Josephine Hjoberg

Mouse Mast Cell Protease 4 Is the Major Chymase in Murine Airways and Has a Protective Role in Allergic Airway Inflammation

It is widely established that mast cells (MCs) have a harmful role in asthma, for example by secreting various proinflammatory substances stored within their secretory granule. However, in this study, we show that one of the substances stored within MC granule, chymase, in fact has a protective role in allergic airway inflammation, indicating that MCs may possess both harmful and protective activities in connection with this type of disease. Wild-type (WT) mice and mice lacking mouse MC protease 4 (mMCP-4), a chymase that is functionally homologous to human chymase, were sensitized and challenged with OVA, followed by the assessment of airway physiology and inflammatory parameters. Our results show that the airway hyperresponsiveness was significantly higher in mMCP-4−/− as compared with WT mice. Moreover, the degree of lung tissue inflammation was markedly higher in mice lacking mMCP-4 than in WT controls. Histological analysis revealed that OVA sensitization/challenge resulted in a marked increased in the thickness of the smooth muscle cell (SMC) layer and, notably, that the degree of SMC layer thickening was more pronounced in mMCP-4−/− animals than in WT controls, thus indicating that chymase may have an effect on airway SMCs. In support of this, mMCP-4-positive MCs were located in the close vicinity of the SMC layer, mainly in the upper airways, and mMCP-4 was shown to be the major chymase expressed in these MCs. Taken together, our results indicate that chymase present in the upper airways protects against allergic airway responses, possibly by regulating SMCs.

Comparisons of effects of intravenous and inhaled methacholine on airway physiology in a murine asthma model.

Airway responses to intravenous (i.v.) and inhaled (i.h.) delivery of methacholine (MCh) in BALB/c and C57BL/6 mouse strains have been compared with and without ovalbumin (OVA)-induced airway inflammation. Bronchial reactivity to MCh was assessed in anaesthetised and tracheostomised animals by using an animal ventilator (flexiVent). We partitioned the response of the lungs into airway and parenchymal components in order to compare the contributions of the airways with those of the lung parenchyma to the pulmonary mechanical responses resulting from different routes of MCh administration. Our results indicate disparate physiological responses. Intravenous MCh delivery induced a higher maximum lung resistance than i.h. MCh in OVA-treated BALB/c mice but not in C57BL/6 mice. Inhaled MCh delivery led to a significantly larger fall in lung compliance and a greater impact on peripheral airways than i.v. MCh in both strains. In conclusion, i.v. and i.h. MCh produced disparate effects in different murine strains and variant responses in inflamed airways and healthy controls. The two methods of MCh delivery have important advantages but also certain limitations with regard to measuring airway reactivity in a murine model of allergic asthma.

Allergen-induced formation of F2-isoprostanes in a murine asthma model identifies oxidative stress in acute airway inflammation in vivo☆

F(2)-isoprostanes have been associated with various forms of oxidant stress. The levels of F(2)-isoprostanes in a murine asthma model were studied both in situ and in vivo and further investigated whether the formation of F(2)-isoprostanes was associated with increased ovalbumin (OVA)-induced airway inflammation after a 17-day (OVA-17) or a 24-day (OVA-24) protocol. Bronchial reactivity was assessed by using a ventilator (FlexiVent). OVA-treated animals had higher lung resistance and lung compliance compared to control groups (P<0.001). 8-Iso-PGF(2)(alpha) levels in bronchoalveolar lavage (BAL) and 8-iso-PGF(2)(alpha) immunoreactivity in lung tissue were analyzed. OVA-17 mice showed a 2.5-fold increased level of 8-iso-PGF(2)(alpha) in BAL compared to PBS-17 mice (P=0.023). Lung tissue from OVA-24 mice had more intense 8-iso-PGF(2)(alpha) staining compared to OVA-17 mice. This study showed an accumulation of F(2)-isoprostanes in acute airway inflammation and a markedly increased tissue damage caused by oxidative stress in an ongoing inflammation.

Hyperosmolarity reduces the relaxing potency of nitric oxide donors in guinea-pig trachea.

Non‐responders to inhaled nitric oxide treatment have been observed in various patient groups. The bronchodilatory effect of inhaled nitric oxide was attenuated when the airway lumen was rendered hyperosmolar in an in vivo study on rabbits. We used a guinea‐pig tracheal perfusion model to investigate the effects of increased osmolarity (450 mOsm, NaCl added) on the relaxing potency of the nitric oxide donors sodium nitroprusside (SNP) and (±)‐S‐nitroso‐N‐acetylpenicillamine (SNAP). Under iso‐osmolar conditions SNP relaxed the carbachol (CCh, 1 μM) contracted trachea by 83±3%. After pretreatment with intraluminal hyperosmolarity SNP relaxed the CCh‐contracted trachea by only 31±7% (P<0.05). When the trachea was contracted to the same extent under untreated and hyperosmolar conditions, the untreated trachea was completely relaxed by SNP but, after hyperosmolar pretreatment, SNP could no longer relax the trachea. SNAP relaxed the CCh contracted trachea by 27±5%. After pretreatment with intraluminal hyperosmolarity, SNAP relaxed the trachea by 11±4%, which was less than in the iso‐osmolar control (P<0.05). Extraluminal hyperosmolarity did not affect carbachol elicited contraction, and SNP administered externally during extraluminal hyperosmolarity was able to relax the trachea (P<0.05). The cell permeable guanosine 3′5′‐cyclic monophosphate analogue 8‐Br‐cGMP relaxed the CCh contracted trachea in both iso‐osmolar (P<0.05) and hyperosmolar conditions (P<0.05). The relaxant effect of nitric oxide donors on tracheal smooth muscle is markedly reduced when the airway epithelium is exposed to hyperosmolar solution.

Different effects of deep inspirations on central and peripheral airways in healthy and allergen-challenged mice

BackgroundDeep inspirations (DI) have bronchodilatory and bronchoprotective effects in healthy human subjects, but these effects appear to be absent in asthmatic lungs. We have characterized the effects of DI on lung mechanics during mechanical ventilation in healthy mice and in a murine model of acute and chronic airway inflammation.MethodsBalb/c mice were sensitized to ovalbumin (OVA) and exposed to nebulized OVA for 1 week or 12 weeks. Control mice were challenged with PBS. Mice were randomly selected to receive DI, which were given twice during the minute before assessment of lung mechanics.ResultsDI protected against bronchoconstriction of central airways in healthy mice and in mice with acute airway inflammation, but not when OVA-induced chronic inflammation was present. DI reduced lung resistance induced by methacholine from 3.8 ± 0.3 to 2.8 ± 0.1 cmH2O·s·mL-1 in healthy mice and 5.1 ± 0.3 to 3.5 ± 0.3 cmH2O·s·mL-1 in acute airway inflammation (both P < 0.001). In healthy mice, DI reduced the maximum decrease in lung compliance from 15.9 ± 1.5% to 5.6 ± 0.6% (P < 0.0001). This protective effect was even more pronounced in mice with chronic inflammation where DI attenuated maximum decrease in compliance from 44.1 ± 6.6% to 14.3 ± 1.3% (P < 0.001). DI largely prevented increased peripheral tissue damping (G) and tissue elastance (H) in both healthy (G and H both P < 0.0001) and chronic allergen-treated animals (G and H both P < 0.0001).ConclusionWe have tested a mouse model of potential value for defining mechanisms and sites of action of DI in healthy and asthmatic human subjects. Our current results point to potent protective effects of DI on peripheral parts of chronically inflamed murine lungs and that the presence of DI may blunt airway hyperreactivity.

Increased airway osmolarity inhibits the action of nitric oxide in the rabbit

Inhalation of nitric oxide (NO) is known to dilate preconstricted airways. In asthmatics, there are large variations in the effect of NO on airway tone. One explanation of these variations may be different degrees of airway wall oedema. The effect of NO inhalation on methacholine (meth)-induced airway constriction was investigated in a rabbit model. Oedema and a change in osmolarity of the airways was achieved by hypertonic saline nebulization and hyperventilation with dry gas. There was an increase in resistance to meth at a concentration of 3 mg x mL(-1), of 86+/-14 cmH2O x L(-1) x s (mean+/-SEM) after oedema formation, compared with 46+/-16 cmH2O x L(-1) x s without oedema. Inhalation of 80 parts per million (ppm) NO failed to counter the increase in resistance due to meth, 92+/-14 cmH2O x L(-1) x s after hypertonic saline nebulization. After hyperventilation of dry gas, the increase in resistance due to meth at 1 mg x mL(-1) was 27+/-11 cmH2O x L(-1) x s with 80 ppm NO and 28+/- 5 cmH2O x L(-1) x s without NO. In conclusion, the relaxant effect of nitric oxide-inhalation on the airway smooth muscle can be blocked by an increase in the osmolarity of the airway surface liquid. The mechanism of this inhibition of nitric oxide remains to be established.

Hyperosmolarity-induced relaxation and prostaglandin release in guinea pig trachea in vitro.

In this study, a tracheal perfusion apparatus was used to investigate the nature of the relaxing factor released by hyperosmolarity on the epithelial side of guinea pig trachea. NaCl induced concentration-dependent relaxation. This relaxation was not affected when the trachea was preincubated with a vasoactive intestinal peptide (VIP) receptor antagonist or with the nitric oxide synthesis inhibitor N(G)-monomethyl-L-arginine (L-NMMA). When the prostaglandin synthesis was prevented by preincubation with the phospholipase A(2)-inhibitor quinacrine, or the cyclooxygenase inhibitor indomethacin, the maximal relaxation induced by NaCl was suppressed by 50% (P<0.05). Moreover, the prostaglandin E(2) concentration was four times higher (P<0.05) in the organ bath during the relaxations, whereas the nitric oxide concentration remained unchanged. In conclusion, increased osmolarity on the airway surface leads to the release of prostaglandins, which are involved in part in the hyperosmolarity-induced relaxation of airway smooth muscle. This might be relevant for asthmatic patients since prostaglandin may modulate the bronchoconstrictive response to hyperosmolar stimuli and exercise.

Dry gas hyperpnea changes airway reactivity and ion content of rabbit tracheal wall.

Dry air hyperventilation provokes airway narrowing in asthmatics. The mechanism is thought to involve the release of inflammatory mediators in response to airway osmolarity. The response to inhaled histamine on lung mechanics and the ion content of the airway subepithelial connective tissue after dry gas hyperventilation was investigated in rabbits. Resistance increased more after histamine given after hyperventilation with dry gas compared to humid gas (P < 0.05). Compliance decreased after histamine for both dry and humid gas hyperventilation. The Na, Cl and K content was decreased in the tissue at 4 min (P < 0.001) and nearly returned to resting content at 8 min of hyperventilation with dry gas. The results suggest that dehydration occurred and was followed by oedema formation in the tracheal wall. Thus, the effects of inhaled histamine on airway resistance was amplified. The mechanism involved may be part of the normal defence of the airway to protect against desiccation during hyperventilation with dry air. This response may be exaggerated in asthmatics.

Both inhaled histamine and hypertonic saline increase airway reactivity in non-sensitised rabbits

Background: Asthmatics react with bronchoconstriction upon a variety of stimuli, i.e. exercise and hypertonic aerosol challenge. We have previously shown that hyperventilation with dry gas in a rabbit model resulted in a change of the ion content of the tracheal wall. This was followed by a hyperreactive response to histamine. Objective: We hypothesised that nebulisation with 3.6% hypertonic saline will be accompanied by a hyperreactive response to histamine in a rabbit model. Methods: Anaesthetised rabbits were given histamine after nebulisation with hypertonic saline. In addition, repeat nebulisation with hypertonic saline was given with or without histamine between these nebulisations. Results: There was a different response to histamine 10 mg·ml–1 whether hypertonic saline had been given or not (p < 0.001). Histamine nebulisation, given after hypertonic saline, caused an increase from baseline in resistance of 65 ± 12 cm H2O·litre–1·s (mean ± SEM, p < 0.001) and a decrease in compliance of 2.3 ± 0.4 ml·cm H2O–1 (p < 0.001). The corresponding values for the control animals were 10 ± 4 cm H2O·litre–1·s (n.s.) and 1.7 ± 0.2 ml·cm H2O–1 (p < 0.001). At a second nebulisation with hypertonic saline, with a histamine challenge 30 min before, the resistance increased from baseline by 35 ± 10 cm H2O·litre–1·s (p < 0.01). This was not observed when no histamine had been given between the hypertonic saline nebulisations. Conclusions: This study in rabbits shows that hypertonic solutions cause an increase in the responsiveness to histamine and that histamine causes an increase in responsiveness to hypertonic saline. This is similar to the response of asthmatics to hypertonic saline.

Effects of hyperosmolarity and airway epithelial ion transport inhibitors on sodium nitroprusside-induced relaxation of guinea pig trachea.

Increased airway surface osmolarity has been shown to abolish the airway relaxant effects of inhaled nitric oxide. We have investigated the effects of increased airway surface osmolarity on airway relaxation induced by nitric oxide. The physiological responses, obtained by a guinea pig tracheal perfusion method, were compared to the ion content of the tracheal tissues, and the effects of amiloride or furosemide were studied. Hyperosmolarity decreased the ability of sodium nitroprusside (SNP) to relax carbachol-constricted trachea. Light microscopy showed shrinkage of the epithelial cells and X-ray microanalysis showed increased epithelial ion content under conditions of intraluminal hyperosmolarity, suggesting dehydration of the epithelium. Amiloride treatment reduced the increase in epithelial ion content but had no effect on shrinkage or SNP-induced relaxation. Furosemide had no effect on the altered ion content, shrinkage, or on SNP-induced relaxation. In conclusion, neither amiloride nor furosemide can counteract the shrinkage of the airway wall induced by increased osmolarity in the lumen of guinea pig trachea in vitro, nor can they affect the reduction in relaxing response to the NO-donor SNP.