Abstract
As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule (MT) stabilization and secretion of MMPs. Macrophages up-regulate MMP-9 expression and secretion upon immunological challenge and require its activity for migration during the inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution as well as the mechanisms responsible for its trafficking are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contained calreticulin and protein-disulfide isomerase in activated RAW 264.7 macrophages. We demonstrated vesicular organelles of MMP-9 aligned along stable subsets of MTs and showed that selective modulation of MT dynamics contributes to the enhanced trafficking of MMP-9 extracellularly. We found a Rab3D-dependent association of MMP-9 vesicles with the molecular motor kinesin, whose association with the MT network was greatly enhanced after macrophage activation. Finally, we implicated kinesin 5B and 3B isoforms in the effective trafficking of MMP-9 extracellularly.
Highlights
Macrophages robustly secrete matrix metalloproteinase (MMP)-9 upon activation, and mechanisms for active delivery of MMP-9 vesicles to the cell surface have not been described
We found a Rab3D-dependent association of MMP-9 vesicles with the molecular motor kinesin, whose association with the MT network was greatly enhanced after macrophage activation
Gelatin zymography is a common tool used to assess the levels of gelatinases [32], to measure the amount of secreted and intracellular MMP-9 protein levels, we turned to immunoblotting
Summary
Macrophages robustly secrete MMP-9 upon activation, and mechanisms for active delivery of MMP-9 vesicles to the cell surface have not been described. Results: MMP-9 is packaged into unique ER protein-containing vesicles that associate with microtubule motors in activated macrophages. Conclusion: Macrophage activation requires enhanced microtubule stabilization for rapid secretion of up-regulated MMP-9. The classical activation of macrophages with LPS and IFN-␥ causes enhanced microtubule (MT) stabilization and secretion of MMPs. Macrophages up-regulate MMP-9 expression and secretion upon immunological challenge and require its activity for migration during the inflammatory response. The dynamics of MMP-9 production and intracellular distribution as well as the mechanisms responsible for its trafficking are unknown. We localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contained calreticulin and protein-disulfide isomerase in activated RAW 264.7 macrophages. We found a Rab3D-dependent association of MMP-9 vesicles with the molecular motor kinesin, whose association with the MT network was greatly enhanced after macrophage activation. We implicated kinesin 5B and 3B isoforms in the effective trafficking of MMP-9 extracellularly
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