The focus of this project is to characterize the nature, genetics and function of a genetically-controlled factor identified by this laboratory that regulates mouse Th1/Th2 cytokine phenotypes and the encephalitogenicity of T-cells in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis in humans. Macrophages from B10.A but not B10.BR mice produce a protein factor, provisionally called cytokine regulatory factor (CYTRF), that down-regulates IFN-gamma and TNF-alpha production by Th1 cells and skews Th1/Th2 commitment to Th2. Preliminary evidence suggests that this factor may be a protein variously called cyclophilin C-associated protein (CyCAP), murine adherent macrophage protein (MAMA), Mac-2 binding protein, and 90K, but which has not previously been implicated in Th1/Th2 cytokine regulation. The proposed studies will test the hypothesis that genetic differences that qualitatively or quantitatively control this protein's activity affect its function in Th1/Th2 cytokine regulation and induction of macrophage-derived CYTRF is CyCAP/90K protein and to identify the basis for the differences in cytokine down-regulatory activity between B10.A and B10.BR mice (through biochemical and molecular analyses, the cloning, expression, and functional characterization of CyCAP from the two strains, and the analyzing the effect of an available CyCAP-/- (knock-out) mutation on CYTRF activity in a B10.ACyCAP-/- congenic mouse whose production will be facilitated by marker-assisted progeny selection protocols); 2) to evaluate the genetic control of CYTRF/CyCAP activity through linkage analyses (using simple sequence length polymorphisms and in other inbred strains, especially strains susceptible to autoimmune diseases); 3) to investigate the mechanisms by which CYTRF/90K both down-regulates IFN-gamma and TNF-alpha production by Th1 cells (using molecular analyses and skews Th1/Th2 commitment to Th2 (in in vitro commitment assays with TCR transgenic mice); and 4) to test the function of CYTRF/90K in autoimmune disease (by crossing different CYTRF/CyCAP alleles into an autoimmune disease-prone strain). These studies may reveal the identity of a new or previously-described autoimmune disease susceptibility gene. The long-range goal of this research is to understand how genetic variation in CYTRF affects cytokine production and susceptibility to autoimmune, inflammatory, and infectious disease in mice and humans.
