The purpose of this study was to explore whether repeated exposure to aerosolized ovalbumin (OVA) in the context of local expression of GM-CSF can initiate a Th2-driven, eosinophilic inflammation in the airways. On day Ϫ 1, Balb/c mice were infected intranasally with an adenovirus construct expressing GM-CSF (Ad/GM-CSF). From day 0 to day 9 mice were exposed daily to an OVA aerosol. Mice exposed to OVA alone did not show any evidence of airway inflammation. Mice receiving both Ad/GM-CSF and aerosolized OVA exhibited marked airway inflammation characterized by eosinophilia and goblet cell hyperplasia. Migration of eosinophils into the airway was preceded by a rise in IL-5 and IL-4. Both IL-5 and class II MHC were critically required to generate airway eosinophilia. After resolution, airway eosinophilia was reconstituted after a single OVA exposure. Flow cytometric analysis of dispersed lung cells revealed an increase in macrophages and dendritic cells expressing B7.1 and B7.2, and expansion of activated (CD69-expressing) CD4 and CD8 T cells in mice exposed to OVA and Ad/GM-CSF. Our data indicate that expression of GM-CSF in the airway compartment increases local antigen presentation capacity, and concomitantly facilitates the development of an antigen-specific, eosinophilic inflammatory response to an otherwise innocuous antigen. ( J. Clin. Invest.

This study demonstrates that an immune response against a replication-deficient adenovirus during the initial exposure to OVA inhibits the development of airways inflammation after antigen aerosol challenge.

Expression of granulocyte macrophage colony-stimulating factor (GM-CSF) in the airway allows allergic sensitization to ovalbumin (OVA) in an experimental protocol that others have shown to induce inhalation tolerance. The ensuing response is characterized by T helper (Th)2 cytokines, marked eosinophilia in the bronchoalveolar lavage fluid (BALF) and the tissue, and goblet-cell hyperplasia. These findings, which underscore the importance of the airway microenvironment in the development of immune responses to airborne antigens, prompted us to investigate whether a Type 1 polarized cytokine milieu in the airway would modulate the allergic sensitization. To this end, we concurrently expressed GM-CSF and interleukin (IL)-12 in the airway, using an adenovirus-mediated gene transfer approach. Coexpression of IL-12 did not prevent the development of an antigen-specific immune inflammatory response, but altered its phenotype. Whereas a similar total cell number was observed in the BALF, airway eosinophilia was abrogated. Histologic evaluation of the tissue corroborated the findings in the BALF and demonstrated that IL-12 coexpression prevented goblet-cell hyperplasia. Expression of IL-12 decreased IL-4 and IL-5 content in the BALF by about 80 and 95%, respectively, and IL-5 in the serum by approximately 80%. In contrast, interferon (IFN)-gamma was increased in both BALF and serum. Similarly, we observed a Th2/Th1 shift in OVA-specific cytokine production in vitro. Recall challenge with OVA in vivo after resolution of the initial inflammatory response demonstrated that the effect of IL-12 was persistent. IL-12-mediated inhibition of airway eosinophilia was mainly IFN-gamma-independent, whereas inhibition of OVA-specific IgE synthesis was IFN-gamma-dependent. Our data underscore the importance of the airway microenvironment in the elicitation of immune responses to environmental antigens.

No abstract

We have characterized the binding of Ca 2+ and Mg 2+ to the anti-inflammatory drug diflunisal in acetonitrile and demonstrated the drug-mediated transport of Ca 2+ across the lipid bilayer in unilamellar vesicles made of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Fluorescence and difference absorption spectral data show that diflunisal undergoes a significant conformational change on binding Ca 2+ and Mg 2+ forming, respectively, 1:2 and 1:1 cation:drug complexes with K~ in the low /~M range. The kinetics of transport showed that Ca 2+ was transported into the vesicle as 1:2 Ca2+:diflunisal sandwich complex . This suggests that the interaction of the drug with the cation in the lipid and the lipid-mimetic solvent are similar. The biological relevance of the Ca2+-binding and translocating abilities of diflunisal is examined in light of the reported ionophoretic properties of several phospholipids as well as cyclooxygenase and lipoxygenase products. a lipid bilayer environment may be important in the bioactivity of many classes of substances (11). Experimentally, members of such diverse classes as peptide hormones (12)(13)(14), adrenergic drugs (15) and calcium channel blockers (16)(17)(18)(19) have been shown to possess such characteristics. We have, in the present study, investigated the interaction of diflunisal with Ca 2÷ in a lipid,mimetic solvent and in aqueous suspensions of lipid bilayer vesicles. Our results, outlined below, show that diflunisal interacts strongly with both Ca 2+ (and Mg 2+) in a non-polar medium to form predominantly 1:2 cation:drug sandwich complexes. They also show that the drug is capable of translocating Ca 2+ across the lipid bilayer in the form of the sandwich complex.