Macrophage migration inhibitory factor (MIF), is a pro-inflammatory cytokine produced by the urothelium. During bladder inflammation, MIF production is upregulated in the bladder and MIF is released into the intraluminal space where it exerts proinflammatory effects on the urothelium by activating downstream inflammatory mediators. A novel finding is that MIF is released, not as an unbound momeric protein, but in high-molecular weight complexes with alpha-1 inhibitor 3, a protease inhibitor in the family of alpha2- macroglobulins. In addition, in another novel finding, MIF-alpha1 inhibitor 3 complexes associate with surface glucose related protein 78 in the bladder which likely results in activation of signal transduction pathways. The overall goal of the present proposal is to continue to investigate the mechanism of MIF- mediated inflammation in the bladder by investigating these two novel findings. Based on our recent experimental evidence, our working hypothesis is that during neurogenic inflammation, MIF is released into the bladder lumen and interacts with specific cell-surface proteins (either CD74 or glucose regulated protein 78) on the urothelium to activate signal transduction pathways resulting in the production of other pro- inflammatory mediators. Thus, even though the effects of tachykinins (e.g. Substance P; SP) on the bladder are short-lived, activation of MIF results in an inflammatory loop that, if left unchecked, regulates other inflammatory mediators and maintains an inflammatory condition. In the new line of research described in the present proposal, we will examine in greater detail the mechanisms of MIF release, the effects of blockade of specific cell-surface molecules associated with MIF in the urothelium and the ability of MIF complexes to activate signal transduction in well-described in vitro systems. Identification of the effect of novel cell-surface proteins associated with MIF will add to knowledge of basic mechanisms of MIF's pro- inflammatory functions, and thus the findings of this proposal may extend to other inflammatory conditions where MIF has been demonstrated to play a role. ? ? ?
