The characterization of gamma-interferon inducible lysosomal thiolreductase (GILT) expression and trafficking in monocytes and macrophages

Gamma-Interferon-inducible Lysosomal Thiolreductase (GILT) is a soluble lysosomal enzyme expressed constitutively in professional Antigen Presenting Cells and induced by interferon-gamma in other cell types. It is synthesized as a precursor protein containing N- and C-terminal pro-peptides that are cleaved as the protein traffics via the mannose-6-phosphate receptor pathway to late endosomes and lysosomas. In B-cells, approximately 20% of GILT escapes lysosomal targeting and is secreted as precursor dimer. Both precursor and mature GILT possess thiolreductase activity that is optimal at acidic pH, consistent with its subcellular localization. GILT thiolreductase activity is required for the processing and presentation of certain epitopes derived from antigens containing disulfide bonds on MHC Class II molecules, a necessary step for the stimulation of the adaptive immune response. Given that GILT is strongly expressed in monocytes and macrophages, important mediators of both innate and adaptive immunity, I sought to characterize GILT expression in these cells, and to determine if GILT played a role in the innate immune response by exposing cultured cells to E. coli-derived lipopolysacchararide or intact bacteria, potent stimulators of Toll-like Receptor 4 signaling.; Using the promonocytic cell line THP-1, an established model for monocyte to macrophage differentiation, I found that exposure to E. coli (or LPS) caused the cells to differentiate to adherent macrophages and subsequently induce robust GILT expression. Unlike GILT induction in response to interferon-gamma, induction by bacteria was found to be dependent on new protein synthesis, Nuclear Factor-kappa-B activity, and the secretion of the proinflammatory cytokines Tumor Necrosis Factor and Interleukin-1-beta. Surprisingly, the majority of GILT synthesized by differentiated THP-1 and primary cells was secreted into the extracellular space as active precursor, rather than matured intracellularly, suggesting a function outside the cell. Secretion likely stems from the differentiation-dependent downregulation of the Uncovering Enzyme, a protein required for tagging soluble lysosomal enzymes with mannose-6-phosphate, thereby preventing mannose-6-phosphate receptor-mediated sorting, and resulting in secretion by default. Precursor GILT was detected in mouse serum, and found to be elevated in response to LPS administration, implying a physiological role for secreted GILT in the context of an innate inflammatory response.