Studies by several laboratories over the last ten years strongly indicate that eosinophils play a central role in the pathophysiology of asthma and other allergic diseases. These cells appear to be selectively recruited to the sites of allergic reactions and them produce mediators which account for many of the histopathologic features of these reactions. The overall goal of this proposal is to advance our understanding of the mechanism of eosinophil recruitment during allergic inflammation by investigating the role of the eosinophil-recruiting chemokine RANTES in different in vivo models of allergen-induced inflammation and the study the effects of glucocorticoids on RANTES production in vitro and in vivo.
Specific aims : 1. To assess whether RANTES is produced in vivo in allergic subjects following experimental antigen challenge in the skin or the lungs. a) Total RNA will be extracted from skin tissue collected up to 24hr after antigen challenge, or from biopsy samples and bronchoalveolar lavage cells derived from the lungs 19 hr after antigen challenge of allergic subjects to determine the presence of mRNA for RANTES. RANTES mRNA will be detected using an RNase protection assay or by semiquantitative polymerase chain reaction (PCR) if required. RANTES generation in vivo will be confirmed by a specific ELISA assay performed on BAL fluids and extracts of skin or lung tissue. A comparison of antigen challenge of non-allergic or control, saline-challenged allergic individuals will be performed. 2. To test the hypothesis that glucocorticoids inhibit RANTES production in vivo and in vitro. For in vivo studies, pretreatment with glucocorticoids will be tested for its effect on RANTES generation in the in vivo models previously described. In the in vitro experiments, cell types known to generate RANTES both constitutively and following activation (including THP-1, rhabdomyosarcoma cells, human synovial fibroblasts and lectin-stimulated human lymphocytes) will be exposed to a glucocorticoid for up to 24 hr prior to stimulation and RANTES mRNA, as well as the mature protein, will be assayed. The results of the experiments described in this proposal will hopefully help advance our knowledge of the pathophysiologic mechanisms of allergic inflammation and of the anti-inflammatory actions of glucocorticoids.
