-induced neutrophil and eosinophil adhesion in rat tracheal venules

The involvement of neurokinin NK1 receptors in cigarette-induced adhesion of neutrophils and eosinophils to venules of the airway mucosa was investigated. Rats were pretreated with the NK1 receptor antagonist CP-99,994 (4 mg/kg IV), its vehicle, or its inactive enantiomer CP-100,263 before exposure to cigarette smoke. Adherent neutrophils and eosinophils were stained histochemically for endogenous peroxidase activity and were counted in tracheal whole mounts. Plasma leakage was quantified be stereological measurements of the extravasation of Monastral blue. Cigarette smoke induced the adhesion of 104 + 17 neutrophils and 10.4 +/- 1.7 eosinophils per square millimeter of mucosa. CP-99,994 reduced neutrophil and eosinophil adhesion by 66 and 61%, respectively, and reduced plasma extravasation by 61% (p < .05), but CP-100,263 had no significant effect. The inhibitory effects of CP-99,994 appeared to be specific because CP-99,994 had no effect on neutrophil and eosinophil adhesion, or on plasma extravasation induced by platelet activating factor, an inflammatory stimulus acting independently of NK1 receptors. These results suggest that NK1 receptors are involved in cigarette smoke-induced adhesion of neutrophils and eosinophils to the endothelium of venules in the rat tracheal mucosa.

Demonstration of the therapeutic potential of non-anaphylactogenic anti-IgE antibodies in murine models of skin reaction, lung function and inflammation

BACKGROUND Allergies and allergic asthma are believed to be mediated by allergen-specific IgE antibodies. We have investigated the therapeutic potential of inhibiting endogenous IgE by a non-anaphylactogenic anti-mouse IgE antibody 1-5 with respect to its effects on antigen-induced skin reaction, lung function changes and lung inflammation in mice. METHODS Mice were immunized with benzylpenicillinoyl-KLH or ovalbumin, and antigen-mediated skin reaction, bronchoconstriction, bronchopulmonary hyperresponsiveness (BHR) and lung eosinophilic inflammation determined in anti-IgE 1-5-treated versus untreated animals. RESULTS Application of anti-IgE 1-5 inhibited (by 90%) the serum IgE and, 3-4 days after onset of treatment, blocked the antigen-induced skin reaction. Furthermore, the antibody also inhibited (by 90%) the antigen-induced infiltration of eosinophils into the lung. This latter effect seems to be mediated by blocking the IgE-CD23 interaction and indicates that lung eosinophilic inflammation also depends on IgE. Moreover, when applied to rats passively sensitized with mouse IgE, antibody 1-5 inhibited the antigen-induced bronchoconstriction. A similar effect could be seen in actively immunized mice, where antibody 1-5 was able to inhibit (by 70%) the ovalbumin-induced bronchoconstriction as well as BHR. CONCLUSIONS In summary, non-anaphylactogenic anti-IgE antibodies can markedly inhibit IgE levels and IgE-mediated allergic reactions. Since bronchoconstriction, BHR and lung eosinophilic inflammation can be suppressed, such antibodies may be attractive principles for the treatment of allergic asthma.