Molecular mechanisms of the unconventional secretion of macrophage migration inhibitory factor (MIF)

Macrophage migration inhibitory factor is a pro-inflammatory cytokine that plays a pivotal role in the pathogenesis of inflammatory disease such as septic shock and rheumatoid arthritis. The release of MIF from inflammatory cells is a common feature in these diseases, and a correlation between the severity of disease and MIF production is observed. MIF is a leaderless protein that is secreted from cells by a specialized, non-classical export pathway, possibly involving the transporter ABCA1. The release of MIF nevertheless is tightly regulated with respect to its production in response to different inflammatory, proliferative, and activating stimuli. In recent studies it was demonstrated that the administration of neutralizing anti-MIF antibodies and small molecule MIF inhibitors is advantageous in inflammatory diseases. These approaches have in common that they all target MIF only after it is released from cells. A more effective approach in treating MIF-related diseases may be to target MIF before it is released from cells. The unconventional secretion pathway of MIF might permit to specifically block the release of MIF without compromising the general secretion mechanism of the cell. To date, little is known about the molecular mechanisms involved in the release of MIF. My thesis demonstrates that MIF associates with the novel MIF interaction partner p115, a Golgi-associated protein that takes part in the ER/Golgi secretion pathway. By confocal microscopy, I was able to demonstrate that MIF and p115 are associating in the cytoplasm; following activation, macrophages redistribute MIF to the plasma membrane together with a portion of p115. This finding agrees with results obtained from Western blot and ELISA analysis, demonstrating that upon LPS stimulation both MIF and p115 are co-secreted, accompanied by a corresponding decrease in intracellular protein concentration. Accordingly, I demonstrated that the depletion of p115 from monocytes/macrophages by RNAi results in an inhibition of MIF release after LPS stimulation. Intriguingly, the secretion of other pro-inflammatory cytokines, both conventionally released (TNF-alpha, IL-6) and unconventionally released (IL-1beta) was not affected by the partial depletion of p115. Similarly, the depletion of p115 in HeLa cells did not affect the conventional (FGF-4) or unconventional (FGF-2) secretion. This observation leads to the assumption that it is possible to specifically target the secretion of MIF without compromising the normal and necessary secretory mechanism of cells. It is important to note that the effect of p115 depletion on MIF secretion was not only observed after LPS stimulation, but also after bacterial infection with Chlamydia trachomatis. Notably, the prototypic small molecule MIF inhibitor, 4-iodo-6-phenylpyrimidine, inhibits MIF secretion by targeting the interaction between MIF and p115. My data reveal p115 to be a critical intermediary component in the regulated secretion of MIF from monocytes/macrophages. After the discovery of GRASP65 in the unconventional secretion of acyl-CoA binding protein in Dictyostelium, p115 is the second protein of the Golgi complex attributed with a dual function in secretion. On one hand p115 is involved in the classical secretion pathway tethering vesicles arriving from the ER to the Golgi, and on the other hand it is involved in the unconventional secretion of MIF. The discovery that 4-IPP specifically blocks the secretion of MIF by interfering with MIF’s interaction with p115 gives hope in the search of a potent approach in treating MIF-related diseases by blocking its release from cells. It seems feasible to perform a screening of MIF inhibitors that specifically target MIF’s ability to interact with p115 and block its release from cells. In addition, in my thesis I report on the initial characterization of the structural homolog of MIF, D-dopachrome tautomerase (DDT). After the successful purification of recombinant, native murine DDT, a specific anti-DDT antibody was produced and utilized for the establishment of a DDT-specific ELISA. It was shown that DDT is released from macrophages in response to LPS in a pattern similar to that of MIF. Furthermore, it was shown that the deletion of the mif gene has no effect on the production of DDT. The precise biological function of DDT remains unclear and the detailed characterization of the secretion pathway of DDT was beyond the scope of this thesis, but the comparable release patterns of MIF and DDT suggest related functions of the two proteins