The long range plan of this research project is to understand the pathomechanisms of human cutaneous hypersensitivity granulomas such as sarcoidosis, metal induced granulomas and tuberculoid leprosy. These diseases create disabling, crippling conditions in patients, and because of a lack of more precise information about the disease process, patients do not receive optimal treatment. This project will be carried out using both in vivo and in vitro murine models in which macrophages aggregate about a nidus, differentiate into epithelioid cells and organize into tightly interdigitating cellular tubercles, closely mimicking the clinical granulomas.
The aim i s to characterize factors responsible for these initial effects on macrophages. A candidate 20 kD protein called Granuloma Initiating Factor (GIF) has been purified from schistosome egg granulomas and a potent monospecific rabbit antisera raised. The tissue, cellular and subcellular distribution will be sought by immuno- histochemistry with light and microscopy, using anti-GIF IgG. Since: a) GIF, interleukin-1 (IL-lbeta) and tumor necrosis factor (TNF-alpha) induce granulomatous reactions in vitro and in vivo when bound to inert beads; and b) IL-1beta and TNF-alpha-induced in vivo granulomas contain many GIF positive cells, macrophages grown in vitro will be examined for he induction of GIF. Further, the interactions between GIF and the cytokines will be studied by use of ELISA and appropriate antibodies to detect secreted cytokines, and by Northern blot and in situ hybridization analysis to detect cytokine gene expression in macrophages with time after GIF stimulation. Also, the cytokines and other lymphokines will be added individually to GIF-induced macrophage cultures in soluble form to determine how they affect granuloma formation. N-terminal eleven amino acid sequence of GIF is identical to mouse cyclophilin and the GIF molecule cross reacts with antisera to some mouse and human cyclophilins. However, GIF differs from known cyclophilin, as it is concentrated in inflammatory cells of the granulomas and not distributed ubiquitously. In order to verify whether GIF is a cyclophilin-like, granuloma associated protein, the gene for GIF will be cloned from a mouse cDNA library made from mouse granuloma RNA and then expressed to produce recombinant GIF for further biological study. The results of studies with this novel granuloma initiating factor will provide: a) new insights into how macrophages are induced to differentiate into epithelioid cells and to organize into space consuming tubercles; and b) a more logical basis for developing strategies to interdict organized epithelioid cell granulomas in man.
Showing the most recent 10 out of 11 publications
