The goal of this project is to better characterize the role of tumor necrosis factor (TNF) in the pathogenesis and treatment of asthma. TNF is capable of up-regulating a variety of important pro-inflammatory gene products that may contribute to asthmatic airway inflammation. Although anti-TNF approaches have been effective in animal models of asthma, the efficacy of an anti-TNF strategy in humans has not been studied. We have initiated a Phase II clinical trial utilizing a soluble, dimeric fusion protein comprised of the extracellular ligand-binding domain of the human p75 TNF receptor linked to the Fc portion of human IgG1 (TNFR:Fc). TNFR:Fc functions by binding TNF and blocking its interaction with cell surface receptors. This randomized, double-blinded, placebo-controlled, proof of concept trial will utilize a bronchoscopic segmental allergen challenge model in mild atopic asthmatics not requiring corticosteroid therapy. Four doses of TNFR:Fc will be administered via subcutaneous injection over a two-week period. Soluble and cellular inflammatory markers, as well as physiological parameters will be assessed pre- and post-TNFR:Fc therapy and following bronchoscopic segmental allergen challenge. The data generated by this study will assess the utility of future trials of anti-TNF therapy in asthma. Laboratory investigations are also being conducted to identify the mechanism via which ectodomain shedding of cell surface TNF receptors is regulated. For example, shedding of TNF receptors may have an anti-inflammatory effect via the generation of soluble TNF binding proteins and by decreasing the number of cell surface TNF receptors available for ligand binding. We hypothesized that the mechanism of TNFR1 shedding might involve interactions with regulatory ectoproteins. Utilizing a yeast two-hybrid approach, we identified ARTS-1 (Aminopeptidase Regulator of TNFR1 Shedding) as a type II integral membrane protein that binds to the TNFR1 extracellular domain. In vivo binding of membrane-associated ARTS-1 to TNFR1 was confirmed by co-immunoprecipitation experiments utilizing human pulmonary epithelial and umbilical vein endothelial cells. A direct relationship exists between membrane-associated ARTS-1 protein levels and concordant changes in TNFR1 shedding. Cells over-expressing ARTS-1 demonstrated increased TNFR1 shedding and decreased membrane-associated TNFR1, while cells expressing anti-sense ARTS-1 mRNA demonstrated decreased membrane-associated ARTS-1, decreased TNFR1 shedding and increased membrane-associated TNFR1. ARTS-1 neither bound to TNFR2 nor altered its shedding, suggesting specificity for TNFR1. Although a GST-ARTS-1 fusion protein demonstrated selective aminopeptidase activity towards non-polar amino acids, multiple lines of negative evidence suggest that ARTS-1 does not possess TNFR1 sheddase activity. These findings indicate that ARTS-1 is a multi-functional ectoprotein capable of binding to and promoting TNFR1 shedding. Therefore, we have proposed that formation of a TNFR1-ARTS-1 molecular complex represents a novel mechanism by which TNFR1 shedding is regulated. Ongoing investigations are assessing the mechanism by which ARTS-1 regulates TNFR1 shedding.
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