Background and PurposeAsthma exacerbations contribute to corticosteroid insensitivity. LPS is ubiquitous in the environment. It causes bronchoconstriction and airway inflammation and may therefore exacerbate allergen responses. This study examined whether LPS and ovalbumin co‐administration could exacerbate the airway inflammatory and functional responses to ovalbumin in conscious guinea pigs and whether these exacerbated responses were insensitive to inhaled corticosteroid treatment with fluticasone propionate (FP).Experimental ApproachGuinea pigs were sensitized and challenged with ovalbumin and airway function recorded as specific airway conductance by whole body plethysmography. Airway inflammation was measured from lung histology and bronchoalveolar lavage. Airway hyper‐reactivity (AHR) to inhaled histamine was examined 24 h after ovalbumin. LPS was inhaled alone or 24 or 48 h before ovalbumin and combined with ovalbumin. FP (0.05–1 mg·mL−1) or vehicle was nebulized for 15 min twice daily for 6 days before ovalbumin or LPS exposure.Key ResultsOvalbumin inhalation caused early (EAR) and late asthmatic response (LAR), airway hyper‐reactivity to histamine and influx of inflammatory cells into the lungs. LPS 48 h before and co‐administered with ovalbumin exacerbated the response with increased length of the EAR, prolonged response to histamine and elevated inflammatory cells. FP 0.5 and 1 mg·mL−1 reduced the LAR, AHR and cell influx with ovalbumin alone, but was ineffective when guinea pigs were exposed to LPS before and with ovalbumin.Conclusions and ImplicationsLPS exposure exacerbates airway inflammatory and functional responses to allergen inhalation and decreases corticosteroid sensitivity. Its widespread presence in the environment could contribute to asthma exacerbations and corticosteroid insensitivity in humans.

Methods BALB/c mice or human ICAM-1 transgenic mice (BALB/c background) were used in all assays. Animals were intranasally inoculated with either human rhinovirus 1B (HRV1B), human rhinovirus 16 (HRV16) or influenza A/Victoria/3/75 (H3N2). The kinetics of viral replication in naive animals and the lung inflammatory profiles in bronchoalveolar lavage (BAL) were assessed for up to seven days after single inoculations. The effects of HRV1B and H3N2 inoculation were further assessed in a model of chronic lung inflammation: animals were intranasally challenged with house dust mite extract (HDM) for up to seven weeks. Virus challenges were given after a robust HDM allergic phenotype developed. Some groups of animals were also treated with fluticasone propionate during and after the viral inoculation. After cessation of the challenge period, airway hyperresponsiveness (AHR) was assessed using whole body plethysmography, followed by BAL, serum and lung tissue analysis. In addition, the arterial oxygen saturation (PO2) of H3N2 treated animals was tested using pulse oximetry. Results In vitro cytopathic effect assays did not demonstrate that either HRV1B or HRV16 were able to replicate in lungs of relevant mice. After viral inoculation, a variable inflammatory response was detectable only in HRV1B treated animals up to 24 hours post inoculation. Animals treated with H3N2 developed a mixed inflammatory lung inflammation and a PO2 profile indicative of a viral effect on lung functionality. H3N2 also replicated well in the mouse. Single inoculations of either HRV1B or H3N2 were given to animals already challenged with HDM. There was no indication that addition of HRV1B to existing phenotype altered the HDM induced AHR or inflammatory profile. Combination H3N2/ HDM treatment resulted in an increase in BAL eosinophils and neutrophils, but no change to steroid responsiveness nor changes in PO2 readouts.

House dust mite (HDM) is the major source of allergen in house dust and is strongly associated with the development of asthma. HDM can evoke a direct, nonallergic inflammatory reaction in vitro. We aimed to determine whether this apparent nonallergic, inflammatory response can be observed in a more complex in vivo setting. Vehicle, Alum or HDM (Dermatophagoides pteronyssinus 5 microg, i.p. with Alum) sensitised Brown-Norway rats were challenged intratracheally with vehicle (saline), HDM (Der p 10 microg) or heat-inactivated HDM on day 21. Lung function changes and the associated inflammatory response were evaluated. Tissue and bronchoalveolar lavage from Alum sensitised Der p challenged animals exhibited strong eosinophilia and neutrophilia associated with an early release of pro-inflammatory cytokines (interleukin-13 and 1beta, eotaxin and thymus and activation-regulated chemokine). This response was not attenuated by removal of HDM-associated protease activity. Interestingly, the vehicle sensitised group (no Alum) lacked this inflammatory response. HDM allergen evokes nonallergic airways inflammation with an inflammatory profile similar to that of the asthmatic airway. This response, independent of the protease activity of the HDM extract, appeared to be linked to prior administration of the adjuvant Alum and the subsequent increase in total immunoglobulin E. This finding could have important implications in the development of future asthma therapies.

Objective and Design: The aim was to determine the time courses for the changes in airway function, airway reactivity, influx of inflammatory cells and levels of the proinflammatory cytokines, interleukin (IL)-5 and IL-8 in bronchoalveolar lavage fluid (BALF), and the plasma levels of cortisol and ACTH after antigen challenge to determine whether a temporal link could be established between these events.Methods: Airway function was measured as specific airway conductance (sG(sw)) in conscious ovalbumin (OvA)-sensitized guinea pigs using whole body plethysmography at intervals after an inhalation challenge with ovalbumin (0.5% for 10 min). Airway responses to the inhaled spasmogen, U46619 (30 ng/ml, 60s), were measured at 3, 6 and 24h after challenge, In separate animals, bronchoalveolar lavage fluid (BALF) was obtained after anaesthetic overdose either before challenge or at 1, 3, 6, 12, or 24h after OvA challenge. Total and differential cell counts of eosinophils and neutrophils were performed on BALF and levels of IL-5 and IL-8 determined by scintillation proximity assays and ELISA, respectively. Plasma cortisol and ACTH levels were determined by RIA kits in blood removed by cardiac puncture at intervals after challenge.Results: An early phase bronchoconstriction occurred which resolved by 3 h and was followed by a late phase between 17 and 24h, Airway hyperresponsiveness to inhaled U46619, was evident at 3, 6 and 24h after antigen challenge. Increased IL-5([BALF]) Was observed by 60 min post challenge implicating a preformed storage site. In contrast, IL-8([BALF]) was not raised until 3 h post challenge. There was a significant infiltration of neutrophils and eosinophils by 3 and 6 h, respectively. IL-5([BALF]) further increased up to 24 h, during the appearance of the late phase of bronchoconstriction and whilst eosinophilia was maximal. Plasma cortisol levels were increased 1 and 3 hours after antigen challenge, thereafter returning to baseline levels.Conclusions: The hyperresponsiveness appears to be dissociated from the appearance of eosinophils in lavage fluid. The early appearance of IL-5, however, could be a trigger for the migration of eosinophils and development of hyperresponsiveness. The increased plasma cortisol levels occurring after antigen challenge were presumably due to the stress involved and these would be expected to exert an endogenous anti-inflammatory effect.

The pig has previously been shown to be a suitable species in which to investigate allergic inflammation of the airways (Fornham et a1 1995). In this study, we have investigated an acute inflammatory response in the lung of the pig following inhalation of aerosolised lipopolysaccharide (LPS). In addition, the effects of fluticasone propionate (FP) administered directly into the lungs as a dry powder was investigated on the LPS-induced inflammatory response. Pigs of either sex, w e i a n g 25-35kg, were anaesthetised, ventilated and surgically prepared to allow measurement of blood pressure and heart rate via a carotid artery, and tracheal inflation pressure. Following an equilibration period of approximately 30 minutes, an aerosol of saline or LPS (lO-lOOOkg/nll) was administered for 10 minutes via a tracheal cannula. Two to six hours later, the lungs were lavaged and the cellular content of the bronchoalveolar lavage fluid (BALF) was determined. Inhaled LPS caused a doseand timedependent lung neutrophilia in the anaesthetised pig (tables 1 and2).

This article describes the development and validation of a scintillation proximity assay (SPA) sensitive for guinea-pig interleukin-5 (IL-5). SPA beads were coated with TRFK-5, a monoclonal antibody directed against mouse IL-5, which is known also to bind guinea-pig IL-5. The assay is a simple competitive binding assay between [125I]-rh-IL-5 and the IL-5, in a sample of guinea-pig bronchoalveolar lavage fluid (BALF), for the binding site on the TRFK-5-coated beads. IL-5 levels in BALF ([IL-5]BALF) were shown to increase in guinea-pigs sensitized to ovalbumin (OvA) and challenged with an OvA inhalation. This occurred at a time (24 h) after challenge when there was also a marked eosinophilia. The assay was validated by treating guinea-pigs with a second antibody, Genzyme 2374-01, directed against IL-5. Treatment with this antibody resulted in a significant reduction of the antigen-induced eosinophilia and concentration of [IL-5]BALF. This observation confirms that the IL-5 identified in BALF also cross-reacts with the antibody Genzyme 2374-01. Interestingly, plasma from sensitized, but unchallenged, guinea-pigs also contained detectable levels of IL-5, and the stimulation of plasma protein extravasation (PPE) within the airways with inhaled histamine also induced a rise in [IL-5]BALF. These observations suggest that the plasma may be an additional source of the IL-5 present in the airways of antigen-challenged guinea-pigs.

Ovalbumin (OvA) inhalation by sensitized guinea-pigs caused a pronounced rise in interleukin (IL)-5 in bronchoalveolar lavage (BAL) fluid at both 3 and 24 h after antigen exposure. The increased levels at 24 h were attenuated by the phosphodiesterase inhibitors Ro 20-1724 and aminophylline and by dexamethasone, all of which also attenuated the concurrent lung eosinophilia. The rise in IL-5 at 3 h was additionally attenuated by the PDE3 inhibitor, siguazodan, which failed to attenuate the eosinophilia at 24 h. These results suggest a pivotal action of these compounds on the later rise in IL-5. Ro 20-1724, aminophylline, siguazodan and dexamethasone attenuated a rise in IL-8 levels in BAL fluid at 3 h and the subsequent neutrophilia at 24 h. There was no increase in plasma ACTH at 3 and 24 h after OvA challenge but cortisol levels were elevated at 3 h. This was inhibited by Ro 20-1724, siguazodan and dexamethasone. Thus, elevation of plasma cortisol does not explain the anti-inflammatory actions of these compounds. Aminophylline, however, did raise plasma cortisol at both 3 and 24 h after antigen challenge which may be an important further mechanism of action for this compound.