TGF-b1 is a potent immunomodulator whose pro-inflammatory and immunosuppressive activities appear to be equally vital. Data indicate that adherence to fibronectin plays a key role in modulating the TGF-b1-dependent activation of IL-8 gene expression in monocytes. The proposed experiments use in vitro and human models to test the hypothesis that transcriptional and post-transcriptional mechanisms play an equally critic al role in controlling the spatial and temporal expression of IL-8 in monocytes during inflammation. Specifically: 1) To test the hypothesis that TGF-b1 acts through Smad dependent signaling pathways to stimulate the delayed and sustained increase in IL-8 mRNA, we will examine the time-dependent onset of IL-8 gene transcription using Nuclear run on assays. Western blotting and ELlSAs will be used to examine the time-dependent activation of Smad proteins and NF-kB. 2) To test the hypothesis that adherence to fibronectin acts a second signal to stimulate the translation of IL-8 transcripts in TGF-b1-primed monocytes, IL-8 and CXC chemokine translation will be studied by measuring their polysomal distribution in TGF-b1-primed cells prior to and following adherence to Fn. The effect of TGF-b1 and Fn on de novo IL-8 and global protein synthesis will also be assessed by measuring the overall rate of incorporation of [35S] methionine at various timepoints. 3) The relative roles played by the 5'-untranslated region (UTR) and 3'-UTR of the IL-8 mRNA in the post-transcriptional control of IL-8 gene expression wiII be examined using luciferase reporter constructs. 4) We hypothesize that the loss of mechanisms restricting IL-8 protein expression to adherent monocytes contributes to AR DS. To test whether there is a cell-specific defect that contributes to the development of ARDS, we will use real-time kinetic PCR and ELISAs to compare the effects of TGF-b1 on the expression of IL-8 and other mediators in monocytes harvested from patients with and without acute lung injury following pulmonary thromboendarterectomy. The results of these studies may lead to the identification of risk factors that render certain patients susceptible to developing lung injury. Elucidating the factors that modulate net TGF-b1 activity is critical to understanding the evolution and resolution of the inflammatory response and will also facilitate the development and usage of novel therapeutic agents for this highly fatal illness. The research plan, in conjunction with didactic seminars and conferences, is designed to foster the continued growth and productivity of the PI as a pulmonary physician scientist.
