Suppression of airway hyperresponsiveness induced by ovalbumin sensitisation and RSV infection with Y-27632, a Rho kinase inhibitor.

BACKGROUND: Smooth muscle contraction is one of the hallmarks of asthma. A recently developed pyridine derivative, Y-27632, a selective Rho kinase inhibitor, has been reported to inhibit the smooth muscle contraction of human and animal trachea in ex vivo systems but its effect in animal models of airway hyperresponsiveness (AHR) has not been examined. The purpose of this study was to evaluate the effect of Y-27632 in a murine model of allergic and virally induced AHR. METHODS: Baseline lung resistance and methacholine induced AHR were measured in mice sensitised to ovalbumin (OVA) and also in mice infected with respiratory syncytial virus (RSV) following ovalbumin sensitisation (OVA/RSV). RESULTS: Time course and dose ranging experiments indicated that 30 mg/kg Y-27632 given by gavage 2 hours before methacholine challenge significantly reduced baseline lung resistance and prevented AHR in OVA sensitised mice. Y-27632 also suppressed AHR induced by the bronchospastic agent serotonin in OVA sensitised mice and prevented methacholine induced AHR in OVA/RSV mice. CONCLUSIONS: These results suggest that the signalling pathway mediated through Rho kinase may have an important role in bronchial smooth muscle tone in allergen induced and virus induced AHR and should be considered as a novel target for asthma treatment.

Immune interaction between respiratory syncytial virus infection and allergen sensitization critically depends on timing of challenges.

Severe respiratory syncytial virus (RSV) infection has been hypothesized to be a risk factor for the development of allergy and asthma, but epidemiologic studies in humans have been inconclusive. By use of a well-characterized murine model of RSV infection and allergic sensitization with ovalbumin, the effect of a preceding severe RSV infection on the development of the pulmonary allergic inflammatory response and airway hyperresponsiveness (AHR) was tested. The impact of prior allergic sensitization on RSV-induced illness, as measured by weight loss, also was evaluated. RSV infection before allergic sensitization decreased allergen-induced AHR, production of interleukin-13 in lung tissue, and lung eosinophilia. In contrast, allergic sensitization before RSV infection increased AHR and decreased RSV-related weight loss and lung levels of interferon-gamma but did not alter viral clearance. These data provide evidence that RSV-associated AHR occurs in hosts with allergic responses and that allergic inflammation is diminished when preceded by RSV infection.

Respiratory syncytial virus (RSV)-induced airway hyperresponsiveness in allergically sensitized mice is inhibited by live RSV and exacerbated by formalin-inactivated RSV.

Respiratory syncytial virus (RSV)-induced disease is associated with recurrent episodes of wheezing in children, and an effective vaccine currently is not available. The use of 2 immunizations (a formalin-inactivated, alum-precipitated RSV vaccine [FI-RSV] given intramuscularly and live RSV given intranasally [LVIN]), with a control immunization, were compared in a well-characterized model of RSV challenge, with or without concomitant allergic sensitization with ovalbumin. FI-RSV caused a significant increase in airway hyperresponsiveness in mice after RSV infection during allergic sensitization, and this was associated with an increase in type 2 cytokine production. In contrast, immunization with LVIN did not change type 2 cytokine production and protected against RSV-induced airway hyperresponsiveness in the setting of allergic sensitization. This study suggests that immune modulation with RSV vaccination can have profound effects on RSV-induced airway disease and that prevention of airway hyperresponsiveness is an important end point in vaccine development.

Cyclooxygenase inhibition increases interleukin 5 and interleukin 13 production and airway hyperresponsiveness in allergic mice.

The immunomodulatory role of arachidonic acid metabolites in allergic sensitization is undefined. Prostaglandin E(2) (PGE(2)), a product of arachidonic acid metabolism through the cyclooxygenase pathway, has been reported to favor Type 2-like cytokine secretion profiles in murine and human CD4(+) T cells by inhibiting the production of Type 1-associated cytokines. On the basis of these in vitro data, we hypothesized that indomethacin, a nonselective cyclooxygenase inhibitor, would diminish allergen-induced production of Type 2 cytokines in mice, and protect against airway hyperresponsiveness (AHR) to methacholine. We found that ovalbumin-sensitized mice that were treated with indomethacin (OVA-indomethacin mice) had significantly greater AHR (p < 0.05) and higher levels of IL-5 (176 +/- 52 versus 66 +/- 4 pg/ml) and IL-13 (1,226 +/- 279 versus 475 +/- 65 pg/ml) in lung supernatants than mice sensitized with ovalbumin alone (OVA mice), while levels of IL-4 and serum IgE were not different. Lung mRNA expression of the C-C chemokine MCP-1 was increased in OVA-indomethacin mice, while there was no difference between the two groups in lung mRNA expression of eotaxin, MIP-1alpha, MIP-1beta, or MIP-2. Histologic examination revealed greater pulmonary interstitial eosinophilia in OVA-indomethacin mice as well. Contrary to our expectations, we conclude that in the BALB/c mouse, cyclooxygenase inhibition during allergen sensitization increases AHR, production of IL-5 and IL-13, and interstitial eosinophilia.

Exposure to hyperoxia decreases the expression of vascular endothelial growth factor and its receptors in adult rat lungs.

Exposure to high levels of inspired oxygen leads to respiratory failure and death in many animal models. Endothelial cell death is an early finding, before the onset of respiratory failure. Vascular endothelial growth factor (VEGF) is highly expressed in the lungs of adult animals. In the present study, adult Sprague-Dawley rats were exposed to >95% FiO2 for 24 or 48 hours. Northern blot analysis revealed a marked reduction in VEGF mRNA abundance by 24 hours, which decreased to less than 50% of control by 48 hours. In situ hybridization revealed that VEGF was highly expressed in distal airway epithelial cells in controls but disappeared in the oxygen-exposed animals. Immunohistochemistry and Western blot analyses demonstrated that VEGF protein was decreased at 48 hours. TUNEL staining demonstrated the presence of apoptotic cells coincident with the decline in VEGF. Abundance of VEGF receptor mRNAs (Flt-1 and KDR/Flk) decreased in the late time points of the study (48 hours), possibly secondary to the loss of endothelial cells. We speculate that VEGF functions as a survival factor in the normal adult rat lung, and its loss during hyperoxia contributes to the pathophysiology of oxygen-induced lung damage.

Vascular endothelial growth factor is expressed in ovine pulmonary vascular smooth muscle cells in vitro and regulated by hypoxia and dexamethasone.

Chronic lung disease in neonates results from both lung injury and inadequate repair processes. Little is known about the growth factors involved in lung injury and repair, but vascular endothelial growth factor (VEGF) has recently been reported in several animal models of lung injury. VEGF is an endothelial cell-specific mitogen, which is also known as vascular permeability factor because of its ability to induce vascular leak in some tissues. Chronic lung disease is complicated by increased vascular permeability, which can be improved by avoidance of hypoxia and in some cases by dexamethasone therapy. In many cells, hypoxia stimulates VEGF expression. Also, in some cases, dexamethasone blocks VEGF expression. This study examined the role of hypoxia and dexamethasone in regulating the expression of VEGF in pulmonary artery smooth muscle cells. An ovine VEGF cDNA fragment (453 bp) was cloned and found to be highly homologous to known human sequences for VEGF165. Sheep pulmonary artery smooth muscle cells were cultured and exposed to room air, hypoxia, and dexamethasone, alone or in combination for 6 h. At baseline these cells expressed VEGF mRNA at approximately 3.9 kb. The half-life of VEGF mRNA in the smooth muscle cells was 171 min, more than 3-fold longer than previous reports for epithelial cells. Exposure to hypoxia caused a 3-fold increase in mRNA abundance, primarily through transcriptional up-regulation. Dexamethasone blocked the hypoxia-induced increase in VEGF mRNA. The results demonstrate that hypoxia and dexamethasone are regulators of VEGF expression in ovine pulmonary artery smooth muscle cells. It is not known whether VEGF derived from these cells is involved in lung injury and/or normal homeostatsis.